CN108478534A - The preparation method and applications of Quercetin liposome - Google Patents

Abstract

The invention discloses the preparation method and applications of Quercetin liposome, preparation method is to wrap up Quercetin not soluble in water with nano liposomes, is prepared into Quercetin liposome, passes through experimental study Quercetin liposome（LQ）To Renal of Diabetic Rats glycosylation end products（AGEs）And its receptor（RAGE）The influence and its influence to DM rat kidney expressed with TGF β 1, and and aminoguanidine（Aminoguanidine, AG）Intervention effects of the LQ to DM kidneys is inquired into comparison.Experiment shows that Quercetin liposome can mitigate the morphopathology change of diabetic nephropathy to a certain extent, improves the general status of STZ DM rats, plays the role of hypoglycemic.The present invention is that the next step clinical application research of Quercetin liposome lays the first stone, while also providing new thinking and theoretical foundation to the clinical Chinese medicine treatment for exploring DM.

Description

The preparation method and applications of Quercetin liposome

Technical field

The present invention relates to Quercetins, the specifically preparation method and applications of Quercetin liposome.

Background technology

Quercetin is natural flavone compound, is widely present in vegetables and fruit.Quercetin has antitumor, anti- Inflammation, antioxidation, and the activity of a variety of enzymes is influenced, it is a kind of potential chemotherapeutics.It is difficult since Quercetin is not soluble in water In absorption, significantly limits it and absorb, be administered and biological utilisation in vivo.External I and II clinical trial phase confirms quercitrin Element can inhibit kinds of tumors to be in progress in vivo, however, its solvent dromisol, can lead to human body haemolysis, hepatorenal damage and unpleasant Smell, limit Quercetin for clinic.One of this method is solved the problems, such as by being chemically modified to Quercetin, however This needs multi-step to chemically react, the Quercetin structural instability after modification, reduces the anti-tumor effect of Quercetin.

Invention content

An object of the present invention is：The preparation method of Quercetin liposome is provided.

The second object of the present invention is to：Open Quercetin liposome application in preparation of anti-tumor drugs.

The third object of the present invention is：Open Quercetin liposome is preparing the application in treating medicine for treating diabetic nephropathy.

The preparation method of Quercetin liposome of the present invention is as follows using rotary evaporation：

（1）Weigh Quercetin:Cholesterol:Lecithin:Macrogol 4000 is by weight 6:4:13:1 is put into round-bottomed flask；

（2）It is added on negative pressure Rotary Evaporators and presses 3:The tri-chlorination methane and methanol of 1 volume ratio, are evaporated, and Huang is formed in bottom of bottle Color membranoid substance；

（3）Be added deionized water into flask, 0 DEG C of ultrasonication, suspension at 4 DEG C aquation for 24 hours, then again at water bath sonicator instrument 20min is managed, is allowed to diffuse to form the liposome that average grain diameter is less than 200nm；

（4）It rotates on constant temperature negative pressure evaporation instrument, dispenses freezen protective, obtain Quercetin liposome.

The present invention wraps up Quercetin not soluble in water with nano liposomes, is prepared into Quercetin liposome, passes through experiment Study Quercetin liposome（LQ）To Renal of Diabetic Rats glycosylation end products（AGEs）And its receptor（RAGE）With TGF-β 1 The influence of expression and its influence to DM rat kidney, and and aminoguanidine（Aminoguanidine, AG）LQ is inquired into comparison To the intervention effect of DM kidneys.Experiment shows that Quercetin liposome can mitigate the form of diabetic nephropathy to a certain extent Pathological change improves the general status of STZ DM rats, plays the role of hypoglycemic.The present invention is under Quercetin liposome One step clinical application research lays the first stone, while also providing new thinking and theoretical foundation to the clinical Chinese medicine treatment for exploring DM.

