CN1377897A - Viva pertusa polyose for regulating blood fat and its preparing method - Google Patents

Viva pertusa polyose for regulating blood fat and its preparing method Download PDF

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CN1377897A
CN1377897A CN 02110254 CN02110254A CN1377897A CN 1377897 A CN1377897 A CN 1377897A CN 02110254 CN02110254 CN 02110254 CN 02110254 A CN02110254 A CN 02110254A CN 1377897 A CN1377897 A CN 1377897A
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polysaccharide
sea lettuce
blood fat
hole sea
regulating blood
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CN1165551C (en
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李智恩
吴志军
王艳梅
张虹
牛锡珍
于鹏展
徐祖洪
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Institute of Oceanology of CAS
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Abstract

The Visa pertusa polyose has an active radical OSO3 content of 15.94-19.20 %, total saccharide content of 49.30-60.8% and characteristic viscosity of 6.2-49.3. The present invention uses the Visa pertusa polyose in preparing marine medicine for regulating blood fat, and it has activity of regulating blood fat, strength immunity, resisting blood coagulation, etc. owing to the active radical OSO3. The Visa pertusa polyose is easy to prepare.

Description

Hole sea lettuce polysaccharide of regulating blood fat and preparation method thereof
(1) technical field
The present invention relates to the research and development of marine drug, specifically is a kind of marine drug of regulating the blood of human body lipids contents---hole sea lettuce polysaccharide and preparation method thereof, it belongs to the marine biotechnology field.
(2) background technology
Chemical composition analytical results to the hole sea lettuce in the prior art is: the chemical composition of hole sea lettuce mainly contains polysaccharide, lipid, protein, amino acid, VITAMIN and inorganic mineral element etc.At present for the application of hole sea lettuce except traditionally a small amount of edible, be used for feed and fertilizer more, raise together with fish, shrimp, can obviously improve the resistance against diseases of fish, do the growth that feed can be urged porgy, abalone, sea cucumber, reduce feed coefficient, save cost, being used for fertilizer can improve the soil.The high-activity component of hole sea lettuce and the latent effect of medical aspect have caused scientific worker's attention, and its applied research DEVELOPMENT PROSPECT is wide.
Hyperlipidemia (hyperlipaemia) is a kind of common metabolic disease, shows as different dyslipidemias accumulations in the blood plasma in the body.Main lipid comprises cholesterol and R-Glyceric acid three fat.LDL and HDL are all synthetic in liver, but how a lot of LDL is than the cholesterol amount that HDL contains, and comparatively speaking HDL then contains more phosphatide, and the big density of particle is little, are difficult for locating retardance at vessel wall etc. and adhere to.So LDL-C content is high more, HDL-C content is low more, and cholesterol content in serum is high more, easy more various hyperlipidemias and the cardiovascular disorder of bringing out.Particularly the cholesterol metabolic disorder is relevant with lipoid in atherosclerotic formation and development, and the cholesterol in serum level increases often makes atherosclerotic sickness rate increase (Shen is equal, 1991), and might cause coronary heart disease.Human life and health in serious threat.The source of cholesterol in serum is mainly food directly picked-up and the interior biosynthesizing of body, some hypolipidemic (as Serotinex) is by regulating the activity of synthesizing relevant enzyme with cholesterol, suppress cholesterol synthetic speed in vivo, thereby reach the effect (Pan Xian etc., 1998) that reduces the cholesterol in serum level.The approach of another kind of reducing blood-fat is the metabolic process that participates in blood fat.Under the normal circumstances, cholic acid enters enteron aisle from liver and gall-bladder secretion, and the fat in the food is played emulsifying effect, promotes the absorption of fat, and most of cholic acid is reuptaked through enterohepatic circulation again.Some drugs (QUESTRAN, chitosan etc.) combines the formation mixture with cholic acid and discharges from ight soil in enteron aisle, thereby has blocked the enterohepatic circulation of cholic acid.Because the continuous combined discharge of cholic acid has caused the precursor-cholesterol of cholic acid constantly to be oxidized to cholic acid, the cholesterol levels in the serum is descended; Similarly, unsaturated fatty acidss such as linolic acid can be combined into ester with cholesterol, make it more easily to transport, metabolism and drainage, and the distribution of change body inner cholesterol, make more being present in beyond the blood vessel of cholesterol, minimizing is in the deposition of vessel wall, thus the cholesterol level (Pan Xian etc., 1998) in the minimizing serum.But part medicine (clofibrate etc.) causes the rising of transaminase level quickening the cholesterol drainage, participating in its metabolic process liver being diminished, and toxic side effect is (Qi Chen etc., 1998) obviously.
