CN102250778A - Lachnum sp., extracellular polysaccharide thereof and application of extracellular polysaccharide in lipid-lowering liver-favoring medicaments - Google Patents
Lachnum sp., extracellular polysaccharide thereof and application of extracellular polysaccharide in lipid-lowering liver-favoring medicaments Download PDFInfo
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Abstract
The invention discloses Lachnum sp. YM-281, an extracellular polysaccharide thereof and application of the extracellular polysaccharide in preparing lipid-lowering liver-favoring medicaments. The invention is characterized in that a Lachnum sp. YM-281 strain with the collection number of CCTCC (China Center for Type Culture Collection) No: M 2011196 is activated and inoculated to a fermentation culture medium, thus preparing the Lachnum sp. YM-281 extracellular polysaccharide. When the administration dosage is 150-300 mg/(kg bw), the prepared Lachnum sp. YM-281 extracellular polysaccharide can significantly lower the levels of serum lipid and liver lipid and atherosis index of high-lipid mice, and increase the level of serum high-density lipoprotein cholesterol and the excretion amount of feces lipid. When the administration dosage is 75-300 mg/(kg bw), the prepared Lachnum sp. YM-281 extracellular polysaccharide can significantly lower the activity of serum aminotransferase and malondialdehyde content in liver tissues of high-lipid mice, and increase the activity of sudismase in the liver tissues. The prepared Lachnum sp. YM-281 extracellular polysaccharide has obvious effects of lowering blood lipid, lowering liver lipid, and treating hepatic injury and dysfunction caused by high lipid, has no toxic or side effects, and can be prepared into tablets or capsules for clinical application.
Description
Technical field
The invention belongs to the applied technical field of a hair disc bacterium and exocellular polysaccharide thereof, be specifically related to a hair disc bacterium YM-281 and exocellular polysaccharide thereof and the application in preparation lowering blood fat and benefiting liver medicine.
Background technology
Along with improving constantly of people's living standard, the absorption of high lipid food increases gradually, and disease incidences such as atherosclerosis, fatty liver and cardiovascular and cerebrovascular disease rise year by year.And according to " food colloid " (Food Hydrocolloids, 2005,19:813-819) introduce, high fat (blood fat, liver fat) is the main reason of atherosclerosis, cardiovascular and cerebrovascular diseases.(2010,28 (8): 1738-1740) introduce, high fat has the potential infringement to liver, can cause liver injury and dysfunction according to another " Chinese Chinese materia medica periodical ".
Often adopt prevention of fibrate and statins and treatment hyperlipemia clinically, but according to " Chinese arteriosclerosis magazine " (2003,11 (3): 223-226) introduce, these medicines have certain side effect, and the liver tissue injury that hyperlipidemia, high liver fat cause is not had alleviation and therapeutic action; Existing in addition fibrate and statins price are higher.Therefore the natural drug majority does not contain harmful composition, has no side effect, and develops the efficient and sharp liver medicine of natural lipopenicillinase (blood fat, liver fat) price economy of low toxicity and receives much concern all the time.
Grain hair disc Pseudomonas (Lachnum Retz.) is the saprophytic property of the class fungi that is distributed in all over the world, and inventor seminar discovered that a hair disc bacterium had important economic value in recent years.Seminar of the present invention is published in " journal of Zhejiang university " (2009,35:153-157) reach " carbon aqueous polymer " (Carbohydrate Polymers, 2011, reported in paper 86:285-290) that some bacterial strains of grain hair disc bacterium can produce a large amount of active polysaccharides under the deep layer culture condition, have stronger anti-oxidant, hypoglycemic isoreactivity.
Chinese patent application numbers 200910145058.4 discloses inventor seminar " a kind of hair disc bacterium and prepare melanic method by its liquid state fermentation ", be to utilize grain hair disc bacterium YM-223 (inventor seminar make the abbreviation of numbering code YeMing-223) the preparation melanochrome of a strain deposit number for CCTCC No:M 209193, this bacterial strain can produce the outer melanochrome of born of the same parents also can produce melanochrome in the born of the same parents; Chinese patent application numbers 201110072469.2 has been introduced inventor seminar " a strain Singh grain hair disc bacterium and liquid state fermentation thereof prepare melanic method in the born of the same parents ", utilizes another strain deposit number to prepare melanochrome in the born of the same parents for Singh's grain hair disc bacterium YM-292 of CCTCC No:M 2011054.
But do not see the report that has the lowering blood fat and benefiting liver effect about new bacterial strain grain hair disc bacterium YM-281 (inventor seminar makes the abbreviation of numbering code YeMing-281 by oneself) and exocellular polysaccharide prepared therefrom at present both at home and abroad as yet.
Summary of the invention
The purpose of this invention is to provide a kind of new bacterial strain grain hair disc bacterium YM-281 and prepare exocellular polysaccharide and the purposes of this exocellular polysaccharide in preparation lowering blood fat and benefiting liver medicine, to satisfy the needs of people the lowering blood fat and benefiting liver medicine by it.
