CN101811956B - Preparation method of trans-crocetin - Google Patents
Preparation method of trans-crocetin Download PDFInfo
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Abstract
The invention relates to the medicine field, and discloses a preparation method of crocetin. Extraction is carried out by adopting diluted alkali, extraction condensation and hydrolysis are combined together, and crocin is hydrolyzed and converted into crocetin during extraction and concentration. The preparation method comprises the steps of: crushing raw materials, carrying out conversion extraction by using a diluted alcoholic solution with pH of 10-14, regulating the pH to be 7-9 by using an extracting solution, condensing by decompression, filtering or centrifuging to remove sediment impurities, regulating the pH to be 2-5, filtering or centrifuging to collect red sediments and obtain a crocetin crude product with crocetin content higher than 50 percent, and recrystallizing and purifying the crocetin crude product to obtain the high-purity trans-crocetin with the crocetin content higher than 95 percent. The trans-crocetin can be used for preparing a medicament for treating fattyliver and has wide application prospect in prevention and control of tumors and other diseases.
Description
Technical field
The invention belongs to field of medicaments, be specifically related to a kind of method for preparing crocetin, is to extract to transform from natural phant to obtain trans-crocetin, and this trans-crocetin has the remarkable efficacy for the treatment of fatty liver.
Background technology
Crocetin is the aglycon of crocin, is one of a kind of effective constituent that separation and Extraction goes out from a kind of Iridaceae crocus plant-style and stigma of Saffron Crocus.It has how unsaturated conjugated olefin(e) acid structure, belongs to the carotenoid material.Crocetin content in the natural phant such as cape jasmine and style and stigma of Saffron Crocus is higher; Except containing crocetin, also contain a large amount of crocins in the natural phant such as cape jasmine, style and stigma of Saffron Crocus, namely crocetin and sugar mixture, such as crocin 1,2,3,4.Be hydrolyzed under certain conditions the glycosidic link of crocin, slough carbohydrate ligands and just can generate crocetin.
Research finds, that crocetin has is anti-oxidant, the protection cardiovascular and cerebrovascular, improve immunologic function and the multiple physiologically active such as antitumor, at aspects such as anti-cardiovascular system diseases, atherosclerosis, control diabetic complications clear and definite biological activity is arranged.Crocetin has anti-tumor activity, and the propagation of anticancer has proof in the related experiment of carcinoma of the pancreas, lung cancer, glioma, mammary cancer effectively.Have and studies have shown that crocetin has important regulating effect for Liver Lipid Metabolism, can effectively improve the insulin resistant situation of obese rat.It also has certain provide protection for aflatoxin B1 and the damage of various acute liver poisoning.
Non-alcohol fatty liver (nonalcoholic fatty liver disease, NAFLD) is a kind of without excessive drinking history, and the pathology main body is stored up as main clinical pathology syndrome take liver cell diffusivity steatosis and fat at liver lobule.The a series of pathology state of the fatty liver of NAFLD indication can be divided into fatty liver, fat hepatitis (Non-alcoholicsteatohepatitis, NASH), liver cirrhosis according to its severity.Wherein, NASH is attended by obvious fatty degeneration of liver, the pathological characters such as hepatic fibrosis.Up to 20%-30%, in the Asia, morbidity approximately has 12%～24% to NAFLD in the morbidity of north America region according to statistics, and this numeral also is being continuous ascendant trend.
Because the change of people's animation, the overnutrition phenomenon becomes more and more general.The physical exertion that day by day reduces adds high fat content food ubiquitous in the life, and obesity, diabetes B, metabolism syndrome have become the common complication of NAFLD.Generally believe that at present NAFLD is a kind of liver performance of metabolism syndrome and insulin resistant.Long-term NAFLD will be by simple fatty degeneration of liver in a single day through worsening, and bringing out becomes hepatitis, and hepatic fibrosis causes liver cirrhosis, hepatocellular carcinoma (hepatocellular carcinoma, HCC) at last, and human life's health is caused great threat.In the study of incident mechanism about NAFLD, " two-hit theory " that Day proposes obtained academia and generally approved.So-called 2 hit theories, refer to that namely first strike is that insulin resistant, the free fatty acids that obesity, diabetes B, disorders of lipid metabolism etc. cause increases, the adipose metabolism obstacle, thereby liver cell synthetic triglyceride (TG) is increased and the output minimizing, cause hepatic cell fattydegeneration; Second strike refers to increase the weight of damage again on the basis of first strike, thereby makes simple fatty liver be developed to nonalcoholic fatty liver disease (NASH) even hepatic fibrosis.Cause the 2nd strike that many factors can be arranged, and wherein oxidative stress and lipid peroxidation play an important role.