Description of the drawings

Fig. 1 is each group rat kidney immunohistochemistry PAS staining conditions in experimental example（×400）Photo；

Fig. 2 is each group rat kidney immunohistochemistry AGEs expressions in experimental example（×400）Photo；

Fig. 3 is 1 expression of each group rat kidney immunohistochemistry TGF-β in experimental example（×400）Photo；

In figure, A-F is respectively N groups, DM groups, LQ-L groups, LQ-M groups, LQ-H groups, AG groups.

Fig. 4 is each group renal tissues of rats RAGE mRNA expressions in experimental example；

Fig. 5 is each group renal tissues of rats TGF-β 1mRNA expressions in experimental example；

Be in figure, on the left of marker the right sides β-actin, marker be RAGE mRNA, 1-6 be respectively N groups, DM groups, LQ-L groups, LQ-M groups, LQ-H groups, AG groups.

Specific implementation mode

The content of present invention is described in further detail with reference to experimental example and attached drawing.

Experimental example：The preparation and its application of Quercetin liposome

1 materials and methods

1.1 experiment materials and instrument

1.1.1 experimental animal

65 male SD healthy adults of SPF grades clean rat, 190~220 g of weight, and 6~8 week old are moved by Medical Colleges Of Guilin Object laboratory（SPF grades）It provides, is raised in Medical Colleges Of Guilin's Animal Lab..Rearing conditions：18~25 DEG C of room temperature keeps air Circulation, relative humidity 55~70%, 12h illumination maintain, and simulate day-night cycle, and daily cleaning cage changes bedding and padding and keeps its life cycle Clean drying in border.

1.2 experimental method

1.2.1 the preparation of Quercetin liposome

This experiment is prepared with rotary evaporation, and specific preparation process is as follows：

（1）Weigh Quercetin：Cholesterol：Lecithin：Macrogol 4000 is by weight 6:4:13:1 is put into round-bottomed flask；

（2）It is added on negative pressure Rotary Evaporators and presses 3:The tri-chlorination methane and methanol of 1 volume ratio, are evaporated, and Huang is formed in bottom of bottle Color membranoid substance；

（4）Be added deionized water into flask, 0 DEG C of ultrasonication, suspension at 4 DEG C aquation for 24 hours, then again at water bath sonicator instrument 20min is managed, is allowed to diffuse to form the liposome that average grain diameter is less than 200nm；

（4）It rotates on constant temperature negative pressure evaporation instrument, dispenses freezen protective, obtain liposome Quercetin, while preparing blank liposome, Method is the same, without Quercetin in formula；

（5）The physico-chemical analysis of liposome Quercetin：The size of liposome Quercetin, high-efficient liquid phase color are detected under an electron microscope Spectrometry（HPLC）Detect liposome Quercetin drugloading rate and encapsulation rate.

1.2.2 Animal Model

65 SD rat feedings 1 week, record every 24 h urine volume of rat.8 are chosen using random digits table to be only used as Normal group, normal diet is fed, remaining all edible high glucose and high fat feed, feed formula reference literature [13].Fasting after 4 weeks 40 mg/kg of 12 h, intraperitoneal injection STZ（It is dissolved in the citric acid-sodium citrates of 0.1 mmol/L, pH4.2~4.5 buffering before use In liquid）Induce diabetes B model.N groups inject the citric acid-sodium citrate buffer solution with dosage.After raising 3 d, daily Early morning 7:00 adopts tail vein detection blood glucose, and continuous 3 blood glucose of survey >=16.7 mmol/L think that DM models are successfully established. 24 h urines are left and taken after the rat for being successfully established 2 patients with type Ⅰ DM models is continued raising 2 weeks, reaches the following conditions and is then set to Diabetic nephropathy model is successfully established：（1）Fasting blood-glucose continues >=16.7 mmol/L；（2）Urinary albumin excretion ratio>20 μg/ min；（3）1.5 times when urine volume is health status；Final 52 Cheng Mo.