Grain hair disc bacterium YM-281 bacterial strain of the present invention, the sample preservation unit of this biomaterial is Chinese typical culture collection center, the address: Lopa Nationality an ancient woman's ornament mountain, wuchang, wuhan life science institute of Wuhan University (postcode 430072), the preservation survival date is on June 18th, 2011; Deposit number is CCTCC No:M 2011196, classification called after grain hair disc bacterium YM-281 (Lachnum sp.YM-281).
The present invention is prepared the method for exocellular polysaccharide by grain hair disc bacterium YM-281 bacterial strain, it is characterized in that: with deposit number is 25-28 ℃ of activation 6-8 days on the solid activation medium of grain hair disc bacterium YM-281 (Lachnum sp.YM-281) inoculation of CCTCC No:M 2011196, this solid activation medium is by 20-25g/L glucose, peptone 4-6g/L, the 4-6g/L yeast extract paste, 14-16g/L agar is formed; Again activatory grain hair disc bacterium YM-281 is inoculated in the 250mL triangular flask that the 100-125mL fermention medium is housed, cultivated 3-4 days with the 180-200r/min isothermal vibration at 24-25 ℃, as the seed liquor of bulk fermentation, this fermention medium is by glucose 45-47g/L, yeast extract paste 14-15g/L, glycine 6-7mg/L, 0.9-1.1g/L MgSO
47H
2O and 0.9-1.1g/L KH
2PO
4Form, and regulate its initial pH at 7.0-8.0 with the NaOH solution of 0.3-0.5mol/L; The 4L fermention medium of in fermentor tank, packing into then, insert the seed liquor of 14-16% volume, the control culture temperature is 24-15 ℃, air flow is 0.9-1.1L/min, mixing speed is 160-180r/min, ferment after 8-10 days, with the fermented liquid suction filtration, be concentrated to 1/5 of original volume, the 90%-100% aqueous ethanolic solution that adds concentrated solution 3-4 times volume again, shake up the back in 4 ℃ of static 12-18h, throw out usefulness dehydrated alcohol that then centrifugation is obtained and acetone difference filtering and washing 3-4 time, with distilled water throw out is dissolved then, again through Sai Wojie (Sevage) method deproteinated, adding mass percentage concentration by the polysaccharide soln 1/3-1/2 volume behind the deproteinated then is the H of 30%-50%
2O
2In 50-55 ℃ of insulation decolouring down, it is in the dialysis tubing of 3500Da that polysaccharide soln after the decolouring is placed molecular weight cut-off, prior to flowing water, the 24-30h that in distilled water, dialyses respectively again, the solution after the dialysis promptly obtains a hair disc bacterium YM-281 exocellular polysaccharide powder through vacuum lyophilization; The Deproteinated operation of wherein said Sai Wojie (Sevage) method is as follows: add the mixed solution (Sevage reagent) that chloroform and propyl carbinol volume ratio by its volume 1/3 are 5: 1 in Crude polysaccharides solution, with the violent jolting 20-30min of this mixture, static 20-30min in the separating funnel packs into, by the time protein denaturation agglutination is present on the interface of water and solvent phase, divides the metaprotein of desolvate phase (lower floor's phase) and water and solvent phase intersection; Repetitive operation to the interface of water and solvent phase does not have till the colloid degeneration protein, and gained aqueous phase solution (upper strata phase) is the coarse grain hair disc bacterium YM-281 exocellular polysaccharide solution behind the deproteinated.
By the pharmacological evaluation that prepared grain hair disc bacterium YM-281 exocellular polysaccharide is carried out the effect of mouse lowering blood fat and benefiting liver, confirm that it has the lowering blood fat and benefiting liver effect, and determine its effective dose: when dosage is 150-300mg/ (kg bw), significantly reduce high fat mice serum lipid, liver lipid level and atheroma index, increase serum high-density LP cholesterol levels and ight soil lipid excretion; Dosage is that 75-300mg/ (kg bw) significantly reduces high fat mice serum transaminase activity and hepatic tissue mda content, and it is active to increase the hepatic tissue sudismase; Have tangible reducing blood-fat, liver lipid lowering, treat high fat and cause liver injury and parafunctional effect, have no side effect, can be made into tablet or capsule for clinical use.
