CN102652786B - Application of total extract composite of Luiwei Dijhuang decoction in preparing medicament for treating diabetic nephropathy - Google Patents
Application of total extract composite of Luiwei Dijhuang decoction in preparing medicament for treating diabetic nephropathy Download PDFInfo
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Abstract
The invention provides application of a total extract composite of Luiwei Dihuang decoction in preparing a medicament for treating diabetic nephropathy. The total extract composite of Luiwei Dijhuang decoction consists of prepared rehmannia, prepared dogwood, yam, rhizoma alismatis, tree peony bark and poria in a weight ratio of 8:4:4:3:3:3, and is characterized by comprising the volatile oil beta-cyclodextrin inclusion complex components of the total extract of the Luiwei Dijhuang decoction, the polysaccharide components of the total extract of the Luiwei Dijhuang decoction, the glucoside components of the total extract of the Luiwei Dijhuang decoction and the fat-soluble components of the total extract of the Luiwei Dijhuang decoction. According to the application, the defect that the fat-soluble effective components of the total extract of the Luiwei Dijhuang decoction are ignored in the prior art can be overcome, and the more complete component composition of the total extract of the Luiwei Dijhuang decoction can be obtained by the extraction process, and the composite is possible to treat the diabetic nephropathy. Based on the new indication and new target treatment of the Luiwei Dijhuang decoction, the composite can be used for treating the diabetic nephropathy due to inhibition of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase.
Description
Technical field
The present invention relates to a kind of application of Chinese medicine composition, be specifically related to the application of a kind of LIUWEI DIHUANG TANG total extract compositions in preparation treatment medicine for treating diabetic nephropathy.
Technical background
LIUWEI DIHUANG TANG comes from the < < key to therapeutic of children's disease > > of Song dynasty physician Qian Yi, the party is total to Six-element Chinese medicine by Radix Rehmanniae Preparata, Fructus Corni (system), Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria and forms, and is the basic prescription of the traditional Chinese medical science " nourishing kidney yin ".Its formula has the advantages that three benefits three are rushed down: LIUWEI DIHUANG TANG is reused Radix Rehmanniae Preparata enriching yin and nourishing kidney, and life essence-filling, marrow-benefitting is monarch drug.Fructus Corni is taken a tonic or nourishing food to build up one's health Liver and kidney, and energy arresting seminal emission, gets the meaning that The liver and the kidney have a common source, and Rhizoma Dioscoreae tonification spleen is cloudy, also can reinforce the kidney, and be ministerial drug altogether.Three medicines coordinate, and kidney liver spleen three is cloudy also to be mended, and be to be three benefits, but Radix Rehmanniae Preparata consumption is Fructus Corni and Rhizoma Dioscoreae sum, therefore still take the kidney invigorating as main.Rhizoma Alismatis dampness removing and to let out kidney turbid, and it is greasy to subtract the taste of Radix Rehmanniae Preparata, the light spleen that oozes of Poria is wet, and helps the strong fortune of Rhizoma Dioscoreae, and to rush down altogether kidney turbid with Rhizoma Alismatis, helps Kidney-Yin its position of must answering, and Cortex Moutan is let out clearly deficiency-heat, and the temperature of Fructus Corni (processed) is puckery.Three medicines are called three and rush down, and are adjuvant drug.Six-element share, and three mend three rushes down, and wherein tonic consumption overweights cathartic, is that to mend be main, and liver spleen kidney three is cloudy and mend, and take tonifying kidney-yin as main.The party is described as the ancestral of Tonifying Kidney Recipe, is mainly used in soreness of the waist and knees, dizziness and tinnitus, the heating of the trick heart, the symptoms such as night sweat of passing out semen that deficiency of the kidney yin causes.Through modern pharmacology research, show, LIUWEI DIHUANG TANG has the renal function of improvement, regulate immunity, defying age, antitumor, resisting fatigue, anti-low temperature, anoxia enduring, blood fat reducing, blood pressure lowering, blood sugar lowering and the effect such as enhance metabolism.Liuwei Dihuang preparation is extensive use in tcm clinical practice, and has obtained good therapeutic effect.But the party exists obvious deficiency in actual applications at present, is exactly that dosage is large, medication is inconvenient, and therefore, improving its preparation technology just has its significance.
According to the literature, the liposoluble constituent in Liuwei Dihuang preparation has good hypoglycemic activity [list of references: Korea Spro's Jing is super, Ji Hui to diabetes model, Xue Chengfeng, Deng. the hypoglycemic activity of Terpenes from Fructus Corni. Chinese natural drug, 2006,4(2): 125-129].In the patent of number of patent application " 200410058600.X ", the preparation method of Liuwei Dihuang preparation has been considered polysaccharide and glucosides effective constituents in LIUWEI DIHUANG TANG, but in preparation process, has ignored the fat-soluble effective site in Liuwei Dihuang preparation.
In the patent of number of patent application " 200410062502.3 ", the preparation method of Liuwei Dihuang preparation has been considered polysaccharide composition and liposoluble constituent, but this method is only that in formula for Liuwei Dihuang, part medicine has adopted alcohol extraction, be not whole extraction, lost the fat-soluble effective ingredient of part.