1.2.3 animal model is grouped

It is divided into following 5 groups at mould rat by choosing 50 using random digits table at the rat of mould：DM groups, LQ- are low（LQ- L）Group, in（LQ-M）Group, height（LQ-H）Group, AG groups, each group are 10.Corresponding drug is given by following dosage：LQ-L 50 mg/ of group (kgd), 150 mg/ of LQ-M groups (kgd), 250 mg/ of LQ-H groups (kgd), AG group aminoguanidines 100mg/ (kgd), DM groups and N groups 100 mg/ of distilled water (kgd).Due to daily about 10:00 gavage.Every 3 d prisons It surveys blood glucose and claims weight 1 time.DM groups and each dead 2 of LQ-L group rats during experiment, LQ-H groups and AG groups are each dead Die 1, when statistical data rejects rat cadavers data.

1.2.4 the collection of sample

After gavage is handled 8 weeks, rat is individually placed in metabolic cage, acquires every 24 h urine of rat, and with 3 000 r/ Min centrifuge 5 min, take 6 mL of supernatant for measure 24 h microdose urine proteins.After 12 h of fasting, measure respectively on an empty stomach Blood glucose and title weight.With 10% chloraldurate intraperitoneal injection of anesthesia, 4 ~ 5 mL of quick abdominal aortic blood takes 2 mL to survey blood urine Plain nitrogen（BUN）, serum creatinine（Scr）, it is remaining with 12 000 r/min, 4 DEG C of 10 min of centrifugation, take supernatant for AGEs, SOD to be measured, MDA.It cuts open the belly and takes bilateral renal sample, reject kidney teleblem, 0.9% NaCl solution is used in combination to clean, clean filter paper absorbs moisture content, Claim kidney quality, calculates renal hypertrophy index（KI）=Bilateral Renal quality/weight × 100%.The left kidney of quick Liquid nitrogen storage, -80 DEG C Refrigerator preserves；Right kidney is cut in half from longitudinal axis face, and 4% paraformaldehyde liquid is fixed, and paraffin embedding is spare, is prepared into 3 μm of thickness and is cut Piece, for PAS dyeing and immunohistochemistry.

1.2.5 the detection of biochemical indicator

Roche blood glucose meter and mating test paper detect blood glucose, and automatic clinical chemistry analyzer detects BUN, Scr；It is examined with spectrophotometer method SOD, MDA are surveyed, serum AGEs expressions and for 24 hours microdose urine protein is detected with ELISA methods, illustrates in strict accordance with kit Book step is operated, and the respective concentration of each index in sample is calculated according to corresponding detection method and calculation formula.

1.2.6 PAS dyeing observation Pathological changes

The diabetic nephropathy wax stone of the rat of early-stage preparations, block sections dewax rinses → 1% periodic acid solution to water → flowing water 10min → flowing water flushing → drying → 37 DEG C of schiffShi liquid is incubated 10min → Jing Guan → flowing water flushing → hematoxylin and contaminates core 5min → flowing water flushing → hydrochloride alcohol differentiation 10-15s → flowing water flushing → weak ammonia liquor returns blue 10-15s → flowing water rinses → and is Row dehydration of alcohol-dimethylbenzene I, II distinguishes transparent 10min or so → gummy sealing, and it is small to observe glomerulus, kidney under the microscope Situations such as pipe, matrix hyperplasia, basement membrane thickness.

1.2.7 the expression of Immunohistochemical Method detection kidney AGEs and TGF-β 1

Renal tissue bakes 2 h of piece after specimens paraffin embedding slices in 60 DEG C of ovens, is sealed with 0.4% gastric enzyme reparation, lowlenthal serum It closes, PBS flushings, primary antibody presses following concentration dilution respectively（AGEs 1: 100, TGF-β 11:200）, secondary antibody（Biotin labeling Goat anti-rabbit igg）And three anti-priority be incubated 20 min at 37 DEG C, DAB colour developings terminate in due course；Redye, break up, being dehydrated, air-drying, Mounting.Positive sample（The atherosclerosis plaque in rat slide that antibody company provides）As positive control, PBS is as cloudy Property control, positive reaction position is brown color under light microscopic, and nucleus is in light blue.Observe low-power field（×100）Lower reaction feelings Condition, 5 high power fields of each example random read take（×400）, the average light of positive expression is obtained by Image-Pro Plus6.0 Density（MOD）.