In the various effects that the discussion crocetin plays, what can not be ignored is its anti-oxidation efficacy.The research discovery, crocetin is for H
2O
2The hydroxy radical qiao (OH) that produces has good scavenging(action), can effectively reduce radical pair red corpuscle and Subcellular membrane structural damage effect, also can significantly suppress the damage of radical pair DNA simultaneously.
Chinese patent application 200710001116.7 discloses a kind of method for preparing crocetin of extracting from cape jasmine, being about to cape jasmine pulverizes through distilled water or the ultrasound-enhanced extraction of ethanolic soln, concentrated behind the extracting liquid filtering, the concentrated solution ethanol of water extraction precipitates rear reconcentration or separates cape jasmine glycoside material through macroporous resin, acid and alkali hydrolysis after the crocin elutriant is concentrated, centrifugal after obtaining the crocetin crude product and being further purified the high purity crocetin; This document provides a kind of batch manufacturing method of crocetin.But, this method all will adopt extraction using alcohol or ethanol precipitation in extraction, and the method for ultrasound-enhanced extraction is subject to equipment and is unsuitable for scale operation, and its extract first concentrate afterwards or upper macroporous resin after elutriant carry out again after concentrated the technique of acid and alkali hydrolysis loaded down with trivial details, have repeatedly concentration process, consume mass energy and be unfavorable for environmental protection.
Summary of the invention
The present invention aims to provide a kind of preparation method of trans-crocetin, in the natural plant raw material leaching process crocin is converted into crocetin.
The present invention also provides the application of above-mentioned trans-crocetin aspect the medicine of preparation treatment fatty liver.
The present invention adopts diluted alkaline to extract, and will extract concentrated and hydrolytic process integrates, and in the extraction concentration process crocin being hydrolyzed changes into crocetin.Be about to raw material pulverizing, transform extraction with diluted alkaline alcohol solution, extracting solution is adjusted to pH7～9 with acid, concentrating under reduced pressure filters or centrifugal removal precipitated impurities, and clear liquid is adjusted to pH2～5, filter or centrifugal collection red precipitate, with weakly acidic water or Diluted Alcohol solution washing precipitation, drying gets crocetin content and is higher than 50% crocetin crude product; Obtain content behind the crocetin crude product recrystallization purifying and be higher than 95% high purity trans-crocetin, concrete grammar is as follows:
(1) plant material is crossed 5～100 mesh sieves and pulverized, extract with 8～16 times of raw material weights, the diluted alcohol aqueous solution that contains alcohol amount 0～60wt%, filter or the centrifugal extracting solution that gets; Described diluted alcohol aqueous solution is 10～14 with alkali with pH regulator;
(2) extracting solution is adjusted to pH 7～9 with acid, and the extracting solution concentrating under reduced pressure is obtained concentrated solution, and resulting concentrated solution volume and material plant weight ratio are 1: 1～5L/Kg;
(3) with concentrated solution filtration or centrifugal removal precipitation, get concentrated clear liquid;
(4) regulate concentrated clear liquid to pH 2～5 with acid, left standstill 0.5～12 hour, centrifugal or filtration obtains the crocetin crude product;
(5) crocetin crude product recrystallization is 1～3 time, namely gets trans-crocetin, and wherein the content of trans-crocetin is more than 95%;
The technique of recrystallization is: the crocetin crude product with the dissolving of the alcoholic solution of 2～20 times of weight parts, pH 7～10, slowly added acid again and is adjusted to pH 2～4 and separates out precipitation, left standstill 0.5～12 hour, and centrifugal or filter and to get precipitation;
Described alcoholic solution is methyl alcohol or the ethanolic soln of mass concentration 50～100%, and regulates the pH value with sodium hydroxide, potassium hydroxide, calcium hydroxide, yellow soda ash or sodium bicarbonate;
Described acid for regulating pH is hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid or oxalic acid.
