CN104398539B - A kind of preparation method of Hericium erinaceus ethanol extract - Google Patents
A kind of preparation method of Hericium erinaceus ethanol extract Download PDFInfo
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- CN104398539B CN104398539B CN201410503816.6A CN201410503816A CN104398539B CN 104398539 B CN104398539 B CN 104398539B CN 201410503816 A CN201410503816 A CN 201410503816A CN 104398539 B CN104398539 B CN 104398539B
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- hericium erinaceus
- ethanol extract
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- ethanol
- water
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
Abstract
The present invention relates to a kind of preparation methods of Hericium erinaceus ethanol extract, including：(1) it takes Hericium erinaceus to be ground into particle, is then dissolved in 1~3h of boiling stirring, standing in water and carries out ultrasonic extraction after being cooled to room temperature, then centrifuged, supernatant is taken after standing, obtain Hericium erinaceus aqueous extract；(2) Hericium erinaceus aqueous extract is added to pretreated macroreticular resin, is adsorbed at room temperature, is then eluted using ethyl alcohol, Hericium erinaceus ethanol extract is obtained after dry.The present invention has the characteristics that good separating effect, stability and recyclable recycling, is suitable for large-scale production；And macroporous absorbent resin adsorption separation method has consumption of organic solvent few recyclable, many advantages, such as can be used repeatedly, regenerate simply, reduce cost, can be used for the concentration and separation of active ingredient and big industrial production in Hericium erinaceus.
Description
Technical field
The invention belongs to edible mushrooms to extract field, more particularly to a kind of preparation method of Hericium erinaceus ethanol extract.
Background technology
China is raw material for treating stomach trouble drug about more than 100 kind using Hericium erinaceus at present, helicobacter pylori
(Helicobacter pylori) is a kind of aerobic-type Gram-negative bacteria, and a large amount of clinical researches show that H.pylori can draw
Play the stomach trouble such as chronic gastritis, peptic ulcer, gastric cancer, gastric mucosa.
Hericium erinaceus (Hericium erinaceus) is applied to gastrointestinal disease treatment with distinct distinct Chinese characteristics, in clinic
On be widely used for treating chronic gastritis, the diseases such as gastroduodenal ulcer, have accumulated a large amount of clinical experience and data.Last century
The success of Hericium erinaceus artificial cultivation is tamed in the sixties, China where Shanghai City academy of agricultural sciences edible mushroom first.The seventies is from civil understanding
Have to Hericium erinaceus and mend stomach function, starts from hedgehog hydnum syrup and clinical trial is made to duodenal ulcer patients, obtain good control
Therapeutic effect.
The method of currently used extraction Hericium erinaceus is mainly the water extract-alcohol precipitation of routine, but the Hericium erinaceus being obtained by carries
It takes object to inhibit helicobacter pylori active constituent few, practical application can not be put into.
Invention content
Technical problem to be solved by the invention is to provide a kind of preparation method of Hericium erinaceus ethanol extract, this method tools
There is the characteristics of good separating effect, stability and recyclable recycling, is suitable for large-scale production；And macroporous absorption
Resin adsorption partition method is recyclable less with consumption of organic solvent, can be used repeatedly, regenerate simply, and it is many excellent to reduce cost etc.
Point can be used for the concentration and separation of active ingredient and big industrial production in Hericium erinaceus.
A kind of preparation method of Hericium erinaceus ethanol extract of the present invention, including：
(1) it takes Hericium erinaceus to be ground into particle, is then dissolved in 1~3h of boiling stirring, standing in water and is cooled to room temperature
After carry out ultrasonic extraction, then centrifuged, supernatant taken after standing, obtain Hericium erinaceus aqueous extract；Wherein Hericium with
The weight ratio of water is 1:5~10；
(2) pretreated macroreticular resin HPD-100 is added in Hericium erinaceus aqueous extract (Hebei Cangzhou treasured grace chemical industry is limited
Company), it adsorbs, is then eluted using 70%~100% ethyl alcohol at room temperature, Hericium erinaceus ethanol extract is obtained after dry.
Centrifugal speed in the step (1) is 8000rpm, centrifugation time 10min.
Macroreticular resin HPD-100 pre-treatment steps in the step (2) are as follows：It is added in resin column and is higher than resin layer
10 centimetres of 90% ethyl alcohol impregnates 4 hours, releases immersion liquid, be washed with distilled water to efflux be diluted with water in test tube it is not muddy
And until eluent uv scan must not detect absorption peak, it is washed with water and washs to ethanol content less than 1%, you can.
It is adsorbed as Static Adsorption in the step (2), is as follows：A concentration of 100mg/ of Hericium erinaceus aqueous extract
The volume ratio of ml, solution and macroreticular resin is 10：1, shaking table 110rpm adsorb 12h.
Elution in the step (2) is static elution or dynamic desorption.
The static state, which elutes, is specially：The macroreticular resin HPD-100 that will have been adsorbed, the distillation through 3 times of bed volumes are washed
After de-, with 95% ethanol solution, 4 times of bed volumes set constant-temperature table 110rpm, 25 DEG C, eluted under conditions of 2h, color by
It is deep to shallow, is washed till colourless；Dynamic desorption is specially：Column is filled by the way of wet method or dry column-packing, chromatographic column specification is that diameter is high
Than 1：8, Hericium erinaceus aqueous extract sample concentration is 0.1kg/L, and resin applied sample amount is 1kg/1L, flow velocity 1.5mL/min, is used
95% ethanol solution is eluted.
Drying in the step (2) is freeze-drying, finally obtains the hedgehog fungus extract product that color is dark brown.