1.2.8 reverse transcription polymerase chain reaction（RT-PCR）Method detects the table of kidney AGEs mRNA and TGF-β 1mRNA It reaches

Extract nephridial tissue total serum IgE according to column method, after UV spectrophotometer measuring RNA concentration and purity, by total serum IgE according to For TaKaRa reverse transcription specification reverse transcriptions at being expanded after cDNA, β-actin are internal reference.PCR amplified reaction systems include 25 2 μ L of μ L Premix Taq, cDNA, upstream and downstream primer（10 μmol/L）Each 19 μ of 2 μ L, RNase-Free Water L, totally 50 μ L.PCR reaction conditions are：94 DEG C of 1 min of pre-degeneration；94 DEG C of 30 s of denaturation, anneal 30 s, 72 DEG C of extensions 30min recycles 30 number left and right；72 DEG C of 1 min of extension.Take a concentration of 1.5% Ago-Gels of 6 μ L of PCR product（Containing 0.5 Mg/L Ethidum Eremides）Electroresis appraisal, with SensiAnsys gel imaging systems photography and quantitative analysis, calculate target gene with it is interior Join the ratio of gene OD value.

1.2.8 statistical method

It is analyzed using SPSS 18.0, measurement data is with mean ± standard deviation（）It indicates, multiple sample standard deviations Several comparisons uses one-way analysis of variance, Multiple range test to be examined using LSD-t, P<0.05 has statistics meaning for difference Justice.

2 results

The physicochemical property of 2.1 Quercetin liposomes

Quercetin liposome observes its particle diameter distribution and exists under the microscope（128.8±18.05）Nm, high performance liquid chromatography detection Its drugloading rate is about（58±7）%, encapsulation rate are（87.1±2.7）%.

The comparison of 2.2 each group rat blood sugars, weight, KI

Compared with N groups, the blood glucose of DM, LQ-L, LQ-M, LQ-H and AG group significantly increases, and weight is substantially reduced, and KI increases （Equal P< 0.05）.It is compared with DM groups, LQ-L group blood glucose no significant differences, LQ-M, LQ-H and AG group blood glucose reduce （Equal P< 0.05）；Compared with DM groups, each dosage group weights of LQ increase, and blood glucose, KI are reduced（Equal P< 0.05）, it is shown in Table 1；

The comparison of table 1 each group rat fasting blood-glucose, weight and KI（）

* P<0.01；A is compared with N groups, P<0.01；B is compared with DM groups, P<0.05.

The comparison of 2.3 each group rat biochemical indicators

Compared with N groups, DM, LQ-L, LQ-M, LQ-H, AG group BUN, Scr, MDA content, serum AGEs, 24 h urine are micro white Protein level increases, and SOD activity levels are substantially reduced in serum（Equal P< 0.05）；Compared with DM groups, LQ-L, LQ-M, LQ- H, AG groups BUN, Scr, MDA contents, serum AGEs, microdose urine protein reduces for 24 hours, SOD activity levels obviously rise in serum Height, wherein being improved with LQ-M groups the most apparent（Equal P< 0.05）, it is shown in Table 2, table 3.

The comparison of table 2 each group rat BUN, Scr, MDA, SOD（）

* P<0.01；A is compared with N groups, P<0.01；B is compared with DM groups, P<0.05.

The comparison of 3 each group rat blood serum AGEs of table, for 24 hours microdose urine protein（）

* P<0.01；A is compared with N groups, P<0.01；B is compared with DM groups, P<0.05.