(3) technology contents
The objective of the invention is to provide a kind of ocean medicinal material of transferring fat, i.e. hole sea lettuce polysaccharide of a kind of regulating blood fat and preparation method thereof.Because the hole sea lettuce is a kind of edible large-scale green alga, polysaccharide fraction wherein has multiple medical actives such as reducing blood-fat, anti-freezing, enhance immunity, and human body is had no side effect.Therefore, the present invention attempts to go to understand the effective constituent of hole sea lettuce accent blood fat and the extraction of effective constituent by the research of modern science and technology.The present invention is expected to be exploited behind deep structure research, for the mankind benefit.
Task of the present invention is finished by following technical scheme, has developed a kind of hole sea lettuce polysaccharide of regulating blood fat, and the typical structure formula of this polysaccharide is: Or
Figure A0211025400042
M wherein, n is the polymerization degree; Active group-the OSO of this polysaccharide 3 -The content of base is: 15.94--19.20%, total sugar content is: 49.30--60.8%, limiting viscosity is: 6.2--49.3.
The preparation method of the hole sea lettuce polysaccharide of regulating blood fat of the present invention, the preparation process of described this polysaccharide is as follows:
(1) takes by weighing quantitative hole sea lettuce green alga of drying in the shade naturally, pulverize;
(2) add the distilled water of 30 times of weight, extracted 1 hour;
(3) said extracted liquid is filtered, the distilled water that filter residue adds 30 times of weight again repeats to extract twice, merges three times filtrate;
(4) filtrate of above-mentioned merging is dialysed with dialysis tubing, got dialyzate;
(5) above-mentioned dialyzate is helped the filtration filter with diatomite;
(6) with above-mentioned filtrate through being evaporated to 1/3 volume;
(7), make the alcohol concn to 75% of this concentrated solution with the ethanol liquid that adds 95% in the above-mentioned concentrated solution;
(8) pulverize the white powder hole sea lettuce polysaccharide that to purify after getting drying precipitate in the above-mentioned ethanol concentrated solution.
The preparation method of the hole sea lettuce polysaccharide of regulating blood fat of the present invention, the extraction temperature in described (2) extract down at 80-100 ℃.
The preparation method of the hole sea lettuce polysaccharide of regulating blood fat of the present invention, dialysing with dialysis tubing in described (4) is to dialyse 24 hours in tap water earlier, dialyses 24 hours in distilled water then.
The preparation method of the hole sea lettuce polysaccharide of regulating blood fat of the present invention, the concentrating under reduced pressure in described (6) is 9.8 * 10 4Under the Pa, carry out under temperature 50--60 ℃.
Hole of the present invention sea lettuce polysaccharide is an important inventive point of the present invention as the application of the marine drug of preparation regulating blood fat.Its experiment effect is as follows:
The accent blood fat experiment I of hole sea lettuce polysaccharide:
1). laboratory animal
The ICR mouse, ♂, body weight 20~24g is provided by Yunnan Province natural drug pharmacology key lab, conformity certification number: No. 9806, the real moving card in Yunnan.
2). main medicine and reagent and equipment
Cholesterol is a Merck company product, and Sodium cholic acid is Shanghai chemical reagents corporation import packing (lot number 980720), and propylthiouracil is German import.Xuezhikang is that the Beijing WBL Peking University Biotech Co., Ltd produces lot number 991113.CL-770 type clinical spectroscopic luminosity is counted Japanese island Tianjin company and is produced.