The purposes of grain hair disc bacterium YM-281 exocellular polysaccharide of the present invention in preparation lowering blood fat and benefiting liver medicine, it is characterized in that: the grain hair disc bacterium YM-281 exocellular polysaccharide of pressing 300-600g, behind pharmaceutical excipient thinner 200-250g and glidant 30-60g mixing, mix with 250-600mL ethanol again and make softwood, with this softwood whole grain behind the wet grain drying that obtains that sieves, add compressing tablet behind the lubricant 3-10g mixing at last, make 1000 in tablet; After perhaps pressing pharmaceutical excipient thinner 200-270g, glidant 40-70g and wetting agent 3-6g mixing, mix the back with the grain hair disc bacterium YM-281 exocellular polysaccharide of 300-600g and divide in 1000 the 1# Capsuleses of packing into, make 1000 of capsules; Described pharmaceutical excipient thinner is selected dextrin or dry starch for use; Described glidant adopts talcum powder or micropowder silica gel or Microcrystalline Cellulose; Described lubricant adopts Magnesium Stearate or Zinic stearas; Described wetting agent adopts sodium lauryl sulphate or dioctyl sulphosuccinate.
The preference of the tablet processing that the present invention recommends is: add 300-500mL ethanol after grain hair disc bacterium YM-281 exocellular polysaccharide 500g, thinner dextrin 250g, the glidant talcum powder 40g that got 80～100 mesh sieves mixes and mix and make softwood, softwood sieves and obtains wet granular, the whole grain in dry back, add compressing tablet behind the magnesium stearate lubricant 10g mixing at last, make 1000, the heavy 0.8g of sheet, polysaccharide content 0.5g/ sheet.
The capsule processing preference that the present invention recommends is: get thinner dry starch 250g, glidant talcum powder 45g, wetting agent sodium lauryl sulphate 5g mixes and mixes 1000 of back dress 1# Capsuleses, polysaccharide content 0.5g/ grain with 500g grain hair disc bacterium YM-281 exocellular polysaccharide.
This polysaccharide fermentation, extraction and purification condition among the present invention in the preparation grain hair disc bacterium YM-281 exocellular polysaccharide method all are to obtain through the contriver of seminar research experiment optimization conclusion of the present invention, and the output of preparation grain hair disc bacterium YM-281 exocellular polysaccharide is bigger under these conditions.
Among the present invention grain hair disc bacterium YM-281 exocellular polysaccharide is carried out pharmacodynamic experiment, dosage this polysaccharide in 150-300mg/ (kg bw) scope has significant blood lipid reducing, the moving arteries and veins gruel type index that reaches of liver lipid level to high fat mouse, increase serum high-density LP cholesterol levels and ight soil lipid excretion, dose-effect relationship is good; Dosage is in 75-300mg/ (kg bw) scope the time, and this polysaccharide has significant transaminase lowering activity and hepatic tissue mda content to high fat mouse, increases hepatic tissue sudismase activity, and is good dose-effect relationship; Show that a hair disc bacterium YM-281 exocellular polysaccharide has tangible reducing blood-fat, liver lipid lowering, treats high fat and causes the parafunctional effect of liver injury, and have no side effect.
The present invention adopts a prepared oral preparations of hair disc bacterium YM-281 exocellular polysaccharide to overcome fibrate and statins has certain side effect, on the high side, and the liver tissue injury that hyperlipemia fat, high liver fat cause do not had alleviate and shortcomings such as therapeutic action.Experimental study confirms that effectively reducing blood-fat of grain hair disc bacterium YM-281 exocellular polysaccharide of the present invention, liver lipid lowering, alleviation and the high fat of treatment cause liver injury and dysfunction of liver, and have no side effect, preparation method's simple economy.
Description of drawings
Fig. 1 is normal control group murine liver tissue pathological section figure;
Fig. 2 is high fat control group mice hepatic tissue pathology slice map;
Fig. 3 is hair disc bacterium YM-281 exocellular polysaccharide 300mg/ (kg bw) group murine liver tissue pathological section figure;
Fig. 4 is hair disc bacterium YM-281 exocellular polysaccharide 150mg/ (kg bw) group murine liver tissue pathological section figure;
Fig. 5 is hair disc bacterium YM-281 exocellular polysaccharide 75mg/ (kg bw) group murine liver tissue pathological section figure;
Fig. 6 be hot he cut down spit of fland control group mice hepatic tissue pathology slice map.