The plant material that step (1) adopts can be selected the gynoecium of cape jasmine fruit or style and stigma of Saffron Crocus (Crocussativus), and wherein cape jasmine fruit comprises the fruit of cape jasmine (Gardenia jasminodes Ellis), water cape jasmine (Gardenia jasminodes Ellis f.longiarpa Z.W.Xie et Okada) or Da Hua cape jasmine (Gardenia jasminodes Ellis var.fortuniana (Lind1) Ham).
The described extracting method of step (1) is backflow, dipping or diacolation.
Alkali described in the step (1) is sodium hydroxide, potassium hydroxide, calcium hydroxide or Calcium hydrogen carbonate.
The acid that is used for adjusting pH described in step (2) and (4) is hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid or oxalic acid.
Alcohol described in the step (1) is methyl alcohol or ethanol.
The present invention adopts diluted alkaline to extract, and will extract and hydrolytic process integrates, and namely in leaching process crocin being hydrolyzed changes into crocetin, makes to extract with one step of conversion and finishes, and method is easy, quick, cost significantly reduces.
Setting up on the fatty liver cell model basis, by the crocetin intervention, mensuration in conjunction with Radical Metabolism and related biochemical indicator carried out proves certain density trans-crocetin intervention, really can improve the effect of fatty liver cell by reducing oxidation level.Experimental results show that this trans-crocetin and the liposome that contains this trans-crocetin by cell levels have remarkable anti-oxidant activity and lipotropic effect.
Description of drawings
Fig. 1 is that embodiment 7 fatty liver cells add before and after the crocetin with attenuate born of the same parents' ratio of Microscopic observation fat after the oil red dyeing
Fig. 2 is the oil red dyeing photo that embodiment 7 fatty liver cells add the crocetin front and back
Fig. 3 is with respect to Normocellular TG content in the embodiment 7 trans-crocetin patients before and after intervention fatty liver cells
Fig. 4 is with respect to Normocellular ROS content in the embodiment 7 trans-crocetin patients before and after intervention fatty liver cells
Fig. 5 is with respect to Normocellular MDA content in the embodiment 7 trans-crocetin patients before and after intervention fatty liver cells
Fig. 6 changes with respect to Normocellular NADPH in the embodiment 7 trans-crocetin patients before and after intervention fatty liver cells
Fig. 7 is with respect to Normocellular GSH content in the embodiment 7 trans-crocetin patients before and after intervention fatty liver cells
Fig. 8 is among the interior SOD activity change Fig. 1,3～8 of embodiment 7 trans-crocetin patients before and after intervention fatty liver cells, histogram is followed successively by blank group, FFA group and FFA+ different concns trans-crocetin and trans-crocetin liposome, the black post is FFA+ trans-crocetin group, and white post is FFA+ trans-crocetin liposome group; Ordinate zou is 10
6Indices is with respect to the value of blank group in the cell; The concentration of FFA is 0.5mmol/L.
*Expression trans-crocetin group is compared p＜0.05 with the FFA group,
*Expression trans-crocetin liposome group is compared p＜0.05 with the crocetin group.
Embodiment
(1) cape jasmine (Gardenia jasminodes Ellis) fruit 10kg, pulverized 40 mesh sieves, use respectively 60L, 40L30% Diluted Alcohol solution (regulating pH=12 with sodium hydroxide) refluxing extraction twice, 3 hours for the first time, 1.5 hours for the second time, filter and obtain extracting solution;
(2) extracting solution is regulated pH=8 with hydrochloric acid, is evaporated to approximately 5L;
(3) filter, get filtrate and obtain concentrated clear liquid;
(4) slowly drip hydrochloric acid soln in the concentrated clear liquid, regulate pH=3, left standstill 4 hours, filter to get brick-red precipitation, with hydrochloric acid Diluted Alcohol solution (alcohol concn the is 20%) washing precipitation of pH=5 2 times, pure water washing 1 time, drying, namely get the crocetin crude product, wherein trans-crocetin content is 81.4%.
