CN101775026B - Spiro alkaloid compound, medicinal composition comprising same and preparation method and application thereof - Google Patents

Abstract

Have the following structural formula compound Acortatarins A (I) and B (II), it is a kind of spiro alkaloid in famous Chinese medicine rhizoma acori graminei, with the morpholine ring structure in natural rare and pharmaceutical chemistry being considered as important pharmacophore, the preparation method of such compound and its application in anti-diabetic nephrosis and chronic kidney disease drug or food is being prepared.

Description

Spiro alkaloid compound and contain its pharmaceutical composition

Technical field: the invention belongs to technical field of pharmaceuticals; Relate to one type and contain the pyrrole ring of aldehyde radical and the spiro alkaloid verivate that the ribose analogue forms; With it is the pharmaceutical composition of effective constituent, its preparation method and in preparation diabetic nephropathy or chronic nephropathy medicine or Application in Food.

Background technology: diabetic nephropathy (diabetic nephropathy; DN) be because the glomerulosclerosis due to the microangiopathies that mellitus cause; It is a kind of occlusive pathology of renal glomerulus; Its take place mainly with the insulin action deficiency due to diabetic Metabolic disorder and active oxygen surplus relevant, clinical manifestation is proteinuria, oedema, hypertension and renal function progressive injury.Current research shows that diabetic nephropathy is to cause renal failure, uremic principal element, but still lacks the efficacious therapy medicine in the world at present, and clinical treatment is main with controlling symptoms.The most scholars of the pathogenesis of DN think relevant with the persistence hyperglycemia.Gp content increases, and can occur the rising of kidney capillary blood vessel basement membrane thickened, ED attenuating and glycolated hemoglobin value in succession.The three all makes nephridial tissue oxygen supply minimizing and damaged.The polyvalent alcohol metabolism is in hyperfunction state under the hyperglycemia state, and under the aldose reductase effect, glucose changes sorbyl alcohol into, and the latter can be changed fructose into again under the SDH effect.Polyvalent alcohol such as sorbyl alcohol and fructose is the hypertonicity material, accumulates the attenuating that in cell, can cause intracellular edema and cell function.Except that hyperglycemia, renal hemodynamics is unusual, not normal, the non-enzymatic glycosylation of pipe ball feedback regulation, polyvalent alcohol metabolic disturbance, prostaglandin(PG) secretion increasing, oxyradical form, extracellular matrix (ECM) metabolic disturbance and various kinds of cell growth factor (like interleukin-) secretion increasing etc. also play an important role in the morbidity of DN.At mesangial cell and renal cells; Produce too much reactive oxygen species in the high sugared inducing cell; Activate active oxygen radical and film and attack complement component; Raise transforming growth factor-beta 1 and ECM gene and proteic expression, cause the damage of cytolemma, it is unusual to cause infringement of renal glomerulus 26S Proteasome Structure and Function and ECM to distribute.High concentration glucose also can directly act on mesangial cell and VSMC; Through effects such as oxyradicals cytoskeleton is destroyed; And blood vessel is reduced the reactivity of the vaso-active substance that contracts, the glomerular arteriole,afferent expansion causes renal glomerulus hypertension.On the other hand; Antioxidant ability of organism such as superoxide-dismutase (SOD), Agifutol px (GSH-PX), katalase isoreactivity descend; The cellular NADPH quantity not sufficient, levels such as plasma antioxidants reduce, and ROS is too much gathered in vivo; Multiple normal protein matter, lipid nucleic acid etc. are caused damage causes diabetic nephropathy, builds consensus.(Nam SM such as Nam; Lee MY, Koh JH etal.Effects of NADPH oxidase inhibitor on diabetic nephropathyin OLETF rats:the role of reducing oxidative stress in itsprotective property.Diabetes Research and Clinical Practice2009; 83:176-82), find that acetovanillon can reduce the secretion of oxidation products MDA, improve oxidative stress protection DN rat in the time spent of doing of research nadph oxidase suppressor factor to the DN rat; (Liu HR such as Liu; Tang XY, Dai DZ et al.Ethanol extractsof Rehmannia complex (Di Huang) containing no Corni fructusimprove early diabetic nephropathy by combining suppression onthe ET-ROS axis with modulate hypoglycemic effect in rats.Journal of Ethnopharmacology 2008; 118:466-72) research also proves; NO and ROS acting in conjunction that inducible nitric oxide synthase (iNOS) produces; Formation is to tissue and the virose peroxynitrite salt of body ONOO-; Activate MAPK MAPK signal path, impel the ETA acceptor to raise, further increased products of oxidative stress.This shows that also activatory endothelin-1 path (ET-1) is all relevant with DN with too much ROS.