Embodiment
Embodiment 1:
The preparation of grain hair disc bacterium YM-281 exocellular polysaccharide:
The preparation process of grain hair disc bacterium YM-281 exocellular polysaccharide is as follows in the present embodiment:
With deposit number is 25-28 ℃ of activation 6-8 days on the solid activation medium of the grain hair disc bacterium YM-281 bacterial strain inoculation of CCTCC No:M 2011196, this solid activation medium is by 20-25g/L glucose, peptone 4-6g/L, the 4-6g/L yeast extract paste, 14-16g/L agar is formed; Again activatory grain hair disc bacterium YM-281 is inoculated in the 250mL triangular flask that the 100-125mL fermention medium is housed, cultivated 3-4 days with the 180-200r/min isothermal vibration at 24-25 ℃, as the seed liquor of bulk fermentation, this fermention medium is by glucose 45-47g/L, yeast extract paste 14-15g/L, glycine 6-7mg/L, 0.9-1.1g/L MgSO
47H
2O and 0.9-1.1g/L KH
2PO
4Form, and regulate its initial pH at 7.0-8.0 with the NaOH solution of 0.3-0.5mol/L; Adopt 5L automatically controlled fermentor liquid submerged fermentation then, the fermentor tank 4L fermention medium of packing into, inoculum size is 14-16%, culture temperature is 24-15 ℃, air flow is 0.9-1.1L/min, mixing speed is 160-180r/min, ferment after 8-10 days the fermented liquid suction filtration, be concentrated to 1/5 of original volume, add the 90%-100% aqueous ethanolic solution of pressing concentrated solution 3-4 times volume, shake up the back in 4 ℃ of static 12-18h, be sub-packed in the centrifugal 8-12min of 5000r/min in the centrifuge tube then, centrifugal sediment is used earlier dehydrated alcohol, use the acetone filtering and washing again 3-4 time, use the dissolved in distilled water throw out then, through Sai Wojie (Sevage) method deproteinated, adding mass percentage concentration by the polysaccharide soln 1/3-1/2 volume behind the deproteinated then is the H of 30%-50% again
2O
2In 50-55 ℃ of insulation decolouring down, it is in the dialysis tubing of 3500Da that polysaccharide soln after will decolouring then places molecular weight cut-off, the 24-30h that dialyses respectively in flowing water, distilled water, the solution after the dialysis promptly get a grain hair disc bacterium YM-281 exocellular polysaccharide powder through vacuum lyophilization.Described Sevage method deproteinated is: add the mix reagent (Sevage reagent) that chloroform and propyl carbinol volume ratio by its volume 1/3 amount are 5: 1 in Crude polysaccharides solution, behind the violent jolting 20-30min of this mixture, static 20-30min in the separating funnel packs into, by the time protein denaturation agglutination is present on the interface of water and solvent phase, and dividing desolvates reaches the metaprotein of water and solvent phase (lower floor mutually) intersection mutually.Repetitive operation to the interface of water and solvent phase does not have till the colloid degeneration protein, and gained aqueous phase solution (upper strata phase) is the coarse grain hair disc bacterium YM-281 exocellular polysaccharide solution behind the deproteinated.
Grain hair disc bacterium YM-281 exocellular polysaccharide is to the effect pharmacological evaluation of mouse lowering blood fat and benefiting liver:
(1) animal: kunming mice, male and female half and half, body weight 20 ± 2g, random packet.
(2) test kit: total cholesterol (TC), tri-glyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), gpt (ALT), glutamic-oxal(o)acetic transaminase (AST), mda (MDA), super oxygen dismutase (SOD), TOTAL BILE ACID (TBA) are measured test kit, build up bio-engineering research institute available from Nanjing.
(3) high lipid food: standard test feed 78.6%, yolk 10%, lard 10%, cholesterol 1%, Sodium cholic acid 0.2%, Thyreostat I 0.2% is mixed thoroughly, adds water and makes high lipid food, dry for standby in right amount.
(4) preparation of grain hair disc bacterium YM-281 exocellular polysaccharide solution: polysaccharide powder is mixed with the solution of 7.5mg/mL, 15mg/mL, 30mg/mL with 0.9% physiological saline, and the administration volume is 10mL/ (kg bw).
(5) method: mouse is divided into 6 groups at random after adaptability under the experimental situation is fed a week: grain hair disc bacterium YM-281 exocellular polysaccharide high dose group, middle dosage group, low dose group, normal control group, high fat control group, positive controls, 15 every group.Wherein the normal control group gives outside the conventional feed, and all the other 5 groups give high lipid food after 2 weeks, and according to dosage 1 filling every day stomach gives relative medicine, and the administration volume is 1.0mL/100g bw, and normal control group and high fat control group give equal-volume physiological saline.The normal control group is irritated stomach 0.9% physiological saline every day, grain hair disc bacterium YM-281 exocellular polysaccharide high dose group, middle dosage group, low dose group be the polysaccharide soln of continuous irrigation stomach 7.5mg/mL, 15mg/mL, 30mg/mL respectively, positive controls continuous irrigation stomach 0.8mg/mL Simvastatin solution is irritated the long-pending 1.0mL/ (100g bw) of being of body of stomach.Give the thing that tried of different volumes according to the mouse body weight, freely ingest and drink water, continuous irrigation fed for 4 weeks.The normal control group gives conventional feed during the administration, and all the other 5 groups still give high lipid food.
(6) index of correlation is measured:
Serum middle finger target is measured:
Each is organized the mouse last and irritates the preceding all fasting 12h of stomach, and last is weighed after irritating stomach 1h, extracts eyeball and gets blood, isolates serum in 4 ℃ of centrifugal 10min of 4000r/min, and refrigerator is preserved (20 ℃), surveyed fully.Use the corresponding reagent box to measure total cholesterol (TC), tri-glyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) content and gpt (ALT), glutamic-oxal(o)acetic transaminase (AST) activity in the serum, concrete operations are by measuring the test kit specification sheets.Atheroma index (AI) is pressed AI=(TC-(HDL-C))/HDL-C and is calculated.