3). experiment grouping and hyperlipidaemia moulding
80 of ICR mouse are divided into 8 groups at random: high fat control group, polysaccharide low dose group (250mg/kg/d), dosage group (500mg/kg/d) in the polysaccharide, polysaccharide high dose group (1000mg/kg/d), and positive controls (Xuezhikang 1.2g/kg/d).All animals normal packets breeding observing changed after three days feeds higher fatty acid, experimentizing property of high cholesterol diet hyperlipidaemia moulding.(W: W) be: cholesterol 2.0%, lard 7.5%, Sodium cholic acid 0.3%, propylthiouracil 0.075%, all the other are basal feed to the high lipid food prescription.When giving high lipid food, each experimental group is irritated stomach with corresponding dosage for test agent physiological saline suspension, and high fat control group is irritated stomach equal volume physiological saline, and irritating the stomach volume is the 0.25ml/10g body weight, moulding administration seven days.
4). lipid determination
After the moulding administration seven days, more than the fasting 12h, pluck eyeball and get blood survey blood fat, serum total cholesterol (TC), triglyceride level (TG), high density lipoprotein cholesterol (HDL-C) are measured and are used enzyme process, give birth to biological high-technology company production enzyme reagent kit in employing Beijing and carry out (production code member is respectively 000501,000709 and 000701), calculate serum low-density LP cholesterol (LDL-C) concentration with the Friedewald formula, experimental data compares between organizing with the t check.
5). test-results sees Table 1.Table 1: hole sea lettuce polysaccharide is to the influence of experimental hyperlipidemia mouse blood lipid level
(n=10,x±s)
With high fat control group ratio *P<0.05, *P<0.01
The TC-total cholesterol, the HDL-C-high density lipoprotein cholesterol
The TG-glyceric acid three ester, the LDL-C-low density lipoprotein cholesterol
As can be seen from the above table, hole sea lettuce polysaccharide low dosage, middle dosage, high dosage and positive controls are compared with high fat control group, total cholesterol (TC) has reduced by 22.1% respectively, 30.2%, 48.1%, 34.3%, triglyceride level (TG) has reduced by 19.0% respectively, 19.8%, 27.6%, 41.4%, low density lipoprotein cholesterol (LDL-C) has reduced by 22.9% respectively, 33.2%, 57.8%, 37.0%, the ratio of high density lipoprotein cholesterol and total cholesterol (HDL-C/TC) has then raise 8.2% respectively, 35.2%, 148.4%, 31.2%.Compare with positive controls, high dose group is except that triglyceride level (TG) is low slightly, all other are as total cholesterol (TC), the effect of the ratio (HDL-C/TC) of low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol and total cholesterol all is better than positive controls, the particularly effect of HDL-C/TC, effect is obvious especially.
As everyone knows, hyperlipidaemia is one of atherosclerosis (Atherosclerosis) primary hazard factor, HDL-C especially ratio and the incidence of atherosclerosis of HDL-C/TC is negative correlation, therefore, sea lettuce polysaccharide in hole has positive meaning to the useful influence of blood fat to the ischemic natural instincts heart, the cerebrovascular disease of preventing and treating atherosclerosis and being caused.
Hole sea lettuce polysaccharide is transferred blood fat experiment II:
1). laboratory animal: ICR mouse ♂, body weight 18-20g.Provide conformity certification number by Yunnan Province laboratory animal room of natural drug pharmacology key lab: No. 99019, the moving pipe of cloud.
2). experiment grouping and hyperlipidaemia moulding: select 50 of ICR mouse, be divided into five groups at random: high fat control group (I group), hole sea lettuce polysaccharide low dose group (II group, 125mgkg -1D -1) dosage group in the sea lettuce polysaccharide of hole (III group, 250mgkg -1D -1), hole sea lettuce polysaccharide high dose group (IV group, 500mgkg -1D -1) and Inositol Nicotinate control group (V group, Inositol Nicotinate, 500mgkg -1D -1).All animals changes after three days with the normal diet breeding observing feeds the moulding of experimentizing property of high lipid food hyperlipidaemia.High lipid food prescription: cholesterol 2%, lard 8%, Sodium cholic acid 0.3%, basal feed 89.7%, when feeding high lipid food, each administration group is irritated stomach, high fat control group equal-volume distilled water filling stomach with the hole sea lettuce polysaccharide or the Inositol Nicotinate aqueous solution of corresponding dosage.