ECM and cytokine have been participated in the generation of DN; What report was many at present is mesangial cell excretory cytokine; Like IL-6, MCP-1 (monocytechemotactic protein-1; MCP-1), nf kappa B (nuclearfactor-κ B, NF-κ B) etc.In the generating process of DN, they act on mesangial cell through autocrine and/or paracrine action.Kidney mesentery ECM comprises multiple composition, like IV Collagen Type VI (CIV), Laminin ELISA (LN), III procollagen type (PIII), mucinase (HA) etc.These compositions are not only the main ingredient of kidney mesentery, also are the important component of kidney basilar membrane simultaneously.Mesangial cell occurred continuing firmly to secrete ECM, mesentery hyperplasia, renal glomerulus hypertrophy when DN took place, and finally cause glomerular sclerosis, so ECM had reflected the metabolism state of GBM.IL-6 is mainly derived from activatory monokaryon or scavenger cell, is a kind of cytokine of polygene manifold effect, can induce expression such as allergy albumen (hs-CRP), Fibrinogen, stimulates proliferation of mesangial cells, and collagen is obviously synthetic, causes increasing of ECM.Research confirms; Human and Rat Mesangial all can be secreted IL-6 and expressed IL-6mRNA; Add the incorporation efficiency that IL-6 can increase tritium-labeled thymidine (3H-TdR) in the supernatant of vitro culture mesangial cell, prompting IL-6 is relevant with the ECM secretion increasing with mesangial cell propagation.MCP-1 (MCP-1) is by a kind of homocysteines of excretory such as monocyte, endotheliocyte and nephridial tissue cells, is a member of inflammation chemokine family, Monocytes is had the chemokine of chemotactic, activation dual function.Research was more in struvite kidney disease in the past; Recently in DN, also be considered to scavenger cell and penetrate into renal glomerulus; Cause diseases associated with inflammation (the Kanamori H of glomerular injury; Matsubara T, Mima A etal.Inhibition of MCP-1/CCR2 pathway ameliorates thedevelopment of diabetic nephropathy.Biochemical andBiophysical Research Communications 2007; 360:772-777), thus the effect of MCP-1 in DN takes place more and more draw attention.Report such as Kanamori DN rat model MCP-1 level obviously increases and is consistent with the kidney function damage degree, the path of blocking-up MCP-1 and acceptor CCR2 mediation thereof, and the expression that can effectively reduce IV Collagen Type VI and TGF-β improves glomerular sclerosis.(Tam FW such as Tam; Riser BL, Meeran K et al.Urinary monocyte chemoattractant protein-1 (MCP-1) andconnective tissue growth factor (CCN2) as prognostic markersfor progression of diabetic nephropathy.Cytokine 2009; 47:37-42) report that also the MCP-1 knock out mice has provide protection to U-9889 inductive diabetic nephropathy model.

To sum up, recent research progress shows that the pathomechanism of dn-incidence and development is excessively to produce with the intracellular reactive chalcogen, cytokine such as IL-6, and the excessive secretion of MCP-1 etc. and extracellular matrix such as IV Collagen Type VI (CIV) is closely related.Observe the influence of medicine, become the universal method of present research medicine for treating diabetic nephropathy the sugared inductive kidney of height mesangial cell ROS and cytokine.

