CN101375867B - Oligosaccharide of mycelium of Hericium erinaceus extracted from fermentation product of mycelium of Hericium erinaceus and uses thereof - Google Patents
Oligosaccharide of mycelium of Hericium erinaceus extracted from fermentation product of mycelium of Hericium erinaceus and uses thereof Download PDFInfo
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- CN101375867B CN101375867B CN2008100512381A CN200810051238A CN101375867B CN 101375867 B CN101375867 B CN 101375867B CN 2008100512381 A CN2008100512381 A CN 2008100512381A CN 200810051238 A CN200810051238 A CN 200810051238A CN 101375867 B CN101375867 B CN 101375867B
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Abstract
The invention relates to hericium erinaceus mycelium oligosaccharides extracted from a fermented product of a hericium erinaceus mycelium and an application thereof. A fermented liquid extract of hericium erinaceus mycelium is diluted by adding water, stirred and centrifugalized, ethanol is added in centrifugal liquid till alcoholization, the centrifugation and the collection of settlement are carried out, distilled water is added for preparing water solution, a dialysis membrane is dialyzed, and the dialysis extracelluler fluid is collected and concentrated to the liquid extract, thereby obtaining the hericium erinaceus mycelium oligosaccharides. The hericium erinaceus mycelium oligosaccharides have the advantages that: the effective parts are definite, the structure is clear, the dose is less, and the hericium erinaceus mycelium oligosaccharides have significant efficacy for treating acute and chronic gastritis, gastric ulcer and chronic atrophic gastritis.
Description
Technical field:
The present invention relates to relate in particular to the application in pharmaceutical field from the Hericium erinaceus (Bull. Ex Fr.) Pers. filament oligosaccharide and the application thereof of the extraction of Hericium erinaceus (Bull. Ex Fr.) Pers. filament tunning; The IPC classification number is A61K36/06.
Background technology:
The alleged Hericium erinaceus (Bull. Ex Fr.) Pers. of the present invention is a hedgehog fungus section fungus Small acupuncture Hericium erinaceus (Bull. Ex Fr.) Pers. (Hericiumcaput-medusae), and its mycelial tunning is applied to clinical for many years as crude drug.About the fermentation of Small acupuncture Hericium erinaceus (Bull. Ex Fr.) Pers., can adopt this area deep layer commonly used to cultivate the air agitation condition; For example rolled up the 198th to 200 page of the 5th phase in 1985 the 12nd referring to " microorganism circular ".
Cultivating extractum with Hericium erinaceus (Bull. Ex Fr.) Pers. filament on the China market is that the medicine that raw material is made has WEILEXIN CHONGJI.Although said preparation demonstrates good curative effect to stomach, duodenal ulcer, chronic gastritis etc., but the problems such as basic chemical composition to extraction, separation, purification process and the active component of effective ingredient in the mycelium are not resolved yet, are restricting the development and utilization of Hericium erinaceus (Bull. Ex Fr.) Pers. filament for a long time always.
Obtained the Hericium erinaceus (Bull. Ex Fr.) Pers. filament oligosaccharide by extraction, separation, purification to effective ingredient in the Hericium erinaceus (Bull. Ex Fr.) Pers. filament, its basic chemical structure is:
The HPGPC analysis result shows that the molecular weight distribution of this chemical compound is in 0.36-1.5 * 10
3, illustrate that this chemical compound is mainly the oligosaccharide constituents; Physicochemical property result shows that the content of sugar accounts for about 90%; Form the glycan analysis result and show, this chemical compound is mainly by Man, and two kinds of monosaccharide of Glc are formed, and mol ratio is 1:5; Liquid-matter result shows that this chemical compound mainly is made of the oligosaccharide that 2-7 monosaccharide connect, and wherein disaccharidase and trisaccharide respectively account for about 20%, and other is tetrose, pentasaccharides, six sugar and seven sugar, also has a spot of monosaccharide.Disaccharidase, tetrose, pentasaccharides all have two kinds of configurations, and six sugar have three kinds of configurations, and trisaccharide and seven sugar have a kind of configuration.Adopt the HPLC method, two pools, trisaccharide that the nh 2 column purifies and separates obtains, utilize NMR, LC-MS, GG-MS, GC and methylation analysis infer that disaccharidase is that (or a-Man1 → 6Glc) two kinds of forms exist, and trisaccharide is to exist with α-Glcl → 4 α-Glcl → 6a-Glc form with α-Glcl → 4a-Glc and a-Glcl → 6Man.
Summary of the invention:
The technical problem to be solved in the present invention provides Hericium erinaceus (Bull. Ex Fr.) Pers. filament oligosaccharide and uses thereof, especially the application in pharmacy.