Embodiment 2
(1) water cape jasmine fruit 10kg pulverized 5 mesh sieves, extracted the centrifugal extracting solution that obtains with 80L 60% dilute methanol solution (regulating pH 10 with potassium hydroxide) diacolation;
(2) extracting solution is regulated pH=7 with acetic acid, is evaporated to 2L;
(3) filter, get filtrate and obtain concentrated clear liquid;
(4) slowly drip acetum in the concentrated clear liquid, regulate pH to 5, left standstill 0.5 hour, filter to get brick-red precipitation, the rare alcohol of the acetic acid of pH=4 (alcohol concn is 40%) washing precipitation 2 times, pure water washing 2 times, drying, namely get the crocetin crude product, wherein trans-crocetin content is 86.2%.
Embodiment 3
(1) the gynoecium 1kg of style and stigma of Saffron Crocus pulverized 100 mesh sieves, used respectively 7L, 5L, 4L water (regulating pH=14 with potassium hydroxide) dipping to extract three times, filtered, and merging filtrate obtains extracting solution;
(2) extracting solution is regulated pH=9 with hydrochloric acid, is evaporated to 1L;
(3) filter, get filtrate, obtain concentrated clear liquid;
(4) slowly drip hydrochloric acid soln in the concentrated clear liquid, regulate pH to 2, static 12 hours, filter to get brick-red precipitation, with the washing precipitation of the rare alcohol of pH=6 (alcohol concn is 30%) 1 time, pure water washing 1 time, drying, namely get the crocetin crude product, wherein trans-crocetin content is 80.5%.
Embodiment 4
With the crocetin crude product recrystallization of embodiment 11 time, obtain crocetin.The step of recrystallization is: the crocetin crude product 100g that gets embodiment 1 is dissolved in 500ml 50wt% aqueous ethanolic solution (pH7.5, regulate with sodium bicarbonate), filter, filtrate is adjusted to pH=3, filter to get the scarlet precipitation, the pure water washing namely gets crocetin to neutral, and wherein trans-crocetin content is 91.2%.
With the crocetin crude product recrystallization of embodiment 12 times, obtain crocetin.The step of recrystallization is: the crocetin crude product 100g that gets embodiment 1 is dissolved in the methanol solution (pH7.5 of 1000ml 70wt%, regulate with sodium bicarbonate), 5000 left the heart 10 minutes, and supernatant liquor is adjusted to pH=2, filter to get the scarlet precipitation, the pure water washing is to neutral; Above-mentioned steps is repeated once again, namely get crocetin, wherein trans-crocetin content is 97.2%.
Embodiment 6
With the crocetin crude product recrystallization of embodiment 13 times, obtain crocetin.The step of recrystallization is: the crocetin crude product 100g that gets embodiment 1 is dissolved in the methanol solution of 1000ml 95wt% (pH9.0,, regulate with NaOH), 5000 left the heart 10 minutes, supernatant liquor is adjusted to pH=2, filters to get the scarlet precipitation, and the pure water washing is to neutral; Repeat above-mentioned steps 2 times again, namely get crocetin, wherein trans-crocetin content is 98.4%.
The research of the anti-oxidant intervention fatty liver cell of embodiment 7 crocetins
(1) material and instrument
Material
L02 human liver cell strain (Zhejiang University's free radical life science center present); RPMI-1640 nutrient solution (GIBCO company); calf serum (Beijing Heng Shengma of unit biotechnology research institute); trans-crocetin (method by embodiment 4 prepares); oleic acid (Chemical Reagent Co., Ltd., Sinopharm Group); phospho-wolframic acid; palmitinic acid; methyl-sulphoxide (Chemical Reagent Co., Ltd., Sinopharm Group); oil red O stain agent (present of histoembryology system of Fudan University); Hematorylin (present of histoembryology system of Fudan University); 50% Virahol (Solution on Chemical Reagents in Shanghai company limited) TG test kit; GSH test kit (company is built up in Nanjing); the ROS test kit; GSH test kit (green skies biotechnology research institute); niacinamide; luminol,3-aminophthalic acid cyclic hydrazide; TBA; MTT; 6PD; G6P (SIGAMA company); TRIS (vast Imtech); Triton X-100, EDTA (Huamei Bio-Engrg Co.).
Instrument
Electronic analytical balance, Bechtop, inverted phase contrast microscope (OLYMPUSCK40), multi-functional microplate reader (ELX 800.BIO-TEK INSTRUMENTS), Panasonic DMC-FX3 photographic camera, 752s ultraviolet spectrophotometer, spectrophotofluorometer (HITACHI F-2500), the 722s spectrophotometer, biochemiluminescence instrument (SHG-1, Shanghai detection technique institute)
(2) method
(a) cell cultures: the LO2 cell within containing the RPMI-1640 nutrient solution of 10% calf serum, at 37 ℃, 5%CO
2Cultivate in the incubator.