Medicinal plant is the important source of developing new drug, and particularly in microbiotic and cancer therapy drug field, the medicine that nearly 60-80% is arranged according to statistics is from the natural product or derivatives thereof.Therefore have especially from nature that to seek effective medicine the historical tcm-of human be possible.Rhizome of Grass leaf Sweelflag (Acorus tatarinowii Schott.) is Araeceae (Araceae) Acorus per nnial herb, main product in Sichuan, ground such as Zhejiang, Jiangsu, be used as medicine with dry rhizome.Rhizome of Grass leaf Sweelflag beginning is stated from Shennong's Herbal, classifies as top gradely, and flavor is hot warm in nature, the gas of tool fragrance, and the power that row looses is strong, is to declare the sensible good merchantable brand of gas.Traditional Chinese medicine thinks that it is have one's ideas straightened out open-minded phlegm, inducing resuscitation intelligence development, hearing-improing and eyesight improving, dampness elimination appetizing and the cold numbness of removing of loosing that the function of Rhizome of Grass leaf Sweelflag cures mainly.Join ginger like these article and smash clothes, control in blinded judgment; Join polygala root and be " not forgetting to loose " (" Standards of Diagnosis and Treatment "), it is forgetful to control fascination; Join the coptis etc. and be " calamus ball " (" Puji Fang "), can tranquillizing by calming the heart, control insomnia forgetfulness; Join the rhizome of Chinese monkshood for " calamus looses " (" Shengji Zonglu "), control cold numbness bodily pain; Join polygala root, Poria cocos, genseng for " happy loosing " (" prescriptions worth thousand gold "), control gloomy mistake will etc.The clinical difficult disease such as treatment epilepsy, coma due to blocking of the respiratory system, pyreticosis coma, forgetful, senile dementia that are widely used in.Can find out that it is the good medicine of treatment encephalopathic that Rhizome of Grass leaf Sweelflag is given the impressive effect of people.Though the treatment that the Chinese medicine compound prescription that contains Rhizome of Grass leaf Sweelflag is used for orthopaedics and dn-was also arranged modern age, still not deeply, effective constituent also can disclose far away in effective substance research.

Acortatarin A; It is one type of spiro alkaloid compound of the therefrom middle discovery of remedies calamus (A.tatarinowii Schott.) with the important drug effect of having of remarkable inhibition kidney mesangial cell active oxygen, the excessive generation of inflammatory factor (like IL-6, MCP-1, Collagen I, Collagen IV) functional group; Do not see its structural formula report at present as yet; Therefore be novel substance, report was not met in the pharmacological action that its inhibition kidney is cytoactive oxygen and multiple inflammatory factor yet.

Summary of the invention: the object of the present invention is to provide new compd A cortatarin A and verivate thereof with pharmaceutical use; The method of from Rhizome of Grass leaf Sweelflag (A.tatarinowii Schott.), extract, separating this compounds; With this compounds is the pharmaceutical composition of effective constituent; This invention compound is preparing anti-diabetic property ephrosis or/and the application in the medicine of chronic nephropathy.

Above-mentioned purpose of the present invention is to be achieved by following technical scheme:

Compd A cortatarins A (I) and B (II) with following structural formula

The method for preparing Acortatarins A (I) and B (II) is got Rhizome of Grass leaf Sweelflag (the Acorus tatarinowii Schott.) rhizome of drying and crushing, uses the water boiling and extraction secondary, filters merging filtrate; Residue with 95% alcohol reflux once filters, and filtrating merges with water liquid, and concentrating under reduced pressure gets extract; Extract mixture is suspended from the water, fully extracts with ETHYLE ACETATE, propyl carbinol successively, and equal-volume respectively extracts three times; N-butyl alcohol extract is through silica gel column chromatography 200-300 order silica gel, chloroform-methanol-water gradient elution: 9: 1, and 8: 2; 8: 2: 0.2,7: 3: 0.1,7: 3: 0.5; Each gradient elution volume is 4L; Every part receives 1000mL, thin-layer chromatography, and the colour developing of 10% sulfuric acid ethanol merges according to spot is identical, obtains eight components, and wherein component IV is through MCI gel CHP 20P column chromatography; The methanol-water gradient elution, 10%, 20%, 30%, 35%, 40%; Each gradient elution quantity of solvent is 2 times of column volumes, and each gradient is collected 2 parts, obtains four component A-D altogether; The component B that wherein collects during the 4th column volume wash-out obtains containing the component of compound I and II through Sephadex LH-20 methanol-eluted fractions, and above-mentioned developer presents yellow spotting; This component is further through half preparative high-performance liquid chromatographic Agilent, 1100 HPLC system, Zorbax SB-C-18, Agilent; 9.4mm * 25cm, 20: 80 purifying of methanol-water get compound I and II.

Pharmaceutical composition wherein contains Acortatarins A (I) and B (II) compound and the pharmaceutically acceptable carrier of treating significant quantity.