Hepatic tissue middle finger target is measured:
After mouse excision eyeball is got blood, dissect and get liver, the physiological saline rinsing, filter paper blots, and weighs, and refrigerator is preserved (80 ℃), is equipped with and surveys.The liver coefficient is calculated as follows, and liver refers to coefficient (g/100g bw)=liver quality (g)/body weight (100g).Measure total cholesterol in the hepatic tissue (TC), tri-glyceride (TG), mda (MDA) content and sudismase (SOD) activity then.Concrete operations are pressed total cholesterol, tri-glyceride, mda and sudismase and are measured the test kit specification sheets.Grind after the hepatic tissue vacuum lyophilization, adopt cable-styled extraction method to measure hepatic fat content then.
The mensuration of bile acide, total cholesterol and fatty excretion in the stool in mice:
Collect last 3 days stool in mice of administration phase, vacuum lyophilization is weighed, and sealing is preserved, and is equipped with and surveys.Then dry stool in mice is ground, measure TOTAL BILE ACID and total cholesterol level in the stool in mice, concrete operations are measured the test kit specification sheets by TOTAL BILE ACID, total cholesterol; Get ight soil dry powder 2g, add 20mL chloroform-methanol solution lixiviate 24 hours, the centrifugal 10min of 4000r/min isolates supernatant liquor then, and supernatant liquor dries up the fat under weighing is residual then with anhydrous sodium sulphate (2g) drying with nitrogen.TOTAL BILE ACID (TC or fat) excretion (mg/mouse/3days)=stool quality * TOTAL BILE ACID (TC or fat) content.
The murine liver tissue pathology section examination:
(2cm * 2cm * 0.2～0.3cm) the freezing embedding medium embedding of OCT of fresh liver tissue piece, with freezing-microtome section (thickness is 8 μ m), fixedly 1min of 10% formalin solution is put in section, carries out conventional H E dyeing, with the observation of optics microscopically, take pictures.
(7) grain hair disc bacterium YM-281 exocellular polysaccharide is to the effect The pharmacological results of mouse lowering blood fat and benefiting liver:
Grain hair disc bacterium YM-281 exocellular polysaccharide is as shown in table 1 to the restraining effect that lipid content in the serum of high fat mouse due to the high fat diet raises.High fat control group mice serum TC, TG, LDL-C and atheroma index (AI) all are significantly higher than the normal control group, and HDL-C and normal control group do not have significant difference.After 4 weeks of administration, compare with high fat control group, high dose group mice serum TC, TG, LDL-C content and AI significantly reduce, and Serum HDL-C content enlarges markedly, and wherein mice serum TC compares with the normal control group with LDL-C does not all have significant difference (table 1).In, the grain hair disc bacterium YM-281 exocellular polysaccharide of low dosage also can be in various degree the high fat mice serum of reduction TC, TG, LDL-C content and AI value, increase Serum HDL-C content, dose-effect relationship is better.Illustrate that grain hair disc bacterium YM-281 exocellular polysaccharide can effectively reduce high fat mice serum lipid level, has reducing blood-fat, suppresses atheromatous effect.
Grain hair disc bacterium YM-281 exocellular polysaccharide is as shown in table 2 to the influence of lipid metabolism in the high fat murine liver tissue.High fat control group mice liver TC, TG, lipid content are significantly greater than the normal control group.After 4 weeks of administration, high, middle dosage group and positive controls mouse liver TC, TG, lipid content all significantly are lower than high fat control group.LEP-1b high dose group mouse liver TG compares no significant difference with lipid content with the normal control group.High dose group murine liver tissue TC, TG and lipid content are lower than positive controls 54.29%, 44.70% and 33.63% respectively.Illustrate that grain hair disc bacterium YM-281 exocellular polysaccharide can effectively reduce high fat murine liver tissue lipid level, have liver lipid lowering, the effect of treatment fatty liver, dose-effect relationship is good, and effect is better than the positive control medicine.
Table 1. a hair disc bacterium YM-281 exocellular polysaccharide is to the influence (mean ± SD, n 〉=10) of high fat mice serum lipid metabolism
In the table 1: TC represents total cholesterol; TG represents triglyceride level; HDL-C represents high density lipoprotein cholesterol; LDL-C represents low density lipoprotein cholesterol; AI represents the atheroma index.