(b) foundation of fatty liver cell model: add 0.5mmol/L lipid acid mixed solution FFA (being formed by oleic acid and the palmitinic acid proportional arrangement by 2: 1) in the nutrient solution, cultivated 48 hours.
(c) drug treating: cell attachment grows to behind the 70%-80% by trysinization, is inoculated in 6 orifice plates, and every porocyte number is 10
5Individual/ml.Be divided into 1. blank group (normal nutrient solution was cultivated 72 hours), 2. lipid acid (FFA) group (is inoculated after 24 hours, the FFA mixed solution that adds 0.5mmol/L was cultivated 48 hours), 3. dosing group (is inoculated the liposome that adds trans-crocetin after 12 hours and contain trans-crocetin, the final concentration that makes trans-crocetin is 2.5 μ mol/L, 5 μ mol/L and 10 μ mol/L, cultivate the FFA mixed solution that adds again 0.5mmol/L after 12 hours, cultivated 48 hours).Liposome-treated adopts film dispersion method.(d) measurement of cytoactive: tetramethyl-azo azoles salt method (mtt assay): 96 orifice plates to be measured, every hole add 20 μ l MTT, cultivate 4 hours in the incubator.Abandon supernatant, every hole adds 150 μ l methyl-sulphoxides, concussion 10min.Select the 490nm wavelength, measure at enzyme-linked immunosorbent assay instrument and respectively manage absorbancy.Cell survival rate=experimental group absorbancy/control group absorbancy * 100%.
(e) cell oil red O stain: cell is inoculated in 6 orifice plates (every hole is placed with cover glass), being added with lipid acid cultivated after 2 days, PBS flushing 3 times (each 5min), 50% Virahol is 1min fixedly, oil red O dye liquor dyeing 10min, distilled water flushing 3 minutes, haematoxylin dyeing 5min, indigo plant is returned in color separation, and camera is collected image.
(f) triglyceride level (TG) is measured in the cell: cell dissociation, and after the centrifugal collection ,-70 ℃ and 37 ℃ of fast freeze-thaws twice, the mirror mirror, if cytoclasis is incomplete, freeze thawing is once again.The concussion mixing, centrifugal 10000rpm, 5min.Draw supernatant, utilize Triglyceride Reagent box (company is built up in Nanjing) to measure.
(g) intracellular reactive chalcogen (ROS) is measured: according to specification sheets operation in the ROS test kit, fluoroscopic examination
(h) measurement of GSH in the cell: cell dissociation to be measured is carried out according to the operation on the specification sheets of GSH test kit after collecting.
(i) NADPH measures in the cell: with reference to the detection method of the people such as Zhiquan Zhang proposition.Cell dissociation, the adding damping fluid is resuspended, 2 freeze thawing smudge cellses.Centrifugal, collect supernatant, carry out immediately the mensuration of NADP, NADPH.
(j) measurement of SOD in the cell: adopt pyrogallol autoxidation method.Collect twice of express delivery freeze thawing behind the cell dissociation.Get clean mensuration pipe, add respectively CBS (pH ≈ 10), luminol,3-aminophthalic acid cyclic hydrazide adds pyrogallol (1mmol/L) at last, begins to measure its value of giving out light behind the reaction 20s.The SOD activity is by the inhibiting rate value representation of the free radical baseline ROS level that produces with respect to the pyrogallol autoxidation in the cell.
(k) measurement of lipid within endothelial cells superoxide (MDA): adopt thiobarbituricacidα-(TBA) fluorescent method.Obtained cell suspension adds respectively H
2SO
4, behind the phospho-wolframic acid, mixing under the room temperature, centrifugal, abandon supernatant.Then add 1%TBA, mixing; Boiling water bath 1h, the flowing water cooling; Add propyl carbinol, the vibration mixing; Centrifugal, get upper strata liquid and measure; Utilizing emitted light (EM) is 553nm, and exciting light (EX) is measured for 515nm.