The pathological change of 2.4 each group rat kidney

PAS dyeing shows that N group renal tubules structure, glomerular basement membrane are normal, and extracellular matrix is uniform；DM groups are shown in glomerulus structure Disorder, the apparent atrophy of glomerular volume, glomerular mesangium territorial matrix increase, basement membrane thickened；Each dosage group kidneys of LQ it is above-mentioned Pathological change makes moderate progress compared with DM groups, most apparent with middle dose group improvement, sees Fig. 1.

2.5 immunohistochemistry detect the expression of each group rat kidney AGEs and 1 albumen of TGF-β

Showed by immune group result, AGEs and TGF-β 1 have a degree of expression in N group rat renal tubules；Compared with N groups, DM groups expression increases, renal cells coating and（Or）Endochylema is in brown color strong positive；Compared with DM groups, each dose of LQ The expression of amount group is reduced, and is reduced with middle dose group most apparent, and middle dose group is similar with AG group effects, AGEs express see Fig. 2, Fig. 3, table 4 are shown in the expression of TGF-β 1.

The case where 4 each group rat kidney immunohistochemistry AGEs albumen of table and 1 protein expression of TGF-β（）

* P<0.01；A is compared with N groups, P<0.01；B is compared with DM groups, P<0.05.

2.6 RT-PCR methods detect the expression of kidney AGEs mRNA and TGF-β 1mRNA

The results show that compared with N groups, DM group RAGE mRNA and TGF-β 1mRNA expression quantity increase obviously RT-PCR；With DM Group compares, and each dosage group expression of LQ is reduced, most apparent with middle dose group reduction, and middle dose group is similar with AG group effects, RAGE mRNA expression is shown in that Fig. 4, TGF-β 1mRNA expression are shown in Fig. 5, table 5.

5 each group rat kidney AGEs mRNA of table and TGF-β 1mRNA expressions

* P<0.01；A is compared with N groups, P<0.01；B is compared with DM groups, P<0.05.

Influence of the 3 Quercetin liposomes to blood glucose, AGEs and TGF-β 1

The pathogenic factors of DN not yet study completely it is clear, wherein generally acknowledged high risk factor is lasting hyperglycemia.Quercetin category day Right flavone compound, because it has the effects that anti-oxidant, hypoglycemic, reducing blood lipid is studied by the favor of extensive scholar.It grinds Study carefully observation and find DM rats after Que is treated, the excretion quantity of urinary protein and AGEs contents of DM rats are decreased obviously.It is passed through in this experiment The expression of AGEs and RAGE significantly reduces in Renal Glomeruli In Rats after Quercetin liposome therapeutic, prompts Quercetin liposome that can press down The generation of kidney non-glycosylation final product and its receptor processed, this is consistent with relevant report result.

The document reports such as Zeng Yunxian, for intraperitoneal injection Que treatment group rats compared with normal group, model group, Que can be significantly DM rat blood sugars, blood fat, insulin level are reduced, but is not made significant difference to normal rats, and also found that the course for the treatment of is more long, Hypoglycemic effect is better.The diabetic mouse model that Gomes etc. is induced with STZ treats through Que and finds within 4 weeks that its albuminuria, uric acid subtract Few and blood glucose reduction slows down the toxicity progress of nephridial tissue.Other correlative studys also indicate that Quercetin can significantly reduce STZ and be lured The blood glucose level for the DM rats led has blood sugar reducing function, mechanism of action and antioxidation, removing free radical, anti-fibrosis Etc. related.One of the goldstandard of DN diagnosis is that microdose urine protein, reduction microdose urine protein can delay DN to send out for 24 hours Exhibition.Find that the blood glucose of DM rats after LQ is treated reduces in this experiment, and the indexs such as BUN, Scr, for 24 hours microdose urine protein, KI Improved, illustrate that LQ complexs also can effectively improve DM renal functions, has the function that mitigate kidney damage, mechanism of action It may be with inhibition protein kinase C（PKC）Activity, aldose reductase activity, reactive oxygen species（ROS）Etc. related.