Bibliographical information, the molecular weight of the active polysaccharide of Poria is 3.8 * 10
4left and right [list of references: Chen Chunxia. the chemical research of carboxymethyl pachyman. edible fungi of china, 1994, (05): 35], Chinese yam polysaccharide molecular weight is 4.22 * 10
4left and right [list of references: Bears is flat, Jiang Lingzhi, Yu Junlong, etc. Chinese yam polysaccharide research comment. Chinese medicine academic periodical, 2003, (11): 53], the molecular weight of skunk bush polysaccharide is 2.47 * 10
4left and right [king bathes inscription for list of references: Yang Yun, Liu Cuiping, etc. the chemical research of skunk bush polysaccharide. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 1999,24(10): 615], the molecular weight of the hypoglycemic active polysaccharide of Cortex Moutan is 1.3 * 10
4[Zhao's flag is flat for list of references: Chen Yan, Shen Yeshou in left and right, Deng. the purification of Cortex Moutan polysaccharide PSM2b and physicochemical property research thereof. laser biology journal, 2004,13(3): 237-241], the molecular weight at visible six drugs containing rehmanniae active polysaccharide position focuses mostly between 10,000～50,000.
summary of the invention
The object of this invention is to provide the application of a kind of LIUWEI DIHUANG TANG total extract compositions in preparation treatment medicine for treating diabetic nephropathy.The present invention will overcome prior art and ignore the deficiency of the fat-soluble effective ingredient of LIUWEI DIHUANG TANG total extract, pass through extraction process, the more complete component that obtains LIUWEI DIHUANG TANG total extract forms, and makes to adopt this compositions treatment treatment diabetic nephropathy to become possibility.The present invention is based on treatment new indication and the novel targets of LIUWEI DIHUANG TANG: stress state is a kind of pathological and physiological condition of complexity.Wherein, the activation of nadph oxidase is most important source.The pharmacological action of " three mend three lets out " of LIUWEI DIHUANG TANG, may come from the activation that suppresses nadph oxidase, thereby, can reduce er stress molecule, alleviate without superimposed proteins react (Unfolded protein response, UPR) LIUWEI DIHUANG TANG treatment diabetic nephropathy, owing to suppressing NADPH oxidase.
Pharmaceutical composition involved in the present invention is, a kind of LIUWEI DIHUANG TANG total extract compositions, crude drug is by Radix Rehmanniae Preparata, Fructus Corni (system), Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria form according to the weight ratio of 10.4 ~ 7.6:5.2 ~ 2.8:5.2 ~ 2.8:3.9 ~ 2.1:3.9 ~ 2.1:3.9 ~ 2.1, it is characterized in that, in described compositions, include: LIUWEI DIHUANG TANG total extract volatile oil beta cyclodextrin inclusion complex component, LIUWEI DIHUANG TANG total extract polysaccharide component, taste Dihuang Decoction total extract glucosides class component, and LIUWEI DIHUANG TANG total extract fat-soluble ingredient.
The optimized proportion of above-mentioned raw materials medicine is: Radix Rehmanniae Preparata, Fructus Corni (system), Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria form according to the weight ratio of 8:4:4:3:3:3.
In above-mentioned raw materials medicine, described Radix Rehmanniae, Fructus Corni and Rhizoma Dioscoreae can be selected respectively raw product; Also can select respectively goods (processed product).
(1) preparation of decocting liquid:
To according to 8:4:4:3:3:3 prescription, be fed intake by Radix Rehmanniae Preparata, Fructus Corni (system), Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria, decocting boils 2 times, reclaims volatile oil simultaneously, merge decocting liquid, decocting liquid is concentrated into clear paste, the relative density of clear paste: 1.10～1.25(50 ℃), medicinal residues are standby;
(2) preparation of volatile oil beta cyclodextrin inclusion complex component:
Adopt the volatile oil of beta-schardinger dextrin-saturated water solution method enclose LIUWEI DIHUANG TANG total extract, the volatile oil of LIUWEI DIHUANG TANG total extract is slowly splashed in beta-schardinger dextrin-saturated aqueous solution and constantly and stirred, cold preservation (4 ℃) sucking filtration after standing 48 hours, 40 ℃ dry, obtains volatile oil beta cyclodextrin inclusion complex component.
(3) ethanol of decocting liquid precipitation:
By adding ethanol in the clear paste in step (1) and being constantly stirred to concentration of alcohol, be 70%, standing 48 hours of cold preservation (4 ℃), separation obtains supernatant and precipitation;
(4) preparation of polysaccharide component:
Precipitation and water in step (3) are made into suspension, centrifugal, remove insoluble matter, adopt the ultrafiltration membrance filter that molecular cut off is 50,000, discard liquid on film, remove the macromolecular substances that relative molecular weight is greater than 50,000, the ultrafiltration membrance filter that under film, liquid is 10,000 by molecular cut off value again, removes relative amount of analysis lower than 10,000 small-molecule substance, liquid on collection membrane, the concentrated material that obtains relative molecular weight 10,000～50,000 molecular weight, the dry polysaccharide component that obtains of spraying;
(5) preparation of glucosides class component:
Supernatant in step (3), through macroporous resin column chromatography, with the gradient elution of water, 20%, 40%, 65% ethanol, is collected respectively to flow point successively, merge 40% and 65% ethanol partial concentration extremely without alcohol taste, the dry glucosides class component that obtains of spraying;
(6) preparation of fat-soluble ingredient:
After medicinal residues in step (1) are dry, add alcohol reflux 2 times, each 1 hour, filter, merging filtrate, filtrate recycling ethanol, is concentrated into without after alcohol taste, the dry fat-soluble ingredient that obtains of spraying;
(7) preparation of LIUWEI DIHUANG TANG total extract:
Volatile oil beta cyclodextrin inclusion complex component in fat-soluble ingredient and step (2) in glucosides class component, step (6) in polysaccharide component, step (5) in step (4) is mixed, obtain LIUWEI DIHUANG TANG total extract compositions.