3). lipid determination: moulding is after 7 days, fasting is more than 12 hours, pluck eyeball and get blood survey serum total cholesterol (TC), glyceric acid three ester (TG), high density lipoprotein cholesterol (HDL-C), calculate the ratio of low density lipoprotein cholesterol (LDL-C) and HDL-C/TC, compare between organizing.Serum TC, TG, HDL-C measure and use enzyme process, give birth to biological high-technology company production enzyme reagent kit in employing Beijing and carry out, and calculate LDL-C content (Friedewald WT et al.1972) with the Friedewald formula.Experimental data is carried out statistical procedures with the t check.The results are shown in Table 2:
Table 2: hole sea lettuce polysaccharide is to the influence of hyperlipidaemia moulding mouse blood lipid level
Table?14. Effect?of?ulvan?on?mice’s?hyperlipaemia
(n=10,x±s)
Figure A0211025400071
Annotate: * * and high fat blank group are than P<0.01
The TC-total cholesterol
The HDL-C-high density lipoprotein cholesterol
The LDL-C-low density lipoprotein cholesterol
TG-R-Glyceric acid three fat
From experimental result:
(1) low dosage (125mgkg -1D -1) the administration group reduces R-Glyceric acid three fat (TG) in the mice serum there were significant differences with the blank group;
(2) dosage (250mgkg in -1D -1) the administration group reduces total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), R-Glyceric acid three fat (TG) and rising HDL-C/TC ratio in the mice serum and blank group there were significant differences;
(3) high dosage (500mgkg -1D -1) the administration group reduces total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), R-Glyceric acid three fat (TG), high density lipoprotein increasing cholesterol (HDL-C) and HDL-C/TC ratio in the mice serum and blank group there were significant differences;
(4) middle and high dosage (250~500mgkg -1D -1) two administration groups have total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) in the remarkable reduction mice serum, high density lipoprotein increasing cholesterol (HDL-C) thus increase the effect of HDL-C/TC ratio;
The hole sea lettuce polysaccharide sample of (5) three dosage all has the effect of remarkable reduction mice serum R-Glyceric acid three fat.
Do not find that in experimentation mouse has toxicity performances such as poor appetite, hair color are undesired, weight loss.
Each dosage group of laboratory sample and the higher fat blank of Inositol Nicotinate positive controls group the results are shown in Table 3 to the treatment improvement of observation index.
Table 3: hole sea lettuce polysaccharide is to the improvement of hyperlipidaemia moulding mouse blood lipid level
Improving?Effect?of?ulvan?on?mice’s?hyperlipaemia
(n=10,X±S)
Figure A0211025400081
Annotate: * * compares P<0.01 with high fat control group
The TC-total cholesterol
The HDL-C-high density lipoprotein cholesterol
The LDL-C-low density lipoprotein cholesterol
The TG-glyceric acid three ester
As can be seen from the above table, high density lipoprotein cholesterol (HDL-C) and HDL-C/TC ratio increase along with the increasing of sample dose, and low density lipoprotein cholesterol (LDL-C) reduces with the increase of dosage, and dose-dependently is clearly all arranged; In dosage sample sets (III group) to fall total cholesterol level the most obvious, and be better than the positive controls (V group) of test dose; Falling R-Glyceric acid three fat effects the most tangible is low-dosage sample group (II group), and effect also is better than the positive controls of test dose; Effect be more or less the same (24.0% and 22.9%) is falling aspect the glyceride effect in middle and high dosage sample sets.
The experimental result of comprehensive all observation indexs, high dosage sample sets (IV group, 500mgkg -1D -1) drug activity the strongest, compare with the positive control drug Inositol Nicotinate of same dose, drug effect is better.