The medicine of prevention and treatment of diabetic nephropathy wherein contains Acortatarins A (I) and B (II) compound and the pharmaceutically acceptable carrier of treating significant quantity.

The application in the medicine of preparation anti-diabetic property ephrosis of Acortatarins A (I) and B (II) compound.

The application in the medicine of the anti-chronic nephropathy of preparation of Acortatarins A (I) and B (II) compound.

The application in the preparation protective foods of Acortatarins A (I) and B (II) compound.

Compounds Acortatarins A and B that the present invention finds from the Rhizome of Grass leaf Sweelflag rhizome have important pharmaceutical use in dn-or chronic nephropathy.

The compounds of this invention can directly be used separately, and Combination application also can comprise the form use of plant milk extract composition compound with other medicines each other, can use different pharmaceutical excipients, processes multiple solid preparation and liquid preparation.Pharmaceutical composition of the present invention is used with the form of per weight dose.But two kinds of form administrations of medicine oral administration of the present invention and injection.Usage quantity can be carried out one or many according to variations such as the type of route of administration, patient's age, body weight, the disease of treating and severity and used.Concerning the adult, 1-100mg every day is proper for dosage.

Description of drawings:

Figure 1A cortatarins A and B suppress high sugared inductive mesangial cell ROS and produce;

A&B, the Acortatarin A and the B of mesangial cell and different concns, or c-SOD preincubate 1 hour stimulated 3 hours with high sugar again; C, the c-SOD preincubate of mesangial cell and 10 μ MAcortatarin A or 100u/mL 1 hour stimulated 1 to 24 hour with normal concentration 5.6mM (NG) or high density 25mM (HG) D-glucose then.With DCFH dyeing, the generation of cells were tested by flow cytometry ROS.Data are represented with the mean ± standard deviation of 3 independent experiments.ANOVA, p＜0.001 is at figure A, B, and C; * p＜0.05vsNG; #p＜0.05vsHG;

Fig. 2 kidney mesangial cell strain inflammatory factor determination experiment: adopt the American ADL ELISA of company test kit to detect Rat Mesangial culture supernatant IL-6, MCP-1, Collagen I, Collagen IV expresses.Fig. 2 A is that IL-6 expresses; Fig. 2 B is that MCP-1 expresses; Fig. 2 C expresses for Collagen I; Fig. 2 D expresses for Collagen IV.

Embodiment:

Further specify essentiality content of the present invention with embodiments of the invention below, but do not limit the present invention with this.Essence according to the present invention all belongs to scope of the present invention to the simple modifications that the present invention carries out.

Embodiment 1:

The preparation of Acortatarins A and B and structure are identified:

Get the Rhizome of Grass leaf Sweelflag rhizome 50kg of drying and crushing, at first use water boiling and extraction secondary (100L * 2), filter merging filtrate; Residue filters with 95% alcohol reflux once (100L * 1), and filtrating merges with water liquid, and concentrating under reduced pressure gets extract 4.7kg.Extract mixture is suspended from the 6L water, fully extracts (equal-volume respectively extracts three times) with ETHYLE ACETATE, propyl carbinol successively.N-butyl alcohol extract 315g through silica gel column chromatography (200-300 order silica gel 2kg, chloroform-methanol-water gradient elution: 9: 1,8: 2,8: 2: 0.2,7: 3: 0.1,7: 3: 0.5; Each gradient elution volume is 4L; Every part receives 1000mL), thin-layer chromatography, the colour developing of 10% sulfuric acid ethanol merges according to spot is identical, obtains eight components, and wherein component IV (10L-14L) is (9.3g) through MCI gel CHP 20P column chromatography (post bed specification: 5.5 * 30cm; The methanol-water gradient elution, 10%, 20%, 30%, 35%, 40%; Each gradient elution quantity of solvent is 2 times of column volumes, and each gradient is collected 2 parts), obtain four components (A-D) altogether, wherein component B (component of collecting during the 4th column volume wash-out) is (1.6g) through Sephadex LH-20 (post specification: 3.0 * 70cm; Methanol-eluted fractions) obtains containing the component of compound I and II; It is characterized by above-mentioned developer and present yellow spotting, and be principal spot, this component is further through half preparative high-performance liquid chromatographic (Agilent 1100HPLC system; Zorbax SB-C-18; Agilent, 9.4mm * 25cm, methanol-water 20: 80) purifying gets compound I (7.3mg) and II (3.4mg).