The further restriction of above compositions, has following characteristics:
(1). the characteristic peak of the infared spectrum of LIUWEI DIHUANG TANG total extract compositions: IR
(cm
-1): 3396,2926,2855,1711,1615,1516,1453,1376,1203,1163,1078,1030,765,714,573; Or,
The characteristic peak of the second dervative of LIUWEI DIHUANG TANG total extract compositions (Second Derivative, is called for short SD, lower same) infrared spectrum: SD-IR
(600～1800cm
-1): 1740,1710,1687,1606,1515,1467,1377,1316,1276,1204,1154,1077,1027,794,768,742,715;
(2). the characteristic peak of the infrared spectrum of the polysaccharide component of LIUWEI DIHUANG TANG extract (position A): IR
(cm
-1): 3400,2930,1718,1618,1417,1372,1238,1152,1079,1024,868,766,604,575,527; Or,
The characteristic peak of the second dervative infrared spectrum of the polysaccharide component of LIUWEI DIHUANG TANG extract (position A): SD-IR
(600～1800cm
-1): 1731,1609,1561,1522,1506,1471,, 1372,1230,1205,1153,1125,1106,1079,1056,1023,995,963,913,866,835,819,799,759,704,658,643,616,604;
(3). the characteristic peak of the infared spectrum of glucosides class component (position B): IR in LIUWEI DIHUANG TANG extract
(cm
-1): 3398,2934,1709,1610,1533,1516,1450,1383,1278,1205,1073,1038,837,765,715,627; Or,
The characteristic peak of the second dervative infrared spectrum of glucosides class component (position B): SD-IR in LIUWEI DIHUANG TANG extract
(600～1800cm
-1): 1747,1715,1653,1631,1603,1534,1515,1490,1468,1451,1418,1396,1384,1349,1316,1280,1202,1176,1119,1098,1072,1038,1005,940,918,897,872,853,821,767,741,715;
(4). the characteristic peak of the infared spectrum of LIUWEI DIHUANG TANG extract fat-soluble ingredient (position C): IR
(cm
-1): 3391,2928,1729,1668,1408,1385,1349,1226,1146,1075,920,868,817,777,629,590,422; Or,
The characteristic peak of the second dervative infrared spectrum of LIUWEI DIHUANG TANG extract fat-soluble ingredient (position C): SD-IR
(600～1800cm
-1): 1736,1667,1609,1560,1523,1498,1457,1419,1384,1341,1315,1195,1150,1105,1079,1054,1026,977,962,917,891,865,817,799,779,700,673,661,631.
The figure spectrum signature of above-described compositions, refers to: the collection of illustrative plates of homogeneous groups compound, its similarity is not less than 0.85.
In other words, the figure spectrum signature of above compositions is:
(1). the Infrared fingerprint spectra of LIUWEI DIHUANG TANG total extract compositions meets: A, the infrared spectrogram of Fig. 8 LIUWEI DIHUANG TANG extract compositions, its similarity is not less than 0.85; Or B, the second dervative infrared spectrogram of Fig. 9 LIUWEI DIHUANG TANG extract compositions;
(2). the infared spectrum of the polysaccharide component of LIUWEI DIHUANG TANG extract meets: A, the infrared spectrogram of the polysaccharide component of Fig. 2 LIUWEI DIHUANG TANG extract (position A); Or B, the second dervative infrared spectrogram of the polysaccharide component of Fig. 3 LIUWEI DIHUANG TANG extract (position A);
(3). in LIUWEI DIHUANG TANG extract, the infared spectrum of glucosides class component meets: A, the infrared spectrogram of glucosides class component (position B) in Fig. 4 LIUWEI DIHUANG TANG extract; Or B, the second dervative infrared spectrogram of glucosides class component (position B) in Fig. 5 LIUWEI DIHUANG TANG extract;
(4). the infared spectrum of LIUWEI DIHUANG TANG extract fat-soluble ingredient meets: A, the infrared spectrogram of Fig. 6 LIUWEI DIHUANG TANG extract fat-soluble ingredient (position C); Or B, the second dervative infrared spectrogram of Fig. 7 LIUWEI DIHUANG TANG extract fat-soluble ingredient (position C).