aRepresentative is compared P＜0.05 with the normal control group,
bRepresentative is compared P＜0.01 with the normal control group;
cRepresentative is compared P＜0.05 with high fat control group,
dRepresentative is compared P＜0.01 with high fat control group;
Table 2. a hair disc bacterium YM-281 exocellular polysaccharide is to the influence of high fat mice serum liver lipid metabolism
In the table 2: TC represents total cholesterol; TG represents triglyceride level;
aRepresentative is compared P＜0.05 with the normal control group,
bRepresentative is compared P＜0.01 with the normal control group;
cRepresentative is compared P＜0.05 with high fat control group,
dRepresentative is compared P＜0.01 with high fat control group;
As shown in table 3, to compare with the normal control group, high fat control group mice ight soil TOTAL BILE ACID, TC and fatty excretion all enlarge markedly.After 4 weeks of administration, compare with high fat control group, high, middle dosage group and positive controls, and only high dose group stool in mice fat excretion enlarges markedly.Low dose group stool in mice TOTAL BILE ACID, TC and fatty excretion are compared with high fat control group not have and are enlarged markedly.High dose group is respectively greater than positive controls 14.91%, 26.91% and 4.75%.Illustrate that a grain hair disc bacterium YM-281 exocellular polysaccharide can effectively increase high fat stool in mice TOTAL BILE ACID, TC and fatty excretion, effectively the result of reducing blood-fat liver fat is consistent with it for this.
Table 3. is respectively organized the drainage situation of TOTAL BILE ACID in the stool in mice, total cholesterol and fat
In the table 3: TC represents total cholesterol;
aRepresentative is compared P＜0.05 with the normal control group,
bRepresentative is compared P＜0.01 with the normal control group;
cRepresentative is compared P＜0.05 with high fat control group,
dRepresentative is compared P＜0.01 with high fat control group;
Fig. 1 has provided normal control group murine liver tissue pathological section figure, liver cell marshalling among Fig. 1, cellular form is normal, fat not occurring becomes. and Fig. 2 has provided high fat control group mice hepatic tissue pathology slice map, the downright bad part of a large amount of individual bigger fat vacuole cellular infiltrations appears among Fig. 2, and tangible diffusivity liver fat becomes and inflammatory becomes, the liver cell that fat becomes loosely organized mixed and disorderly, expand distortion, show that this group mouse has been tangible fatty liver symptom, and more serious hepatomegaly, the necrosis of liver cell wetting property have occurred; It is 300 and 150mg/ (kg bw) group murine liver tissue pathological section figure that Fig. 3 and Fig. 4 have provided a grain hair disc bacterium YM-281 exocellular polysaccharide dosage respectively, liver cell arranging situation among Fig. 3 and Fig. 4, and cellular form is all close with the normal group murine liver tissue; It is 75mg/ (kg bw) group murine liver tissue pathological section figure that Fig. 5 has provided grain hair disc bacterium YM-281 exocellular polysaccharide dosage, Fig. 6 be hot he cut down spit of fland control group mice hepatic tissue pathology slice map, still have the little fat cavity among Fig. 5 and Fig. 6, but comparatively small amt, individual less.Because the formation of hyperlipidemia and fatty liver causes peroxide injury and dysfunction of liver to murine liver tissue, thereby cause high fat control group mice Serum ALT, AST activity, hepatic tissue MDA content and liver coefficient significantly to raise, active significantly reduce (table 3) of hepatic tissue SOD.After 4 weeks of administration, than high fat control group, each dosage group mice serum ALT of grain hair disc bacterium YM-281 exocellular polysaccharide, AST activity, hepatic tissue MDA content and liver coefficient significantly reduce, and hepatic tissue SOD is active significantly to raise.The fatty liver symptom that mouse can significantly be alleviated, be treated to above presentation of results grain hair disc bacterium YM-281 exocellular polysaccharide, and promote the liver tissue injury reparation, dose relationship is good, and effect is better than the positive control medicine.
Table 4. a hair disc bacterium YM-281 exocellular polysaccharide is to the repair (mean ± SD, n 〉=10) of high fat mouse liver damage
In the table 4: ALT represents gpt; AST represents glutamic-oxal(o)acetic transaminase; MDA represents mda; SOD represents sudismase;
aRepresentative is compared P＜0.05 with the normal control group,
bRepresentative is compared P＜0.01 with the normal control group;
cRepresentative is compared P＜0.05 with high fat control group,
dRepresentative is compared P＜0.01 with high fat control group;
Above-mentioned pharmacological experiment assay determination blood fat, the most representative index that liver fat and liver injury are relevant, comprehensive The above results shows, the grain hair disc bacterium YM-281 exocellular polysaccharide that makes has blood lipid reducing (TC to hyperlipemia mouse due to the high fat diet, TG, LDL-C) and liver lipid (TC, TG, fat) level, increase the Serum HDL-C level, increase ight soil lipid (bile acide, TC, fat) output, reduce atheroma index (AI), serum transaminase (ALT, AST) activity, hepatic tissue MDA content, improve the active effect of hepatic tissue SOD, and be certain dose-effect relationship, illustrate that grain hair disc bacterium YM-281 exocellular polysaccharide has reducing blood-fat preferably, the effect that liver lipid lowering and promotion liver tissue injury are repaired, and effect obviously is better than the positive control medicine.