Hole sea lettuce polysaccharide has significant accent blood fat, infer that its mechanism of action may be polysaccharide and combine with lipid material in the serum, participate in cholesterol, lipoprotein metabolism activity as a kind of carrier, make the lipid material in the serum transport with comparalive ease and drain, influence its pathways metabolism.Hole sea lettuce polysaccharide is more special because of its syndeton, is difficult for by intravital colon bacterium degraded (B.Ray ﹠amp; M.Lahaye, 1995b), molecular weight is much bigger than cholesterol, lipoprotein and their derivative, and is survivable in small intestine with the combination that blood fat forms, the change of molecular weight may influence the heavily absorption of bile acide etc., impels the more bile acides that are oxidized to quickly of cholesterol; The hole sea lettuce polysaccharide that contains more sulfate active group may affect the quantity of low density lipoprotein receptor, played the effect of adjusting low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) level indirectly, thereby the content of rising total cholesterol middle-high density lipoprotein cholesterol improves the content and the distribution of blood fat under the pathological condition.
Hole sea lettuce polysaccharide is commonly considered as the effect of active group sulfate wherein in the activity that aspects such as reducing blood-fat, strengthening immunity, anticoagulation demonstrate; And think that in certain scope along with the increase of activity group content, activity also strengthens.Simultaneously, active power also is subjected to the influence of polysaccharide molecular weight size: molecular weight is more little within the specific limits, and activity is strong more.
(3) accompanying drawing and embodiment: protection scope of the present invention not only is confined in following examples.
Fig. 1 is the infrared spectrogram of hole sea lettuce polysaccharide.
Fig. 2 is a hole sea lettuce polysaccharide 13The C-NMR collection of illustrative plates.
Fig. 3 is a hole sea lettuce polysaccharide 1The H-NMR collection of illustrative plates.
The extracting method of the marine drug of regulating blood fat of the present invention-hole sea lettuce polysaccharide is as follows:
Raw material hole sea lettuce picks up from peaceful angle, Qingdao in March, 1999, and tap water cleans, and removes silt particle and other weeds, and sample dries in the shade naturally, preserves under the plastic bag sealing room temperature.Take from each 100 gram of three parts of hole sea lettuce green algas that so dry in the shade, pulverize; Extracted one hour in 80--100 ℃ with 30 times distilled water respectively; Filter, filter residue repeats to extract twice, merges three times filtrate; Filtrate was dialysed one day in tap water earlier with dialysis tubing, dialysed in distilled water twice then; Make flocculating aids with diatomite and filter dialyzate; Filtrate is through reducing pressure 9.8 * 10 4Under the Pa, be concentrated into 1/3 volume under temperature 50--60 ℃; Adding 95% ethanol to strength of solution is 75%, three parts of precipitations of gained; Obtain the polysaccharide sample of purifying after drying, weigh 15 grams, 15.7 grams, 14.4 grams, yield average out to 15%.The preparation method of this polysaccharide is simple, and running cost is low, pollutes and lacks, the efficient height.The physicochemical property of the hole sea lettuce polysaccharide of purifying sees Table 4.
Table 4, the physicochemical property of the hole sea lettuce polysaccharide of purification:
The atlas analysis of hole sea lettuce polysaccharide:
(1) the infrared spectrogram analytical results of hole sea lettuce polysaccharide:
The main absorption peak of collection of illustrative plates is all very similar, illustrates that monosaccharide component and the main functional group kind in each laboratory sample do not have too big difference.3425-3445cm -1It is the absorption peak of the small amount of moisture that contains in hydroxyl on the polysaccharide and the sample; Saturated hydrocarbon stretching vibration γ C-HAt 2935cm -1About; The absorption peak of carboxyl appears at 1640cm in the uronic acid residue -1Near, the absorption peak position of each sample is slightly variant; 1050-1113cm -1Between strong broad absorption band be the vibration peak of glycosidic link C-O-C between each saccharide residue of polysaccharide, owing to differing, each saccharide residue mode of connection and order cause difference between the glycosidic link, cause this broad absorption band peak shape irregular, peak position slightly changes with the difference of ratio between the glycosidic link; 1225-1265cm -1Strong peak and 840cm -1, 782-786cm -1Two spikes are absorption signals of sulfate.