(3) result
(a) impact of content is dripped in the intervention of different concns trans-crocetin on fatty liver cell fat
With after the oil red dyeing, attenuate born of the same parents' ratio of Microscopic observation fat, the result as shown in Figure 1 before and after the fatty liver cell dosing).The cell that adds FFA, the fat born of the same parents' ratio that attenuates is 28.7%, adds behind the lower concentration crocetin fat born of the same parents' ratio that attenuates and drops to 16%, and increase with the increase fat of the intervening concentration born of the same parents' fall that attenuates.Add behind the lower concentration liposome trans-crocetin fat born of the same parents' ratio fall that attenuates and increase, difference is more obvious during lower concentration.
Fig. 2 is oil red dyeing photo, and the result shows, blank group cell (A) edge clear, and nuclear is large, and rare dyeing fat drips in the cell.Singly adding the visible most cells of lipid acid group (B) includes than greasiness and drips.Visible stain fat drips to count and lacks than only adding the lipid acid group in lower concentration crocetin group (C) cell, only has a few fat in the large many cells and drips, and cell state also shows like blank group.The intracellular fat of liposome trans-crocetin intervention group (D) drips number still less.The dosing intervention group is dripped number with respect to the fat of simple FFA group and is obviously reduced, and further specifies the cellular fat that trans-crocetin can be caused by mixed fatty acid in the cell levels prevention.The liposome of trans-crocetin can further improve the fat of cell.
(b) variation of TG content in the fatty liver cell after the trans-crocetin intervention
Triglyceride level TG is the important indicator of cell lactones content, and what Fig. 3 showed is that each group is with respect to the content of TG in the normal group cell.After FFA processes, cause the content of TG in the cell obviously to rise, increased by 363%; And the TG content that has risen is significantly reduced, only risen 230% with respect to normal cell TG value; The increase TG fall of intervening concentration with trans-crocetin obviously increases.Illustrate that certain density trans-crocetin processing can obviously improve the accumulation of fat in the cell.And after adding the trans-crocetin of liposome-treated, the fall of TG obviously increases than simple trans-crocetin, and difference is more obvious during lower concentration.Illustrate that the trans-crocetin liposome also can make the crocetin lipotropy improve.
(c) variation of oxidative stress level in the fatty liver cell after the trans-crocetin intervention
ROS and MDA content are the important indicators of Free Radical Level in the reflection cell.The height of ROS also represents the height of cellular oxidation level to a certain extent, and Fig. 4 can find out, add FFA after, ROS obviously increases, and has raise 44%, illustrates that in the process of fatty acid-induced fatty liver, the oxidation level of cell increases; The ROS content of cell behind the adding lower concentration trans-crocetin, the increase ROS fall of intervening concentration with trans-crocetin obviously increases, the intervention of middle concentration trans-crocetin has only risen 11% with respect to normal cell ROS value, illustrates that trans-crocetin has the effect of removing free radical, improving the Cellular Oxidation level.Behind the trans-crocetin that adds liposome-treated, the fall of ROS increases than simple trans-crocetin intervention group, and difference is more obvious during lower concentration.
MDA is the product of lipid peroxide, can promote free radical lock reaction, and then protein, nucleic acid, lipid etc. are damaged, make the microbial film sex change, thereby cause cell mutation, aging or dead, the variation of MDA also can reflect the snperoxiaized degree of body inner lipid, can indirectly reflect the degree of cellular oxidation damage.Intracellular MDA content reduces (Fig. 5) than lipid acid groups of cells after adding crocetin.The crocetin intervention presents dosage effect, along with the also increase of increase MDA decline of crocetin concentration.Show that the crocetin drug intervention can alleviate the snperoxiaized level of lipid within endothelial cells, thereby alleviate the oxidative damage of fatty liver cell.Behind the crocetin that adds liposome-treated, the fall of MDA increases than simple crocetin intervention group, and difference is more obvious during lower concentration.
(d) variation of antioxidant levels in the fatty liver cell after the trans-crocetin intervention
The important indicator that NADPH also can regulate antioxidant system in the antimer, Fig. 5 shows that NADPH is with respect to the content of blank group, after adding the FAA modeling, intracellular NADPH content obviously reduces (descending 30%) than blank group, add that the NADPH content of cell raises than the FAA group behind the crocetin, middle and high concentration trans-crocetin intervention group has approached or has surpassed normal level.Illustrate trans-crocetin to the esterified process of liver cell except the certain restraining effect of tool, and tool is regulated the balance of Cellular Redox.Behind the trans-crocetin that adds liposome-treated, the ascensional range of NADPH also increases than simple trans-crocetin intervention group, and difference is more obvious during lower concentration.