The processing of grain hair disc bacterium YM-281 polysaccharide formulation:
1, tablet processing:
250-600mL ethanol behind grain hair disc bacterium YM-281 exocellular polysaccharide and the pharmaceutical excipient thinner 200-250g of 300-600g, the glidant 30-60g mixing mixed make softwood, softwood sieves and obtains wet granular, the whole grain in dry back, add compressing tablet behind the lubricant 3-10g mixing at last, make 1000 tablets of tablets.Described pharmaceutical excipient thinner is selected dextrin or dry starch for use; Described glidant adopts talcum powder or micropowder silica gel or Microcrystalline Cellulose; Described lubricant adopts Magnesium Stearate or Zinic stearas.
The preference of the tablet processing that the present invention recommends is: add 300-500mL ethanol after grain hair disc bacterium YM-281 exocellular polysaccharide 500g, thinner dextrin 250g, the glidant talcum powder 40g that got 80～100 mesh sieves mixes and mix and make softwood, softwood sieves and obtains wet granular, the whole grain in dry back, add compressing tablet behind the magnesium stearate lubricant 10g mixing at last, make 1000, the heavy 0.8g of sheet, polysaccharide content 0.5g/ sheet.
2, capsule processing
With mixing back dress 1# Capsules with the grain hair disc bacterium YM-281 exocellular polysaccharide of 300-600g behind pharmaceutical excipient thinner 200-270g, glidant 40-70g, the wetting agent 3-6g mixing, make 1000 of capsules.Described pharmaceutical excipient thinner is selected dextrin or dry starch for use; Described glidant adopts talcum powder or micropowder silica gel or Microcrystalline Cellulose; Described wetting agent adopts sodium lauryl sulphate or dioctyl sulphosuccinate.
The capsule processing preference that the present invention recommends is: get thinner dry starch 250g, glidant talcum powder 45g, wetting agent sodium lauryl sulphate 5g mixes and mixes 1000 of back dress 1# Capsuleses, polysaccharide content 0.5g/ grain with 500g grain hair disc bacterium YM-281 exocellular polysaccharide.
Claims (5)
1. a grain hair disc bacterium YM-281 (Lachnum sp.YM-281) bacterial strain, the sample preservation of this biomaterial is numbered CCTCC No:M 2011196.
2. method for preparing exocellular polysaccharide by grain hair disc bacterium YM-281 bacterial strain, it is characterized in that: with deposit number is 25-28 ℃ of activation 6-8 days on the solid activation medium of grain hair disc bacterium YM-281 (Lachnum sp.YM-281) inoculation of CCTCC No:M 2011196, this solid activation medium is by 20-25g/L glucose, peptone 4-6g/L, the 4-6g/L yeast extract paste, 14-16g/L agar is formed; Again activatory grain hair disc bacterium YM-281 is inoculated in the 250mL triangular flask that the 100-125mL fermention medium is housed, cultivated 3-4 days with the 180-200r/min isothermal vibration at 24-25 ℃, as the seed liquor of bulk fermentation, this fermention medium is by glucose 45-47g/L, yeast extract paste 14-15g/L, glycine 6-7mg/L, 0.9-1.1g/L MgSO
47H
2O and 0.9-1.1g/L KH
2PO
4Form, and regulate its initial pH at 7.0-8.0 with the NaOH solution of 0.3-0.5mol/L; The 4L fermention medium of in fermentor tank, packing into then, insert the seed liquor of 14-16% volume, the control culture temperature is 24-15 ℃, air flow is 0.9-1.1L/min, mixing speed is 160-180r/min, ferment after 8-10 days, with the fermented liquid suction filtration, be concentrated to 1/5 of original volume, the 90%-100% aqueous ethanolic solution that adds concentrated solution 3-4 times volume again, shake up the back in 4 ℃ of static 12-18h, throw out usefulness dehydrated alcohol that then centrifugation is obtained and acetone difference filtering and washing 3-4 time, with distilled water throw out is dissolved then, again through the Sai Wojiefa deproteinated, adding mass percentage concentration by the polysaccharide soln 1/3-1/2 volume behind the deproteinated then is the H of 30%-50%
2O
2In 50-55 ℃ of insulation decolouring down, it is in the dialysis tubing of 3500Da that polysaccharide soln after the decolouring is placed molecular weight cut-off, prior to flowing water, the 24-30h that in distilled water, dialyses respectively again, the solution after the dialysis promptly obtains a hair disc bacterium YM-281 exocellular polysaccharide powder through vacuum lyophilization; The Deproteinated operation of wherein said Sai Wojiefa is as follows: add the mixed solution that chloroform and propyl carbinol volume ratio by its volume 1/3 are 5: 1 in Crude polysaccharides solution, with the violent jolting 20-30min of this mixture, static 20-30min in the separating funnel packs into, by the time protein denaturation agglutination is present on the interface of water and solvent phase, and dividing desolvates reaches the metaprotein of water and solvent phase intersection mutually; Repetitive operation to the interface of water and solvent phase does not have till the colloid degeneration protein, and the gained aqueous phase solution is the coarse grain hair disc bacterium YM-281 exocellular polysaccharide solution behind the deproteinated.