In fact the invention provides the Hericium erinaceus (Bull. Ex Fr.) Pers. filament oligosaccharide as the application in the medicine of preparation treatment disease of stomach.
In order to understand the present invention better, provide the extracting method of Hericium erinaceus (Bull. Ex Fr.) Pers. filament oligosaccharide below, and pharmacological testing illustrates its purposes in pharmaceutical field.
Method 1
With the Hericium erinaceus (Bull. Ex Fr.) Pers. filament extractum 10kg that ferments, add water 1-2 and doubly dilute, stir, centrifugal, centrifugal liquid adds ethanol to determining alcohol and is 80%, and is centrifugal, collecting precipitation, adding distil water is made into aqueous solution, to the dialysis of dialyzer molecular weight ranges 8,000-10,000 water, collect extracellular fluid dialysis, be concentrated into extractum 1kg, lyophilization gets Hericium erinaceus (Bull. Ex Fr.) Pers. filament oligosaccharide 800g.
Method 2
Get hericium mycelium fermentation extractum 10kg, add water 1-2 and doubly dilute, stir, centrifugal, centrifugal liquid adds ethanol to determining alcohol and is 80%, and is centrifugal, collecting precipitation 1kg, adding distil water is made into aqueous solution, by the DEAE-SephadexA50 ion exchange resin column, uses the distilled water eluting respectively, collect eluent, be concentrated into extractum, lyophilization gets Hericium erinaceus (Bull. Ex Fr.) Pers. filament oligosaccharide 780g.
Method 3
Get hericium mycelium fermentation extractum 10kg, add water 1-2 and doubly dilute, stir, centrifugal, centrifugal liquid adds ethanol to determining alcohol and is 80%, and is centrifugal, collecting precipitation 1kg, and adding distil water is made into aqueous solution.At 8 thousand-1 ten thousand Hollow Fiber Ultrafiltration, obtain Hericium erinaceus (Bull. Ex Fr.) Pers. filament oligosaccharide 800g by molecular weight ranges.
Through the pharmacological experiment screening, above-claimed cpd has the acute and chronic gastritis of treatment, gastric ulcer, chronic atrophic gastritis effect.
Press practice of pharmacy, the Hericium erinaceus (Bull. Ex Fr.) Pers. filament oligosaccharide can prepare various clinical pharmaceutical preparation as the acute and chronic gastritis of treatment, gastric ulcer, the medicine of chronic atrophic gastritis, said medicament is a said dosage form on any pharmaceutics, particularly as various peroral dosage forms.
It is active component that medicine of the present invention contains the above-claimed cpd for the treatment of effective dose, and contains one or more pharmaceutically acceptable carriers.
Carrier above is meant that the medicine of pharmaceutical field routine carries not, comprises diluent, figuration punishment, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier.
Medicine of the present invention can be applied to the patient of this treatment by oral, rectum, vein, intramuscular injection or parenteral mode.Conventional production method according to pharmaceutical field prepares various dosage forms such as tablet, granule, electuary, capsule, suppository, spray, slow releasing agent and injection.Also can make its active component or compositions and variety carrier or medicament mixed, make required dosage form.
Following pharmacological evaluation has confirmed that the pharmaceutical preparation of Hericium erinaceus (Bull. Ex Fr.) Pers. filament oligosaccharide has the acute and chronic gastritis of treatment, gastric ulcer, the pharmacologically active of chronic atrophic gastritis.One, Hericium erinaceus extract is to the influence of damage type gastric ulcer due to the acetic acid
Summary: this laboratory observation Hericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide is to the influence of damage type gastric ulcer rat due to the acetic acid.The continuous irrigation stomach gives damage type gastric ulcer rat due to the acetic acid respectively
480mg/kgHericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide 14 days all has significant inhibitory effect to damage type gastric ulcer due to the acetic acid.
Medicine: positive drug is a WEILEXIN, is produced lot number: 041205 by Bethune medical university pharmaceutical factory.
Animal: 220-260g male rat.Available from 208 animal housing of hospital of the Chinese People's Liberation Army.
Method: get the rat fasting and can't help water 48 hours, etherization, it is fixing to face upward the position, abdominal part unhairing, iodine tincture, alcohol disinfecting, xiphoid-process hits exactly the otch of opening about 2.5cm down, duodenum is ticked gently below right liver with curved hemostat, stomach is pulled out outside the abdomen gently, at the facies ventralis body of stomach and the pyloric antrum intersection of stomach, thrust 0.4-0.5mm under the rat stomach serous coat with the 0.25ml syringe, inject 10% acetum 0.05ml, stomach is sent back to gently, sew up abdominal muscle and skin, the sterilization back is conventional raises, Rhizoma Atractylodis Macrocephalae rises next day, grouping, successive administration 14 days, last administration 2h, broken end, ligation pylorus and cardia, stomach are dipped in 1% formalin, fix 10 minutes, cut off along greater gastric curvature, the washing of turning up is observed the ulcer situation at the mucosal surface of injection acetum corresponding site, as the ulcer diameter, calculate ulcer index with the average of major diameter of ulcer and minor axis.See Table 1.