Also studies have reported that, Quercetin can reduce the generation of superoxides in mitochondria, while by stablizing mitochondrial membrane Antioxidant ability of organism, suppression can be improved to reduce superoxide level in mitochondria in the superoxides generated with removing The signal path of TGF-β 1 processed, to play its protective effect to renal function.Kim etc. is demonstrate,proved in the observation of mouse messangial cell It is real, it, can be by activating different accesses since Que structures are different（PKA、PKC）To play its antioxidant activity, and then inhibition The generation of collagen caused by TGF-β 1.Effect of quercetin Fibrosis parameters and its antioxidant activity are closely related.Kedziora Deng the content correlation of the once excretion and MDA of report DM rat Urine proteins.In this experiment, the antioxygen in DM group rats Change enzyme SOD activity to be decreased obviously, and MDA contents are significantly raised, 1 albumen of renal tissue TGF-β and TGF-β 1mRNA expression increase, Prompt kidney under diabetic disease states that apparent oxidative stress occurs, after Quercetin liposome therapeutic, DM rat kidney SOD activity is bright Aobvious to improve, MDA contents are remarkably decreased, and 1 albumen of renal tissue TGF-β and TGF-β 1mRNA expression decline, and show Quercetin lipid Body can protect the antioxidant activity of DM rats, remove free radical, anti-lipid peroxidation reaction, reduce 1 albumen of TGF-β and gene Expression, to delay the occurrence and development of DM rat kidney lesions.In addition, DM renal tissues of rats AGEs, RAGE mRNA are expressed Level has and obviously increases, similar to the results of study such as Sun Fengjuan；And express and be remarkably decreased in each dosage groups of LQ, wherein again in Dosage group decline is the most apparent, shows that Quercetin liposome is also possible to the expression by inhibiting AGEs to lower RAGE and its albumen To delay the occurrence and development of DN.

4. conclusion

（1）The morphopathology that Quercetin liposome can mitigate diabetic nephropathy to a certain extent changes, and improves STZ The general status of DM rats plays the role of hypoglycemic；

（2）Quercetin liposome lowers RAGE by effectively reducing AGEs in BUN, Scr, for 24 hours microdose urine protein, nephridial tissue The expression of mRNA intervenes the occurrence and development of diabetic nephropathy that is, by inhibiting protein non-enzyme glycosylation reaction；

（3）Quercetin liposome increases the Antioxidant Indexes such as the oxidation activity of SOD by the content of reduction MDA, inhibits inflammatory The expression of cell factor there is protection to make diabetes rat nephrosis to reduce 1 albumen of nephridial tissue TGF-β and gene expression With.

This experiment is that the clinical application research of Quercetin liposome lays the first stone, while also controlling the clinical Chinese medicine for exploring DM It treats and new thinking and theoretical foundation is provided.

Claims (4)

1. the preparation method of Quercetin liposome, using rotary evaporation, which is characterized in that be as follows：

Weigh Quercetin:Cholesterol:Lecithin:Macrogol 4000 is by weight 6:4:13:1 is put into round-bottomed flask；

It is added on negative pressure Rotary Evaporators and presses 3:The tri-chlorination methane and methanol of 1 volume ratio, are evaporated, and yellow film is formed in bottom of bottle Shape object；

Deionized water is added into flask, 0 DEG C of ultrasonication, for 24 hours, then water bath sonicator instrument is handled aquation suspension again at 4 DEG C 20min is allowed to diffuse to form the liposome that average grain diameter is less than 200nm；

（4）It rotates on constant temperature negative pressure evaporation instrument, dispenses freezen protective, obtain Quercetin liposome.

2. preparation method described in claim 1 obtains Quercetin liposome.

3. the Quercetin liposome application in preparation of anti-tumor drugs described in claim 2.

4. the Quercetin liposome described in claim 2 is preparing the application in treating medicine for treating diabetic nephropathy.