The structure appraising datum of Acortatarins A and B:

The digital polarimeter of JASCO DIP-370 type; Bio-Rad FTS-135 type IR (KBr compressing tablet); UV210A type ultraviolet-visible protractor; VG AUTO Spec-3000 and Finnigan MAT 90 mass spectrographs; Bruker DRX-500 NMR, TMS are as interior mark, and δ is ppm, and J is Hz; Fusing point is measured with X-4 digital micro-analysis fusing point appearance (not calibrated).Silica gel is that Haiyang Chemical Plant, Qingdao produces; MCI-gel CHP-20P, Sephadex LH-20 are General Corporation's product; Developer is 10%H ₂SO ₄Ethanolic soln.

Acortatarin A structural formula is as follows:

Acortatarin A: clear crystal, fusing point 164-166 ℃; [α] ²⁷ _D+ 253.1 (c 0.10, MeOH); UV (MeOH) λ max (log ε): 296 (4.25), 254 (3.75) nm; IR (KBr) ν max:3426,2928,2855,1734,11713,1641,1462,1185cm ^{-1 1}H and ¹³C NMR data are seen Table I; FABMS:m/z 254 [M+H] ⁺(100), 206 (9); HRESIMS m/z 276.0843 (calcd for [M+Na] ⁺276.0847).

Acortatarin B structural formula is as follows:

Acortatarin B: white amorphous solid, fusing point 151-153 ℃; [α] ²⁷ _D-92.73 (c0.10, MeOH); UV (MeOH) λ max (log ε): 295 (4.05), 255 (3.63) nm; IR (KBr) ν max:3427,2923,2852,1640,1033cm ^{-1 1}H and ¹³C NMR data are seen table 1; FABMS:m/z270 [M+H] ⁺(81), 255 (23), 225 (100), 207 (31), 193 (10), 179 (6), 163 (33), 115 (35), 74 (22); HRESIMS m/z 292.0803 (calcd for [M+Na] ⁺292.0797).

Table I. ¹H (500MHz) and ¹³C NMR (125MHz) Data of I and II

Embodiment 2: the method by embodiment 1 makes Acortatarins A or B, adds the injection water by conventional method, and smart filter can be made into injection liquid after the embedding sterilization.

Embodiment 3: the method by embodiment 1 makes Acortatarins A or B, and it is dissolved in the sterile water for injection, filter with aseptic funnel, and packing, aseptic sealing by fusing promptly gets powder injection behind the frozen drying.

Embodiment 4: the method by embodiment 1 makes Acortatarins A or B, is equipped with various pharmaceutical excipients by conventional method and can be made into tablet:

Acortatar ins A or B medicinal composition formulation-tablet:

Use Acortatarins A or B as active constituents of medicine; Use the adjunct ingredient of several kinds of vehicle as preparation medicinal composition tablet; Proportioning is processed every tablet samples that contains Acortatarins A or B pharmaceutical cpd 5～60mg according to a certain percentage, and table 2 provides the formula rate of conventional tablet.

The bulk drug and the accessory formula of table 2 Acortatarins A or B medicinal composition tablet

The method that some amount Acortatarins A or B raw material and vehicle auxiliary material is prepared into the various dose tablet formulation is with several kinds of vehicle auxiliary materials and bulk drug uniform mixing; Add 1% sodium cellulose glycolate solution and process soft material in right amount; The granulation of sieving; The wet grain oven dry and the whole grain that sieves, adding Magnesium Stearate and talcum powder mix the back compressing tablet and promptly get.

Embodiment 5: the method by embodiment 1 makes Acortatarins A or B, is equipped with various pharmaceutical excipients by conventional method and can be made into capsule:

Acortatarins A or B medicinal composition formulation-capsular preparation:

Contain Acortatarins A or B preparation as the drug regimen capsule preparations of effective constituent; Use Acortatarins A or B as active constituents of medicine, use the adjunct ingredient of several kinds of vehicle as preparation medicinal composition capsule; Proportioning is processed the capsule preparations that contains Acortatarins A or B pharmaceutical cpd 5～50mg in every capsules according to a certain percentage, and table 3 provides the formula rate of conventional capsule preparation:

The bulk drug and the accessory formula of table 3 Acortatarins A or B medicinal composition capsule preparations

The method that the Acortatarins A of some amount or B and vehicle auxiliary material is prepared into capsule preparations is: several kinds of vehicle auxiliary materials and Acortatarins A or B bulk drug are mixed; It is an amount of to add 1% sodium cellulose glycolate solution; Process wet grain oven dry and sieve whole; Add Magnesium Stearate and mix, insert capsule and make; Or do not use granulation step, and and directly Acortatarins A or B bulk drug and several kinds of vehicle auxiliary materials are mixed, after sieving, directly incapsulate and make.