3. the purposes of described hair disc bacterium of claim 2 YM-281 exocellular polysaccharide in preparation lowering blood fat and benefiting liver medicine, it is characterized in that: the grain hair disc bacterium YM-281 exocellular polysaccharide of pressing 300-600g, behind pharmaceutical excipient thinner 200-250g and glidant 30-60g mixing, mix with 250-600mL ethanol again and make softwood, with this softwood whole grain behind the wet grain drying that obtains that sieves, add compressing tablet behind the lubricant 3-10g mixing at last, make 1000 in tablet; After perhaps pressing pharmaceutical excipient thinner 200-270g, glidant 40-70g and wetting agent 3-6g mixing, mix the back with the grain hair disc bacterium YM-281 exocellular polysaccharide of 300-600g and divide in 1000 the 1# Capsuleses of packing into, make 1000 of capsules; Described pharmaceutical excipient thinner is selected dextrin or dry starch for use; Described glidant adopts talcum powder or micropowder silica gel or Microcrystalline Cellulose; Described lubricant adopts Magnesium Stearate or Zinic stearas; Described wetting agent adopts sodium lauryl sulphate or dioctyl sulphosuccinate.
4. as the purposes of a hair disc bacterium YM-281 exocellular polysaccharide as described in the claim 3 in preparation lowering blood fat and benefiting liver medicine, it is characterized in that described tablet is processed as: add 300-500mL ethanol after grain hair disc bacterium YM-281 exocellular polysaccharide 500g, thinner dextrin 250g, the glidant talcum powder 40g that got 80～100 mesh sieves mixes and mix and make softwood, softwood sieves and obtains wet granular, the whole grain in dry back, add compressing tablet behind the magnesium stearate lubricant 10g mixing at last, make 1000, the heavy 0.8g of sheet, polysaccharide content 0.5g/ sheet.
5. as the purposes of a hair disc bacterium YM-281 exocellular polysaccharide as described in the claim 3 in preparation lowering blood fat and benefiting liver medicine, it is characterized in that described capsule is processed as: get thinner dry starch 250g, glidant talcum powder 45g, wetting agent sodium lauryl sulphate 5g mixes and mixes 1000 of back dress 1# Capsuleses, polysaccharide content 0.5g/ grain with 500g grain hair disc bacterium YM-281 exocellular polysaccharide.
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| CN102949409A (en) * | 2012-11-08 | 2013-03-06 | 合肥工业大学 | Application of using lachnum exopolysaccharide to prepare medicine for preventing gastric ulcer |
| CN103319619A (en) * | 2013-06-25 | 2013-09-25 | 合肥工业大学 | Application of lachnum extracellular polysaccharide phosphorylated derivative and application thereof in preparation of antitumor drugs |
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| CN106832026A (en) * | 2016-12-09 | 2017-06-13 | 合肥工业大学 | Carboxy methylation Lachnum exocellular polysaccharide and its purposes as hypoglycemic drug |
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| CN102746705A (en) * | 2012-06-28 | 2012-10-24 | 合肥工业大学 | Method for modifying lachnum extracellular melanin into water-soluble melanin |
| CN102949409A (en) * | 2012-11-08 | 2013-03-06 | 合肥工业大学 | Application of using lachnum exopolysaccharide to prepare medicine for preventing gastric ulcer |
| CN102949409B (en) * | 2012-11-08 | 2014-10-08 | 合肥工业大学 | Application of using lachnum exopolysaccharide to prepare medicine for preventing gastric ulcer |
| CN103319619A (en) * | 2013-06-25 | 2013-09-25 | 合肥工业大学 | Application of lachnum extracellular polysaccharide phosphorylated derivative and application thereof in preparation of antitumor drugs |
| CN104974270A (en) * | 2015-07-14 | 2015-10-14 | 合肥工业大学 | Sulfated Lachnum extracellular polysaccharide and application thereof in anticoagulant drugs |
| CN104974270B (en) * | 2015-07-14 | 2017-03-08 | 合肥工业大学 | A kind of Sulfation Lachnum exocellular polysaccharide and its purposes in terms of anticoagulation medicine |
| CN106832026A (en) * | 2016-12-09 | 2017-06-13 | 合肥工业大学 | Carboxy methylation Lachnum exocellular polysaccharide and its purposes as hypoglycemic drug |
| CN106924297A (en) * | 2017-03-30 | 2017-07-07 | 合肥工业大学 | A kind of Lachnum intracellular melanin as acute liver damage medicine purposes |
| CN107011452A (en) * | 2017-03-30 | 2017-08-04 | 合肥工业大学 | A kind of acetylation Lachnum exocellular polysaccharide and its prepare cardiovascular drugs purposes |
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