Table 1. Hericium erinaceus extract is to the influence of damage type gastric ulcer due to the acetic acid
Annotate: n=8, X ± SD.※P<0.05，※※※P<0.001。
Brief summary: 480mg/kg Hericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide 14 days all has significant inhibitory effect to damage type gastric ulcer due to the acetic acid.480mg/kg is especially remarkable to the inhibitory action of damage type gastric ulcer due to the acetic acid.
Two, Hericium erinaceus extract is to the influence of stress gastric ulcer
Summary: adopt the stress gastric ulcer model to compare the antiulcer action of Hericium erinaceus extract and WEILEXIN.
The continuous irrigation stomach gives rat 480mg/kg Hericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide 15 days respectively, and the Hericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide has tangible antiulcer action as a result.
Animal: 32 of Wister rats, available from Norman Bethune Medical University animal housing.
Medicine: positive drug is a WEILEXIN, is produced lot number: 041205 by Bethune medical university pharmaceutical factory.
Method: get 50 of rats, be divided into 5 groups at random, 10 every group, irritate every day stomach give 480mg/kg Hericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide and WEILEXIN each once, matched group gives ordinary water, give 15 days continuously, with the hungry 32h of rat, at 16 o'clock the next afternoon was used etherization after the administration in the 14th day, the rat extremity are tied up on the ferrum cage with fly, the rat that to tie up is vertically placed 18h upside down simultaneously, draws neck to put to death animal.With the cardia and the pylorus ligation of its stomach, get full stomach, and after inwardly injecting 1% formalin fixed 5min, cut off along greater gastric curvature, lie on glass and pull, observe and judge grade.
The result: matched group is 3.87 ± 0.35, and 480mg/kg Hericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide is 2.38 ± 1.51 (P＜0.05), and WEILEXIN is 2.13 ± 1.13 (P＜0.001).
This shows that the Hericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide has very significantly antiulcer action.
Three, Hericium erinaceus extract is to rat chronic atrophic gastritis experimentation
Summary: the employing active immunity adds the integrated approach that gavages bile and hot water and observes the therapeutical effect of Hericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide to rat chronic atrophic gastritis CAG, the result: X mg/kgHl, X mg/kgL2 can obviously improve the attenuation of CAG gastric mucosa of rat, and the intrinsic body of gland of gastric mucosa is increased, the effect of good curing CAG is arranged.
1. experiment material and method:
1.1 animal: the Wistar male rat, qualified 10-5112 available from Changchun High-technology Medical Animal Experiment Research Center, body weight 220-260g.
1.2 medicine: positive drug is a WEILEXIN, is produced lot number: 041205 by Bethune medical university pharmaceutical factory.
1.3 Preparation of model:
1.3.1 the antigenic preparation of adjuvant: get 160-180gWistar rat, get gastric mucosa with putting to death behind its hungry 24h, every Mus gastric mucosa adds normal saline 3ml, utilizes tissue refiner that its dozen is even, add the equal-volume white oil then and be prepared into uniform Emulsion, standby.
1.3.2 Preparation of model: get 90 of 220-260g rats, at 2 backs of every rat subcutaneous injection adjuvant antigen 2ml, every some 1ml, 2 week back repetitions 1 time repeat again after 1 week 1 time again, and immunity is 3 times altogether; Gavage only (bile: glycerol=1:1) of Fel Sus domestica 2ml/ for the people Mus from immunity every 3 days; Simultaneously ought be beyond the highest heavens except that gavaging Fel Sus domestica, gavage 35 days 55 ℃ of hot water every day continuously.
1.3.3 animal is handled: first immunisation was put to death 10 rats in 35 days and is got stomach and make tectology and detect, visible gastric mucosa body of gland attenuation, and the gastric gland body reduces, and as seen indivedual stomaches have a large amount of cellular infiltrations under ulcer, hemorrhage, the mucosa.
1.3.4 grouping and administration: remaining rat is divided into six groups at random, and promptly model control group only gives ordinary water 4ml/ every day, the 480mg/kg WEILEXIN, and 480mg/kg Hericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide, administration every day 1 time, successive administration 30 days, and in weighing weekly 1 time.
1.3.5 to the influence that changes in the pathology of gastric mucosa tissue: animal curved is drawn materials embedded section to cardia from gastric antrum along little.Adopt x
2Check.