The content of reduced glutathion (GSH) in Fig. 6 showed cell, the content of GSH can reflect the reduced level state of some systems in the cell, and the ability of removing lipid peroxidation.After FFA processed cell, GSH significantly descended in the cell, dropped to only have originally 11%, and this has further confirmed lipid acid in the process of the fatty liver cell of inducing, and can make that oxidation level improves in the born of the same parents.And the GSH level of lower concentration trans-crocetin group obviously rises (be blank group 65%) than FFA group, and middle and high concentration trans-crocetin group GSH content has approached or above normal group.Behind the trans-crocetin that adds liposome-treated, more obvious to keeping in the born of the same parents GSH reduced level effect.
Reduced glutathion (GSH) but appellation is the first barrier that opposing ROS generates, in tissue, play a part the cytophylaxis factor.Thereby the variation of GSH level is widely used to the index of oxidative damage tool protective effect as estimating exogenous material.Superoxide-dismutase (SOD) is main antioxidase in a kind of organism, can remove superoxide radical, removing superoxide anion that can be special, produce thereby reduce the material that hydroxy radical qiao etc. has more detoxifying function, in the toxicity of defence oxygen, suppress geriatric disease and the important role such as pre-anti-aging.Experimental result shows that cell quasi-SOD activity that FFA processes reduces to originally 71%, and the SOD level of trans-crocetin group intervention group obviously rises than the FFA group, and increases with the increase SOD level of intervening concentration.After adding the trans-crocetin intervention, the SOD activity is replied even above normal, the liposome of trans-crocetin can make intracellular SOD antioxidase level further improve.See Fig. 8.
This experiment shows, GSH and SOD content obviously reduce in the fatty liver cell model, the cell resistance of oxidation relatively a little less than, the oxidative stress level is higher.Adopt the trans-crocetin intervention group of present method preparation then can improve this redox nonequilibrium state, thereby can improve the survival rate of cell.The trans-crocetin intervention effect is strengthened.By changing the trans-crocetin intervention time, namely the different time intervenes before and after induced lipolysis liver cell model, shows that crocetin brings out for fatty liver and has certain prevention and control action kou.
Experimental data adopts mean value ± standard deviation, and (mean ± SD), relatively adopt the T check between group, p＜0.05 expression difference has significance.
The trans-crocetin of the results show present method preparation can effectively improve the oxidative stress status of fatty liver cell by its oxidation resistant effect.Also point out the trans-crocetin of present method preparation to change for fatty liver and have certain preventive and therapeutic effect.
According to " the two-hit theory " of fatty liver, in second strike, oxidative stress and lipid peroxide have been brought into play Main Function.Lipid peroxidation mainly refers to a kind of free chain reaction of occuring in polyunsaturated fatty acid, it is mainly caused by free radical and produces, and its feature is the generation lipid peroxide of chain type, the disorganize structure and function.Be accompanied by plastosome impaired, liver carries fatty function also significantly to descend, and these variations can be induced the gene alteration of coding lipid metabolism enzyme, thereby further cause liver injury so that fatty acid peroxidase and triglyceride level are piled up.Continue the superfluous accumulation of superfluous ROS and can make the interior antioxidant of body pursue exhaustion, thereby form the vicious cycle of free chain reaction.These Biochemical processes activate proinflammatory cytokine, cause liver cell be inflamed infiltrations, necrosis, even progress are hepatic fibrosis, liver cirrhosis.
Mda (MDA) is the product of lipid peroxidation, and it can reflect directly that free radical causes the degree of injury of lipid peroxidation.In the Models of Fatty Liver, the remarkable rising of ROS, MDA content has verified that also the high-caliber oxidative stress status in the fatty liver cell makes the interior redox equilibrium of hepatic tissue by havoc.And and the crocetin intervention of present method preparation can effectively reduce the oxidative stress level, thereby prevent the generation of " second strike ".