The present invention carries out the processing of science by the LIUWEI DIHUANG TANG to traditional, under the guidance of traditional Chinese medical science Overall View theory, keep on the basis of each medicine drug effect of former LIUWEI DIHUANG TANG, has increased fat-soluble effective site in six drugs containing rehmanniae.As above as described in " background technology ": the molecular weight of Poria active polysaccharide is 3.8 * 10
4left and right, the molecular weight of skunk bush polysaccharide is 2.47 * 10
4left and right, the molecular weight of the hypoglycemic active polysaccharide of Cortex Moutan is 1.3 * 10
4left and right, the molecular weight at visible six drugs containing rehmanniae active polysaccharide position focuses mostly between 10,000～50,000; Therefore this method adopts ultrafiltration to extract the active polysaccharide class position of having refined six drugs containing rehmanniae 10,000～50,000 molecular weight.This method adopts macroporous resin column chromatography method to extract and has refined the glucosides active component in six drugs containing rehmanniae, greatly reduces the use volume of LIUWEI DIHUANG TANG.
LIUWEI DIHUANG TANG extract preparation technology's flow process of the present invention is shown in Fig. 1.
The polysaccharide component of LIUWEI DIHUANG TANG extract according to the present invention (position A) is mainly comprised of 10,000～50,000 molecular weight polysaccharide components.
According to the present invention, in LIUWEI DIHUANG TANG extract, glucosides class component (position B) is mainly comprised of glucosides constituents.
The LIUWEI DIHUANG TANG extract fat-soluble ingredient according to the present invention (position C) is mainly comprised of liposoluble constituent.
The scheme that completes the present application task is: the application of above-mentioned LIUWEI DIHUANG TANG total extract compositions in preparation treatment treatment medicine for treating diabetic nephropathy.
Treatment new indication and the novel targets of LIUWEI DIHUANG TANG:
The application of LIUWEI DIHUANG TANG is very extensive, and sales volume is very large, by comparison, clinical indication and pharmacological research is seemed to very not enough, cures mainly not clear, machine-processed unclear.Clinical a large amount of use and pharmaceutical research and contemporary molecular biology and biomedical research disconnect.Therefore, the pharmacological action understanding to it, and to the curing mainly of disease, act on main target spot and should give further investigation.
Violent irritability (muscle power or spirituality) often causes cardiac function to worsen suddenly, vascular function infringement, cerebral lesion, meeting grievous injury body in the short time, threat to life.Blocking the cardio cerebrovascular affection that acute serious stress causes, is the important measures that ensure people ' s health.
In addition, in mid-aged population, the morbidity of male sexual dysfunction is high, presents growth trend.Under the state of acute anxiety, the gonadal hormone of testis is generated and there will be obvious inhibitory state, blood Sex Hormone Levels obviously reduces.Prompting: men's health is obviously affected.Nigeria army report, nervous military training, can make the content of testosterone in male soldier's blood obviously reduce, and proves too heavy military training, can damage the function of testis.
Stress state is a kind of pathological and physiological condition of complexity.In occlusion body, generation and the burst size of active oxygen (reactive oxygen species, ROS) increase greatly.In body, the generation of oxygen-derived free radicals has multiple source, and wherein, the activation of nadph oxidase is most important source.The pharmacological action of " three mend three lets out " of LIUWEI DIHUANG TANG, may come from the activation that suppresses nadph oxidase, thereby, can reduce er stress molecule, alleviate without superimposed proteins react (Unfolded protein response, UPR); We find: LIUWEI DIHUANG TANG is treated the multiple complications of stress, are mainly to suppress the activity of nadph oxidase.
Discovered in recent years, it is early stage that unfolded albumino reaction (unfolded protein response, UPR) increases, it is compensatory reaction, along with PD, er stress molecule constantly increases, and Inflammatory substances and proinflammatory material that nucleus is transcribed generation are more and more many.Final cell cannot overcome, and causes cell death.In protein, peptide chain will carry out-SH, and the condensation of-SH key forms the three-dimensional conformation in 3 degree spaces, just obtains biological activity.The building-up reactions of protein is very responsive to oxygen-derived free radicals, too much the building-up reactions of oxygen-derived free radicals interferencing protein.Stress is active by activating nadph oxidase, ROS is generated sharply and rise, and brings out er stress, and cell suffers serious injury.LIUWEI DIHUANG TANG, by the activation of blocking-up nadph oxidase, suppresses er stress, makes irritability pathological changes obviously alleviate or suppress completely.LIUWEI DIHUANG TANG blocking-up nadph oxidase, just likely prevents the generation of the infringements such as serious stress cardiomyopathy, brain, or obviously alleviates.
LIUWEI DIHUANG TANG is alleviated cardiovascular complication, the treatment diabetic nephropathy of irritability reaction, owing to suppressing NADPH oxidase:
nadph oxidase, under pathological conditions, becomes the main source that generates ROS.The structure of nadph oxidase, can be divided into two major parts: catalytic subunit: NOX2, and NOX4, etc., and regulation and control subunit: p22phox, p47phox, p67phox, etc.Stress state, and diabetes, nadph oxidase is highly activated, and produces numerous ROS, causes histologic lesion; Irritability reaction causes: cardiomyopathy, angiopathy, nephropathy, testopathy and encephalopathy.Nadph oxidase is the main target spot of LIUWEI DIHUANG TANG, is activated the many diseases that mediate by nadph oxidase, and LIUWEI DIHUANG TANG all can be effective.