Embodiment 6:

The anti diabetes and kidney disease of The compounds of this invention Acortatarins A or B and the pharmaceutical composition formed with pharmaceutical excipient thereof or the pharmacological action of chronic nephropathy.

Anti-kidney mesangial cell active oxygen experiment:

Reference literature method (Wei XF; Zhou QG, Hou FF et al.Advancedoxidation protein products induce mesangial cell perturbationthrough PKC-dependent activation of NADPH oxidase.Am J PhysiolRenal Physiol 2009; 296:F427-F437), Rat Mesangial strain (HBZY-1, Life-Science Academy of Wuhan University, Wuhan; China) under 37 ℃ of conditions, cultivation is 7.4 DMEM (Invitrogen, Carlsbad in the pH value; CA) in the nutrient solution, add 10% foetal calf serum (Invitrogen, Carlsbad in the nutrient solution; CA), 2mM Stimulina, 100U/ml penicillium mould and 100 μ g/ml Streptomycin sulphates.Cytogamy reaches at 80% o'clock, adopts hungry 24 hours of nutrient solution containing 0.5% foetal calf serum, makes cell synchronization in the G0 phase, is used for subsequent experimental.

For the effect of detection architecture I and II, mesangial cell at first with the structure I of different concns, II or endochylema type Cu/Zn superoxide dismutase (c-SOD; An intracellularsuperoxide scavenger, Sigma Chemical Co., St.Louis; MO) preincubate is 1 hour; Then with 5.6mM (normal glucose, NG) or 25mM (highglucose, D-glucose (Xia L HG); Wang H; Munk S et al.Reactiveoxygen species, PKC-β 1, and PKC-ζ mediatehigh-glucose-induced vascular endothelial growth factorexpression in mesangial cells.Am J Physiol Endocrinol Metab2007; 293:E1280-E1288) stimulate different time, observe each item index.

Cell viability is measured:

Adopt trypan blue to repel experiment and detect cell viability.Reference literature (Frank S; Zacharowski K, Wray GM et al.Identification of copper/zincsuperoxide dismutase as a novel nitric oxide-regulated gene inrat glomerular mesangial cells and kidneys of endotoxemic rats.FASEB J 1999; 13:869-882), tryptic digestion is collected cell, and (CA) with 1: 1 volume mixture, room temperature reaction 3 minutes is with the cell counter counting cells for Life Technologies, Carlsbad for mesangial cell suspension and 0.4% trypan blue.The trypan blue exclusion cell is a viable cell, and the painted cell of trypan blue is a dead cell.

Intracellular reactive oxygen (reactive oxygen species, ROS) assay:

ROS content adopts fluorescent probe carboxymethyl-H2-dichlorofluorescein diacetate (CM-H2-DCF-DA in the cell; Sigma Chemical Co.; St.Louis; MO) dyeing; Flow cytometer detects (XiaL, Wang H, Goldberg HJ et al.Mesangial cell NADPH oxidaseupregulation in high glucose is protein kinase C dependent andrequired for collagen IV expression.Am J Physiol Renal Physiol2006; 290:F345-F356).Cell is collected in digestion, hatches 30 minutes with CM-H2-DCF-DA (1 μ M).(BD FACSCalibur system, FranklinLakes NJ) measure fluorescence intensity (excitation wavelength lambda=488nm, emission wavelength lambda=515nm) with flow cytometer.Every group of ROS content is with the ratio value representation of each group cell fluorescence intensity with normal sugared concentration cultured cells fluorescence intensity; The result proofreaies and correct (Rygiel TP with total protein of cell content; Mertens AE, Strumane K et al.The Rac activator Tiaml preventskeratinocyte apoptosis by controlling ROS-mediated ERKphosphorylation.J Cell Sci 2008; 121:1183-1192).