2. result:
2.1 influence to the gastric mucosa tectology:
As seen from Table 2, the negative rate of phenomenons such as the attenuation of model group gastric mucosa, body of gland minimizing is still apparently higher than the administration group, and thickens and the Hericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide all has the obvious suppression effect to above-mentioned phenomenon and do not see that the flesh layer thickens phenomenon from the flesh layer with a large amount of cell infiltration.
2.2 the influence to the variation of chronic atrophic gastritis rat body weight: see Table 3.Table 3 shows that the variation of chronic atrophic gastritis rat body weight is not had influence.
Brief summary: The above results shows that the Hericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide has certain therapeutical effect to the rat chronic atrophic gastritis.
The table 2. pair morphologic influence of chronic atrophic gastritis mucosa tissue
Annotate: with table 1.
The influence that table 3. pair chronic atrophic gastritis rat body weight changes
Can draw from above result and the invention has the advantages that: effective site is clear and definite, and structure is clear, and dose is few, for the acute and chronic gastritis of treatment, and gastric ulcer, chronic atrophic gastritis has significant curative effect.And be better than positive control drug.
The specific embodiment:
Embodiment 1
With the Hericium erinaceus (Bull. Ex Fr.) Pers. filament extractum 10kg that ferments, add water 1-2 and doubly dilute, stir, centrifugal, centrifugal liquid adds ethanol to determining alcohol and is 80%, and is centrifugal, collecting precipitation, adding distil water is made into aqueous solution, and dialyzer molecular weight ranges 8,000-10,000 water is dialysed, collect extracellular fluid dialysis, be concentrated into extractum 1kg, lyophilization gets powdery Hericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide 800g, packing into seals in the medicine bottle, and product is made in sterilization.
Embodiment 2
Get hericium mycelium fermentation extractum 10kg, add water 1-2 and doubly dilute, stir, centrifugal, centrifugal liquid adds ethanol to determining alcohol and is 80%, and is centrifugal, collecting precipitation 1kg, adding distil water is made into aqueous solution, by the DEAE-SephadexA50 ion exchange resin column, use the distilled water eluting respectively, collect eluent, be concentrated into extractum, lyophilization, get Hericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide 780g, seal in the medicine bottle of packing into, product is made in sterilization.
Embodiment 3
Get hericium mycelium fermentation extractum 10kg, add water 1-2 and doubly dilute, stir, centrifugal, centrifugal liquid adds ethanol to determining alcohol and is 80%, and is centrifugal, collecting precipitation 1kg, and adding distil water is made into aqueous solution.By molecular weight ranges at 8 thousand-1 ten thousand Hollow Fiber Ultrafiltration, Hericium erinaceus (Bull. Ex Fr.) Pers. oligosaccharide 800g, seal in the medicine bottle of packing into, product is made in sterilization.
Claims (1)
1. the extracting method of a Hericium erinaceus (Bull. Ex Fr.) Pers. filament oligosaccharide is characterized in that: get hericium mycelium fermentation extractum 10kg, add water 1-2 and doubly dilute, stir, centrifugal, it is 80% that centrifugal liquid adds ethanol to determining alcohol, centrifugal, collecting precipitation 1kg, adding distil water is made into aqueous solution, by the DEAE-SephadexA50 ion exchange resin column, use the distilled water eluting respectively, collect eluent, be concentrated into extractum, lyophilization gets the Hericium erinaceus (Bull. Ex Fr.) Pers. filament oligosaccharide.
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| CN102731365B (en) * | 2011-04-06 | 2014-04-09 | 中国科学院上海生命科学研究院 | Hericium erinaceum biological micro-molecules for inhibiting helicobacter pylori and use of hericium erinaceum biological micro-molecules in treatment of digestive tract diseases |
| CN107916215B (en) * | 2017-10-17 | 2021-05-25 | 大兴安岭北极鄂伦春生物工程有限责任公司 | Low-temperature fermentation extraction device and extraction method for hericium erinaceus mycelium microorganisms |
| CN109438528A (en) * | 2018-10-17 | 2019-03-08 | 浙江工业大学 | A method of Hericium erinaceus oligosaccharide is prepared based on membrane separation technique |
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Non-Patent Citations (2)
| Title |
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| 中华人民共和国卫生部药典委员会.《胃乐新颗粒》.《药品标准 中药成方制剂 第十一册》.1996,125. * |
| 姜瑞芝等.《猴头菌寡糖的分离及其结构确定》.《高等学校化学学报》.2007,第28卷(第7期),1313-1315. * |
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Address after: 130011 No. 358 Innovation Road, Changchun High-tech Zone, Jilin Province Patentee after: Changchun Bethune Pharmaceutical Co., Ltd. Address before: 130012 No. 358 Innovation Road, Changchun High-tech Zone, Jilin Province Patentee before: Pharmaceutical Factory, Bethune Medical Univ. |