The activation of nadph oxidase, also can cause er stress (ER stress).One of endoplasmic reticulum function, synthesizes and has bioactive protein.If any more ROS, exist, protein cannot be normally superimposed, or wrong superimposed, the protein non-activity of generation.Be referred to as without protein building-up reactions (UPR).Now, the dialogue between endoplasmic reticulum and nucleus is abnormal, generates the molecule of a large amount of ER stress, as PERK, and Bip, CHOP, etc., impel a large amount of generation inflammatory factors, or proinflammatory inflammation factor.Causing apoptosis,, there is cardiovascular diseases in cell death, encephalopathy, nephropathy, testopathy etc.Cause ER stress main mechanism, be due to the oxidasic activation of NADPG.
The present invention, finds the very strong blocking effect of LIUWEI DIHUANG TANG to nadph oxidase first, is its pharmacological action basis, antagonism irritability and diabetes and its complication.Tool using medicine selective N ADPH oxidase blocker acetyl Vanillin of the present invention (APO) is as positive control, and APO is a kind of instrument medicine in pharmaceutical research just, and APO is not the medicine of clinical use.With its contrast, just illustrate: the drug effect of six drugs containing rehmanniae decoction formulation is by the mechanism of blocking-up nadph oxidase.Six drugs containing rehmanniae decoction formulation to act on APO similar, prove by same mechanism of action.This is effect and the mechanism that proves that first six drugs containing rehmanniae decoction formulation has.Therefore, six drugs containing rehmanniae decoction formulation can suppress the source that oxygen-derived free radicals produces, and can control the formed pathological changes of all oxygen free radicals.
By the treatment of LIUWEI DIHUANG TANG, in very strong irritability reaction, suppress the nadph oxidase of activation, resist er stress, make every observation index of organ, all get back to or approach normal state.
The infringement of LIUWEI DIHUANG TANG treatment diabetic nephropathy:
Method: SD rat, clean level.With thiourea mycin (STZ, streptozotocin, 65 mg/kg, an ip) shot.After 8 weeks, form diabetic complication (in early days).Use the new technology preparation of LIUWEI DIHUANG TANG.Latter 4 weeks pharmaceutical intervention, give heavy dose of (LW-H, 2.4mg/kg, po), middle dosage (LW-M, 1.2mg/kg, po), and pure liposoluble extract (LW-ST, is equivalent to 2.4mg/kg, po), with positive drug aminoguanidine (AMG, 100mg/kg, po, double blocking AGEs, and the medicine of iNOS, the curative effect for the treatment of diabetic complication is fine) and APO(acetyl Vanillin 80mg/kg, po.Selective N ADPH oxidase blocker), do dual comparing.
Treatment diabetic nephropathy
1, improve renal function:
Renal failure (uremia) approximately 50% is due to diabetic nephropathy.The morbidity of diabetic nephropathy
The course of disease length of diabetes is relevant, carries out sexual development and deterioration, may with activate nadph oxidase, er stress is relevant with p66Shc gene.We find: the preparation of LIUWEI DIHUANG TANG, and can be abnormal to the above-mentioned target spot in anti diabetes and kidney disease tissue.
With reference to Figure 11-A ~ Figure 11-E, in the serum of diabetes rat and urine, BUN and Cr all rise, and in 24h urine, trace albumin increases.Six drugs containing rehmanniae decoction formulation and positive drug, all alleviate significantly.N=10, X ± SD; * P<0.01, vs. normal; #P<0.05, ##P<0.01, vs. DM (diabetes).
8 weeks rats of diabetes, non-protein nitrogen in blood (A) BUN (C) and Cr(D) also rise (BUN) and in the urine of creatinine (B) (creatinine, Cr) rising 24h, trace albumin (E) increases.Show that renal function is obviously low.LW-H, LW-ST gets back to normally renal function, and LW-H is better than JT; The drug effect of LW-M is slightly poor.AMG and APO are all effective.
2, correct the interior biological targets of kidney abnormal:
With reference to Figure 12-A ~ Figure 12-H: in the nephridial tissue of diabetic nephropathy, the mRNA of MMP-9 (A) (Matrix Metalloproteinase-9) and Cx43 (B) (gap junctions albumen) lowers; The mRNA(C of the p22 of nadph oxidase) and albumen (D) obviously raise; Body internal control oxygenerating stress important gene p66Shc mRNA (E) and albumen (F) raise significantly; The albumen of er stress molecule PERK raises (G), and the mRNA of endothelin receptor A (H) raises.Six drugs containing rehmanniae decoction formulation makes obvious alleviation, similar with the effect of positive drug.
Accompanying drawing explanation
Fig. 1 is process chart prepared by LIUWEI DIHUANG TANG extract of the present invention;
Fig. 2 is the infrared spectrum of the polysaccharide component (position A) of LIUWEI DIHUANG TANG extract;
Fig. 3 is the second dervative infrared spectrum of the polysaccharide component (position A) of LIUWEI DIHUANG TANG extract;
Fig. 4 is the infrared spectrum of the glucosides class component (position B) of LIUWEI DIHUANG TANG extract;
Fig. 5 is the second dervative infrared spectrum of the glucosides class component (position B) of LIUWEI DIHUANG TANG extract;
Fig. 6 is the infrared spectrum of the fat-soluble ingredient (position C) of LIUWEI DIHUANG TANG extract;
Fig. 7 is the second dervative infrared spectrum of the fat-soluble ingredient (position C) of LIUWEI DIHUANG TANG extract;
Fig. 8 is the infrared spectrum of LIUWEI DIHUANG TANG extract;
Fig. 9 is the second dervative infrared spectrum of LIUWEI DIHUANG TANG extract;
Figure 10 is the infrared spectrum stack collection of illustrative plates of 10 batches of LIUWEI DIHUANG TANG extract;
Vertical coordinate in accompanying drawing is absorbance (A).