Statistical analysis:

All test triplicate.Continuous variable is expressed as mean ± standard deviation, adopts one-way analysis of variance to compare, and SPSS 13.0 carries out statistical analysis.Adopt the Student-Newman-Keuls method to compare when variance is neat, adopt Dunnett ' s T3 method to compare during heterogeneity of variance.Two-tailed test P value thinks to have significant difference less than 0.05.

The result:

Acortatarins A and B have suppressed high sugared inductive mesangial cell ROS and have generated.

This experimental result shows; High sugar has induced mesangial cell ROS to generate; This and previous report (Xia L, Wang H, Munk S et al.Reactive oxygen species; PKC-β 1, and PKC-ζ mediate high-glucose-induced vascular endothelialgrowth factor expression in mesangial cells.Am J PhysiolEndocrinol Metab 2007; 293:E1280-E1288.Xia?L，Wang?H，Goldberg?HJ?et?al.Mesangial?cell?NADPH?oxidase?upregulationin?high?glucose?is?protein?kinase?C?dependent?and?required?forcollagen?IV?expression.Am?J?Physiol?Renal?Physiol?2006；290：F345-F356。Ha H; Yu MR, Choi YJ et al.Role of highglucose-induced nuclear factor-κ B activation in monocytechemoattractant protein-1 expression by mesangial cells.J AmSoc Nephrol 2002; 13:894-902) unanimity.Can suppress the excessive generation that high sugar is induced mesangial cell ROS in order to detect Acortatarins A and B, mesangial cell be at first hatched with the Acortatarins A of different concns and B or c-SOD (positive control) in advance, stimulates 3 hours with high sugar then.As scheme shown in the Figure 1A&B, the generation of high sugared inductive mesangial cell ROS is obviously suppressed by Acortatarin A, and Acortatarin B also shows slight retarding effect.The anti-oxidant activity of Acortatarins A and B is dose-dependently, and reaches the largest inhibition effect at 10 μ M (Acortatarin A) and 50 μ M (Acortatarin B) respectively.

For further proof Acortatarin A is at the anti-oxidative effect of mesangial cell, the Acortatarin A of mesangial cell and 10 μ M or the c-SOD of 100u/ml were hatched 1 hour in advance, then with 5.6 or the D-glucose incubation of 25mM 1 to 24 hour.As scheme shown in the Figure 1C, high sugar induces mesangial cell ROS to produce with the time-dependent mode.And show that through the ROS result who detects different time points Acortatarin A has almost reduced the generation of half ROS.

In order to get rid of Acortatarins A and the B toxic effect for cell, each experiment finishes all to adopt trypan blue repulsion experiment to detect cell viability.The result shows between each group does not have significant difference (data not shown).This result shows that the mesangial cell that Acortatarins A and B cultivate for high sugar has anti-oxidative effect.

Acortatarins A and B suppress high sugared inductive mesangial cell ROS and produce (seeing shown in Figure 1):

A&B, the Acortatarin A and the B of mesangial cell and different concns, or c-SOD preincubate 1 hour stimulated 3 hours with high sugar again; C, the c-SOD preincubate of mesangial cell and 10 μ MAcortatarin A or 100u/mL 1 hour stimulated 1 to 24 hour with normal concentration 5.6mM (NG) or high density 25mM (HG) D-glucose then.With DCFH dyeing, the generation of cells were tested by flow cytometry ROS.Data are represented with the mean ± standard deviation of 3 independent experiments.ANOVA, p＜0.001 is at figure A, B, and C; * p＜0.05vs NG; #p＜0.05 vs HG.

Kidney mesangial cell strain inflammatory factor determination experiment (seeing shown in Figure 2):

Adopt the American ADL ELISA of company test kit to detect Rat Mesangial culture supernatant IL-6, MCP-1, Collagen I, Collagen IV expresses.Fig. 2 A is that IL-6 expresses; Fig. 2 B is that MCP-1 expresses; Fig. 2 C expresses for Collagen I; Fig. 2 D expresses for Collagen IV.