This experiment is by setting up the fatty liver cell model, study the trans-crocetin of present method preparation to the effect of fatty liver cell state at cell levels, for utilizing natural phant antioxidant component control fatty live lesions that theoretical foundation is provided, also provide experiment basis for clinical application research.
Claims (6)
1. a method for preparing trans-crocetin is characterized in that, may further comprise the steps:
(1) plant material is crossed 5～100 mesh sieves and pulverized, extract with 8～16 times of raw material weights, the diluted alcohol aqueous solution that contains alcohol amount 0～60wt%, filter or the centrifugal extracting solution that gets; Described diluted alcohol aqueous solution is 10～14 with alkali with pH regulator; Described plant material is the fruit of cape jasmine, the fruit of water cape jasmine, the fruit of Da Hua cape jasmine or the gynoecium of style and stigma of Saffron Crocus;
(2) extracting solution is adjusted to pH 7～9 with acid, and the extracting solution concentrating under reduced pressure is obtained concentrated solution, and resulting concentrated solution volume and material plant weight ratio are 1:1～5L/Kg;
(3) with concentrated solution filtration or centrifugal removal precipitation, get concentrated clear liquid;
(4) regulate concentrated clear liquid to pH 2～5 with acid, left standstill 0.5～12 hour, centrifugal or filtration obtains the crocetin crude product;
(5) crocetin crude product recrystallization is 1～3 time, namely gets trans-crocetin.
2. the method for preparing trans-crocetin claimed in claim 1 is characterized in that, the described extracting method of step (1) is backflow, dipping or permeating extraction.
3. the method for preparing trans-crocetin claimed in claim 1 is characterized in that, the alkali that is used for pH regulator in the described step (1) is sodium hydroxide, potassium hydroxide or calcium hydroxide.
4. the method for preparing trans-crocetin claimed in claim 1 is characterized in that, described step (2), (4) described acid for regulating pH are hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid or oxalic acid.
5. the method for preparing trans-crocetin claimed in claim 1 is characterized in that, the described alcohol of described step (1) is methyl alcohol or ethanol.
6. the method for preparing trans-crocetin claimed in claim 1 is characterized in that, the technique of recrystallization is in the described step (5):
The crocetin crude product with the dissolving of the alcoholic solution of 2～20 times of weight parts, pH 7～10, is slowly added acid again and is adjusted to pH 2～4 and separates out precipitation, left standstill 0.5～12 hour, centrifugal or filter and to get precipitation;
Described alcoholic solution is methyl alcohol or the ethanolic soln of mass concentration 50～100%, and regulates the pH value with sodium hydroxide, potassium hydroxide, calcium hydroxide, yellow soda ash or sodium bicarbonate;
Described acid for pH regulator is hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid or oxalic acid.
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| US9211298B2 (en) * | 2012-11-16 | 2015-12-15 | Song Gao | Compositions containing enriched natural crocin and/or crocetin, and their therapeutic or nutraceutical uses |
| JP7355394B2 (en) | 2018-05-03 | 2023-10-03 | エル．イー．エー．エフ． ホールディングス グループ エルエルシー | Carotenoid compositions and their uses |
| CN109180469B (en) * | 2018-09-05 | 2021-04-13 | 嘉文丽（福建）化妆品有限公司 | Separation method of cis-trans isomeric crocetin |
| TWI695827B (en) * | 2018-10-09 | 2020-06-11 | 鴻元生技股份有限公司 | The preparation method of trans-crocetin |
| CN109528729A (en) * | 2018-12-20 | 2019-03-29 | 浙江九如堂生物科技有限公司 | Application of the crocetin in prevention nonalcoholic steatohepatitis |
| WO2020125194A1 (en) * | 2018-12-20 | 2020-06-25 | 浙江工业大学 | Use of crocetin for prevention and treatment of non-alcoholic steatohepatitis |
| CN109620831A (en) * | 2018-12-20 | 2019-04-16 | 浙江工业大学 | Application of the crocetin in preparation treatment nonalcoholic steatohepatitis drug |
| CN110538186A (en) * | 2019-09-05 | 2019-12-06 | 浙江工业大学 | Application of crocin-I in preparation of medicine for reducing fat accumulation |
| CN110599485B (en) * | 2019-09-19 | 2022-04-15 | 北京大学人民医院(北京大学第二临床医学院) | Hepatitis C liver fibrosis characteristic information extraction method and device |
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