A in Figure 11, B, C, D, E figure, represent respectively: in the serum of diabetes rat and urine, BUN and Cr all rise, and in 24h urine, trace albumin increases.Six drugs containing rehmanniae decoction formulation and positive drug, all alleviate significantly.N=10, X ± SD; * P<0.01, vs. normal; #P<0.05, ##P<0.01, vs. DM (diabetes);
A in Figure 12, B, C, D, E, F, G, H figure, represent respectively: in the nephridial tissue of diabetic nephropathy, biological targets is obviously abnormal, nadph oxidase activates, and er stress molecule rises, and p66Shc raises.The therapeutical effect of six drugs containing rehmanniae decoction formulation is obvious, is to get back to normal range.N=10, X ± SD; * P<0.01, vs. normal; #P<0.05, ##P<0.01, vs. DM (diabetes).
The specific embodiment
Embodiment: the preparation of LIUWEI DIHUANG TANG extract
Radix Rehmanniae Preparata, Fructus Corni (system), Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria in LIUWEI DIHUANG TANG are fed intake according to 8:4:4:3:3:3 ratio, add 10 times of water gagings, soak 3h, decoct twice, reclaim volatile oil simultaneously, each 30min, by filtered through gauze, be evaporated to clear paste, the relative density of clear paste is 1.10～1.25(50 ℃), after filtering, remaining medicinal residues are standby; Volatile oil anhydrous alcohol solution, beta-schardinger dextrin-is made saturated aqueous solution, volatile oil alcoholic solution is injected to beta-schardinger dextrin-saturated aqueous solution and constantly stirs, then standing 48 hours of cold preservation (4 ℃), filter, add a small amount of petroleum ether (30～60 ℃) washing, 40 ℃ dry, obtains volatile oil beta cyclodextrin inclusion complex.The amount that adds ethanol in clear paste and be constantly stirred to ethanol is 70%(V/V), be placed on that at 4 ℃, refrigerator, cold preservation is after 48 hours, separation obtains supernatant and precipitation, 70% washing with alcohol 3 times for precipitation; Precipitation is except alcohol, precipitation is made into suspension with water, centrifugal, removes insoluble matter, adopt the ultrafiltration membrance filter that molecular cut off is 50,000, discard liquid on film, remove the macromolecular substances that relative molecular weight is greater than 50,000, the ultrafiltration membrance filter that under film, liquid is 10,000 by molecular cut off value again, remove relative amount of analysis lower than 10,000 small-molecule substance, liquid on collection membrane, the concentrated material that obtains relative molecular weight 10,000～50,000 molecular weight, the dry position A that obtains of spraying.Merge supernatant and be evaporated to clear paste, through D101 macroporous resin column chromatography, according to every kilogram of crude drug, adopt the ratio loading of the macroporous resin of 1.5Kg, then water, 20% ethanol, 40% ethanol and 65% ethanol eluting successively, water and 20% ethanol elution discard, merge 40% ethanol and 65% ethanol elution concentrating under reduced pressure, the dry position B that obtains of spraying.After medicinal residues are dry, add 5 times of amount alcohol reflux 2 times, 1 hour/time, filter, merging filtrate, after concentration and recovery ethanol, the dry position C that obtains of spraying.Merge A, B, C and cyclodextrin of volatile oil clathrate, obtain LIUWEI DIHUANG TANG total extract.
The infrared analysis of LIUWEI DIHUANG TANG extract
Adopt the FTIR Fourier transform infrared spectrometer of Perkin Elmer company, use KBr pressed disc method to measure the infrared spectrogram that obtains each effective site.
The infrared spectrogram of each effective site adopts Perkin Elmer Spectrum v6.0 function software of company (smoothly count is 13) to obtain the second derivative spectra figure of each effective site.
By adopting infrared spectrum software to carry out similarity computing to the IR spectrum scanning data of 10 batches of LIUWEI DIHUANG TANG extract, the infrared spectrum stack collection of illustrative plates of 10 batches of LIUWEI DIHUANG TANG extract of final gained is as figure, take LIUWEI DIHUANG TANG extract (lot number: 10111001) be reference, the similarity value of all the other LIUWEI DIHUANG TANG extract infrared spectrograms in Table, result shows that the similarity value of different batches LIUWEI DIHUANG TANG extract is all greater than 0.85, shows that this preparation technology is more stable.