Experimental principle:

Adopt double antibody sandwich method to measure Rat Mesangial culture supernatant MCP-1 (IL-6, Collagen IV, Collagen I) level in the sample.Anti--MCP-1 (IL-6, Collagen IV, Collagen I) antibody sandwich microwell plate with purifying; Process insolubilized antibody; In the micropore that encapsulates monoclonal antibody, add rat MCP-1 (IL-6, Collagen IV, CollagenI) successively; With MCP-1 (IL-6, Collagen IV, the Collagen I) antibodies of HRP mark, form antibody-antigen-hrp-antibody complex again, through thoroughly adding substrate TMB colour developing after the washing.TMB changes into blueness under the catalysis of HRP enzyme, and under the effect of acid, changes into final yellow.The depth of color and the MCP-1 in the sample, IL-6, Collagen IV, Collagen I are proportionate.Measure absorbancy (OD value) down with ELIASA 450nm wavelength, through rat MCP-1, IL-6, Collagen IV, CollagenI concentration in the typical curve calculation sample.

Sample process:

1) cells and supernatant: sterile tube is collected, and 2,000rpm/min, centrifugal 20min.The careful supernatant of collecting.Packing is frozen in-80 ℃.

2) scrape and get the diapire cell, the cracking of total protein lysate, 12,000rpm/min, 4 ℃ of centrifugal 10min collect supernatant, and the Bradford method is measured total protein content.

ELISA detects, and operation is undertaken by the test kit explanation:

1) dilution of standard substance and application of sample: the enzyme mark encapsulates plate and establishes the standard substance hole, and doubling dilution (each hole application of sample amount of dilution back all is 50 μ l) adds standard substance MCP-1 (IL-6, Collagen IV, Collagen I).

2) application of sample: establish blank well (the blank hole does not add sample and enzyme marking reagent, and all the other each rapid operations are identical), testing sample hole (the diluted sample degree is 5 times) respectively.Rock mixing gently.

3) incubation: 37 ℃, 30min.

4) washing: discard liquid in the plate, dry, washing lotion is washed 5 times, 30 seconds/inferior.Thieving paper is clapped and is done.

5) enzyme-added: every hole adds 50 μ l enzyme labelling liquid, except the blank well.

6) incubation: 37 ℃, 30min.

7) washing: discard liquid in the plate, dry, washing lotion is washed 5 times, 30 seconds/inferior.Thieving paper is clapped and is done.

8) colour developing: every hole adds substrate A, each 50 μ l/ hole of B liquid.37℃，15min。Lucifuge.

9) stop: every hole adds each 50 μ l/ hole of stop buffer.

10) measure: with the blank well zeroing, ELIASA 450nm reads absorbancy (OD value).

11) the experiment repetition is 3 times.

The result calculates: Curve Expert1.3 matched curve analytical calculation sample concentration is also proofreaied and correct (MCP-1 & IL-6:pg/mg cell protein with total protein of cell content; CollagenIV & Collagen I:ng/mg cell protein).The result is as shown in the figure.SCP-88 in the Acortatarin A corresponding diagram wherein.

Above presentation of results Acortatarin A (I) is to the sugared inductive kidney of height mesangial cell strain inflammatory factor IL-6; MCP-1; Collagen I and Collagen IV have the obvious suppression effect; And these cytokines play an important role in the evolution of dn-pathology, and therefore above-mentioned pharmacological evaluation shows that compound of the present invention is worth the control of dn-.

The compounds of this invention says that from structure comparison is novel, and contains the important drug effect functional group that generally acknowledge in the pharmaceutical chemistry field in the structure, and the entire compound structure has into the property of medicine.Take to screen targetedly the generally acknowledged high sugar of dn-and induce kidney mesangial cell strain model; Find The compounds of this invention ability remarkable inhibiting activity oxygen and several kinds of inflammatory factor (IL-6; MCP-1; CollagenI, Collagen IV), show that therefore this invention compound has important value in dn-and chronic nephropathy control, can be used for the control of dn-and chronic nephropathy.

Claims (6)

1. the spiro alkaloid compound Acortatarins A (I) and the B (II) that have following structural formula,

2. pharmaceutical composition wherein contains the compound and the pharmaceutically acceptable carrier of the claim 1 of treating significant quantity.

3. the prevention and the medicine of treatment of diabetic nephropathy wherein contain the compound and the pharmaceutically acceptable carrier of the claim 1 of treating significant quantity.

4. the application of compound in the medicine of preparation anti diabetes and kidney disease in the claim 1.

5. the application of compound in the medicine of the anti-chronic nephropathy of preparation in the claim 1.

6. the application of compound in the preparation protective foods in the claim 1.

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