(2) hole sea lettuce polysaccharide 13C-NMR spectroscopic analysis result:
Chemical shift (ppm)
C 1 C 2 C 3 C 4 C 5 C 6
GA (glucuronic acid) 102.91 75.66 77.89 78.56 73.62 175.11
R (rhamnosyl residue) 97.32 71.23 70.18 78.56 73.62 16.58
X (xylose residues) 99.59 73.62 77.89 67.53
G (glucosyl residue) 100.80 71.23 73.62 78.56 70.18 60.33/57.18
(3) hole sea lettuce polysaccharide 1H-NMR spectroscopic analysis result:
H1 H2 H2 H4 H5 H6
R 5.38 4.18 4.75 3.80/3.74 3.96 1.29
GA 4.75 3.46/3.42 3.65 3.62 3.80/3.14
X 4.75 3.21 3.59 3.74 3.32

Claims (5)

1, a kind of hole sea lettuce polysaccharide of regulating blood fat, the typical structure formula of this polysaccharide is:
Figure A0211025400021
Or
Figure A0211025400022
M wherein, n is the polymerization degree; It is characterized in that: the active group-OSO of this polysaccharide 3 -The content of base is: 15.94--19.20%, total sugar content is: 49.30--60.8%, limiting viscosity is: 6.2--49.3.
2, according to the preparation method of the hole sea lettuce polysaccharide of the described regulating blood fat of claim 1, it is characterized in that: the preparation process of described this polysaccharide is as follows:
(1) takes by weighing quantitative hole sea lettuce green alga of drying in the shade naturally, pulverize;
(2) add the distilled water of 30 times of weight, extracted 1 hour;
(3) said extracted liquid is filtered, the distilled water that filter residue adds 30 times of weight again repeats to extract twice, merges three times filtrate;
(4) filtrate of above-mentioned merging is dialysed with dialysis tubing, got dialyzate;
(5) above-mentioned dialyzate is helped the filtration filter with diatomite;
(6) with above-mentioned filtrate through being evaporated to 1/3 volume;
(7), make the alcohol concn to 75% of this concentrated solution with the ethanol liquid that adds 95% in the above-mentioned concentrated solution;
(8) pulverize the white powder hole sea lettuce polysaccharide that to purify after getting drying precipitate in the above-mentioned ethanol concentrated solution.
3, according to the preparation method of the hole sea lettuce polysaccharide of the described regulating blood fat of claim 2, it is characterized in that: the extraction temperature in described (2) is to extract down at 80-100 ℃.
4, according to the preparation method of the hole sea lettuce polysaccharide of the described regulating blood fat of claim 2, it is characterized in that: in described (4) with the dialysis tubing dialysis, be dialysis 24 hours in tap water earlier, dialysis 24 hours in distilled water then.
5, according to the preparation method of the hole sea lettuce polysaccharide of the described regulating blood fat of claim 3, it is characterized in that: the concentrating under reduced pressure in described (6) is 9.8 * 10 4Under the Pa, carry out under temperature 50--60 ℃.
CNB021102546A 2002-04-05 2002-04-05 Viva pertusa polyose for regulating blood fat and its preparing method Expired - Fee Related CN1165551C (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105050608A (en) * 2012-12-11 2015-11-11 阿玛德特公司 Algal extract comprising sulphated and non-sulphated polyanionic polysaccharides and uses thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106749723A (en) * 2016-12-09 2017-05-31 潍坊医学院 U. pertusa separation of polysaccharides product and its isolation and purification method

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105050608A (en) * 2012-12-11 2015-11-11 阿玛德特公司 Algal extract comprising sulphated and non-sulphated polyanionic polysaccharides and uses thereof
CN105050608B (en) * 2012-12-11 2021-06-08 阿玛德特公司 Algae extract containing sulfurized and unsulfurized polyanionic polysaccharides and its application

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