The similar kilsyth basalt of LIUWEI DIHUANG TANG extract infrared spectrogram
| Lot number | Similarity |
| 10111001 | 1 |
| 10111002 | 0.9901 |
| 10111003 | 0.9843 |
| 10111004 | 0.9822 |
| 10111005 | 0.9784 |
| 10111006 | 0.9571 |
| 10111007 | 0.9426 |
| 10111008 | 0.9275 |
| 10111009 | 0.9247 |
| 10111010 | 0.8576 |
pharmacological evaluation raw material A
By the dry pharmacological evaluation raw material A that obtains of supernatant in (3).
pharmacological evaluation raw material B
Position B and position C and volatile oil are merged and obtains pharmacological evaluation raw material B.
The infringement of LIUWEI DIHUANG TANG treatment diabetic nephropathy:
Method: SD rat, clean level.With thiourea mycin (STZ, streptozotocin, 65 mg/kg, an ip) shot.After 8 weeks, form diabetic complication (in early days).Use the new technology preparation of LIUWEI DIHUANG TANG.Latter 4 weeks pharmaceutical intervention, give heavy dose of (LW-H, 2.4mg/kg, po), middle dosage (LW-M, 1.2mg/kg, po), and pure liposoluble extract (LW-ST, is equivalent to 2.4mg/kg, po), with positive drug aminoguanidine (AMG, 100mg/kg, po, double blocking AGEs, and the medicine of iNOS, the curative effect for the treatment of diabetic complication is fine) and APO(acetyl Vanillin 80mg/kg, po.Selective N ADPH oxidase blocker), do dual comparing.
Treatment diabetic nephropathy.1, improve renal function: 8 weeks rats of diabetes, non-protein nitrogen in blood (A) BUN (C) and Cr(D) also rise (BUN) and in the urine of creatinine (B) (creatinine, Cr) rising 24h, trace albumin (E) increases.Show that renal function is obviously low.LW-H, LW-ST gets back to normally renal function, and LW-H is better than JT; The drug effect of LW-M is slightly poor.AMG and APO are all effective.2, correct the interior biological targets of kidney abnormal: in the nephridial tissue of diabetic nephropathy, the mRNA of MMP-9 (A) (Matrix Metalloproteinase-9) and Cx43 (B) (gap junctions albumen) lowers; The mRNA(C of the p22 of nadph oxidase) and albumen (D) obviously raise; Body internal control oxygenerating stress important gene p66Shc mRNA (E) and albumen (F) raise significantly; The albumen of er stress molecule PERK raises (G), and the mRNA of endothelin receptor A (H) raises.Six drugs containing rehmanniae decoction formulation makes obvious alleviation, similar with the effect of positive drug.
Claims (2)
1. a LIUWEI DIHUANG TANG total extract compositions is treated the application in medicine for treating diabetic nephropathy in preparation, it is characterized in that, described LIUWEI DIHUANG TANG total extract compositions, its crude drug is by Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria form according to the weight ratio of 10.4～7.6:5.2～2.8:5.2～2.8:3.9～2.1:3.9～2.1:3.9～2.1, in described compositions, include: LIUWEI DIHUANG TANG total extract volatile oil beta cyclodextrin inclusion complex component, LIUWEI DIHUANG TANG total extract polysaccharide component, LIUWEI DIHUANG TANG total extract glucosides class component, and LIUWEI DIHUANG TANG total extract fat-soluble ingredient,
Described compositions has following characteristics:
(1). the characteristic peak of the infrared spectrum of LIUWEI DIHUANG TANG total extract compositions:
(cm
-1): 3396,2926,2855,1711,1615,1516,1453,1376,1203,1163,1078,1030,765,714,573; Or,
The characteristic peak of the second dervative infrared spectrum of LIUWEI DIHUANG TANG total extract compositions:
(600～1800cm
-1): 1740,1710,1687,1606,1515,1467,1377,1316,1276,1204,1154,1077,1027,794,768,742,715;
(2). the characteristic peak of the infrared spectrum of the polysaccharide component of LIUWEI DIHUANG TANG extract:
(cm
-1): 3400,2930,1718,1618,1417,1372,1238,1152,1079,1024,868,766,604,575,527; Or,
The characteristic peak of the second dervative infrared spectrum of the polysaccharide component of LIUWEI DIHUANG TANG extract:
(600～1800cm
-1): 1731,1609,1561,1522,1506,1471,, 1372,1230,1205,1153,1125,1106,1079,1056,1023,995,963,913,866,835,819,799,759,704,658,643,616,604;
(3). the characteristic peak of the infared spectrum of glucosides class component in LIUWEI DIHUANG TANG extract:
(cm
-1): 3398,2934,1709,1610,1533,1516,1450,1383,1278,1205,1073,1038,837,765,715,627; Or,
The characteristic peak of the second dervative infrared spectrum of glucosides class component in LIUWEI DIHUANG TANG extract:
(600～1800cm
-1): 1747,1715,1653,1631,1603,1534,1515,1490,1468,1451,1418,1396,1384,1349,1316,1280,1202,1176,1119,1098,1072,1038,1005,940,918,897,872,853,821,767,741,715;
(4). the characteristic peak of the infared spectrum of LIUWEI DIHUANG TANG extract fat-soluble ingredient:
(cm
-1): 3391,2928,1729,1668,1408,1385,1349,1226,1146,1075,920,868,817,777,629,590,422; Or, the characteristic peak of the second dervative infrared spectrum of LIUWEI DIHUANG TANG extract fat-soluble ingredient:
(600～1800cm
-1): 1736,1667,1609,1560,1523,1498,1457,1419,1384,1341,1315,1195,1150,1105,1079,1054,1026,977,962,917,891,865,817,799,779,700,673,661,631;
Described compositions refers to the compositions preparing by the following method:
(1) preparation of decocting liquid:
To according to 8:4:4:3:3:3 prescription, be fed intake by Radix Rehmanniae Preparata, Fructus Corni (processed), Rhizoma Dioscoreae, Rhizoma Alismatis, Cortex Moutan and Poria, decocting boils, and reclaims volatile oil simultaneously, merges decocting liquid, and decocting liquid is concentrated into clear paste, and medicinal residues are standby;
(2) preparation of volatile oil beta cyclodextrin inclusion complex component:
Adopt the volatile oil of beta-schardinger dextrin-saturated water solution method enclose LIUWEI DIHUANG TANG total extract, the volatile oil of LIUWEI DIHUANG TANG total extract is slowly splashed in beta-schardinger dextrin-saturated aqueous solution and constantly and stirred, the standing rear sucking filtration of cold preservation, dry, obtain volatile oil beta cyclodextrin inclusion complex component;
(3) ethanol of decocting liquid precipitation:
By adding ethanol in the clear paste in step (1) and being constantly stirred to ethanol weight ratio concentration, be 70%, cold preservation is standing, and separation obtains supernatant and precipitation;
(4) preparation of polysaccharide component:
Precipitation and water in step (3) are made into suspension, centrifugal, remove insoluble matter, adopt the ultrafiltration membrance filter that molecular cut off is 50,000, discard liquid on film, remove the macromolecular substances that relative molecular weight is greater than 50,000, the ultrafiltration membrance filter that under film, liquid is 10,000 by molecular cut off value again, removes relative molecular weight lower than 10,000 small-molecule substance, liquid on collection membrane, the concentrated material that obtains relative molecular weight 10,000～50,000 molecular weight, the dry polysaccharide component that obtains of spraying;
(5) preparation of glucosides class component:
Supernatant in step (3), through macroporous resin column chromatography, with the gradient elution of the ethanol of water, variable concentrations, is collected respectively to flow point successively, merge ethanol partial concentration to without alcohol taste, the dry glucosides class component that obtains of spraying;
(6) preparation of fat-soluble ingredient:
After medicinal residues in step (1) are dry, add alcohol reflux, filter, filtrate recycling ethanol, is concentrated into without after alcohol taste, the dry fat-soluble ingredient that obtains;
(7) preparation of LIUWEI DIHUANG TANG total extract:
In volatile oil beta cyclodextrin inclusion complex component in step (2), step (5), in glucosides class component and step (6), fat-soluble ingredient mixes, by polysaccharide component mixing granulation in proper proportion and step (4), dry, tabletting or embedding capsule, obtain LIUWEI DIHUANG TANG total extract compositions;
In step (1), described decocting liquid is that decocting boils 2～3 times; Described decocting liquid is concentrated into clear paste, is the relative density that is concentrated into 50 ℃ of clear paste: 1.10～1.25;
In step (2), described cold preservation, refers to standing 12～48 hours of 4 ℃ of cold preservations; Described is dry, is dry at 30～60 ℃;
In step (3), the ethanol of decocting liquid precipitation: described cold preservation, refers to standing 12～48 hours of 4 ℃ of cold preservations;
In step (5), the immobile phase in described gradient elution is acceptable macroporous resin in pharmacy; Gradient elution is successively with the ethanol elution of water, weight ratio concentration 20%, discards eluent; Ethanol gradient elution with 40%～75%, collects flow point, merges eluent;
In step (6), described alcohol reflux, refers to alcohol reflux 2 times, each 1 hour, and merging filtrate, then reclaim ethanol, concentrated.
2. the application of LIUWEI DIHUANG TANG total extract compositions according to claim 1 in preparation treatment medicine for treating diabetic nephropathy, is characterized in that, the consumption in rat experiment is:
Middle dosage group: the dry extract of 1.2g/kg LIUWEI DIHUANG TANG; High dose group: the dry extract of 2.4g/kg LIUWEI DIHUANG TANG, is equivalent to respectively raw medicinal herbs 7g/kg and 14g/kg.
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| CN1565494A (en) * | 2003-06-25 | 2005-01-19 | 毛友昌 | Chewing tablet of |
| CN1626228A (en) * | 2003-08-19 | 2005-06-15 | 中国人民解放军军事医学科学院毒物药物研究所 | Extractive from decoction of rehmannia including six elements, combination of medication, and medical use |
| CN101002906A (en) * | 2006-01-16 | 2007-07-25 | 上海清风阁医药科技有限公司 | Compounding traditional Chinese medicine, and its use |
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| CN1565494A (en) * | 2003-06-25 | 2005-01-19 | 毛友昌 | Chewing tablet of |
| CN1626228A (en) * | 2003-08-19 | 2005-06-15 | 中国人民解放军军事医学科学院毒物药物研究所 | Extractive from decoction of rehmannia including six elements, combination of medication, and medical use |
| CN101002906A (en) * | 2006-01-16 | 2007-07-25 | 上海清风阁医药科技有限公司 | Compounding traditional Chinese medicine, and its use |
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