Advantageous effect
(1) present invention has the characteristics that good separating effect, stability and recyclable recycling, is suitable for scale
Metaplasia is produced；And macroporous absorbent resin adsorption separation method is recyclable less with consumption of organic solvent, can be used repeatedly, regenerate letter
Singly, many advantages, such as reducing cost, it can be used for the concentration and separation of active ingredient and big industrial production in Hericium erinaceus；
(2) for the present invention compared with before unused macroporous resin adsorption, which improves 20 times,
Better than untreated Hericium erinaceus alcohol extract.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, people in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Embodiment 1
Water extraction prepares hedgehogt fungus crude active material：
The Hericium erinaceus dry powder 0.2kg that pulverizer is ground into particle is weighed, 1~3h is stirred in boiling soluble in water, it
It stands afterwards and carries out ultrasound 2h with ultrasonic wave after being cooled to room temperature and extract, then carry out centrifugation 8000rpm, 10min takes after standing
Clear liquid obtains Hericium erinaceus water extract；Wherein the weight ratio of Hericium and water is 1:5~10.
Embodiment 2
The screening of macroreticular resin performance
(1) pretreatment of macroreticular resin
HPD-100, HPD-400, D101 are taken, AB-8 (is purchased from Hebei Cangzhou Bao En Chemical Co., Ltd.s), in resin column
90% ethyl alcohol being added higher than 10 centimetres of resin layer impregnates 4 hours, releases immersion liquid, is washed with distilled water to efflux in test tube
It is diluted with water until not muddy and eluent uv scan must not detect absorption peak, is washed with water and washs to ethanol content
Less than 1%, you can use.
(2) absorption of resin
Each 3 parts of pretreated HPD-100, HPD-400, D101, AB-8 macroreticular resin is measured, every part of 10mL is set respectively
In the triangular flask of 250mL, the sample liquid 100mL of embodiment 1, i.e. sample liquid is added：Resin=10：1.It sets in shaking table 110rpm, 25
DEG C, 12h makes fully to adsorb.
(3) elution of resin
The resin of adsorption saturation is rinsed with 3 times of column volume (BV) distilled water, to wash away impurity and not adsorb into
Point, 95% ethyl alcohol that 50mL is then added sets constant-temperature table 110rpm, 25 DEG C, is eluted under conditions of 2h, and eluent is collected,
Concentration recycles ethyl alcohol, obtains condensed cream, dilute certain multiple.
Embodiment 3
Inhibit the lowest bacteria fogging-resistant concentration determining of helicobacter pylori
(1) preparation of test sample
The ethanol extract sample that the accurate Hericium erinaceus water extract for weighing the preparation of embodiment 1 and embodiment 2 are prepared in
In sterilized eppendorf pipes, certain density solution is configured to 75% ethyl alcohol.The ultrasound 15min under ultrasound.Blank pair
According to for 75% ethyl alcohol, positive controls metronidazole is configured to 2mg/mL.
(2) preparation of helicobacter pylori bacteria suspension
Picking cultivates the bacterium colony of 72h in 1mL brucella broth culture mediums in right amount, and ultrasound about 10s is made and is equivalent to
2.0McFarland standard (contain bacterium 1 × 107To 1 × 108C.f.u./L) bacteria suspension, the bacteria suspension matching while using.
(3) preparation of sheep blood cloth culture medium
It weighs Brucella culture mediums 28.g and adds in 1000mL distilled water, it is 7.0 ± 0.2 to adjust pH value, is packed as
Every bottle of 100mL, separately plus agar 2g is in 121 sterilizing 15min, adds 7% sterile sheep blood before use.
(4) lowest bacteria fogging-resistant concentration determining (MIC)
It takes 75% ethyl alcohol of 40 μ L to be added in 96 orifice plate A1-A12 and is used as negative control, in B12-H12 plus metronidazole conduct
Positive control.It takes isometric test sample to be added in B1-B11,2 times of serial dilutions is carried out to each sample liquid by row with liquid-transfering gun,
Plate lid is covered, after being volatilized under ultraviolet, 100 μ L sheep blood Bu Shi cultures is inhaled based in 96 hole plates with liquid-transfering gun, is placed in 4 DEG C of refrigerators
Ultraviolet light irradiates about 15min after for 24 hours.Next helicobacter pylori bacteria suspension is dipped with sterile swab stick, is inoculated in each hole successively
In media surface, be put into incubator (O2：5%；CO2：15%；N2：80%), 37 DEG C of constant temperature incubation 72h.With stereoscopic aobvious
Micro- each hole media surface of microscopic observation, no bacterial growth are the minimum i.e. MIC of drug dilution concentration.
(5) experimental result
The MIC of Hericium erinaceus water extract prepared by embodiment 1 to helicobacter pylori>10mg/mL, the monkey that embodiment 2 obtains
Head bacterium ethanol extract to the MIC of helicobacter pylori be respectively 0.5-1mg/mL (HPD-100), 1-2mg/mL (HPD-400),
0.75-1.5mg/mL (D101) and 0.6-1.2mg/mL (AB-8), the results showed that using after macroreticular resin HPD-100 absorb-elutes
Obtained Hericium erinaceus ethanol extract to the minimum inhibitory concentration of H.pylori ATCC be less than using macroreticular resin HPD-400,
The minimum inhibitory concentration for the ethanol extract that AB-8 and D101 are obtained, and compared with water extraction before absorption, bacteriostatic activity difference
10 times or more are improved, adsorption activity effect is preferable.
Embodiment 4
The elution effect of different volumes score ethyl alcohol
(1) static elution
Take resin HPD-100, the AB-8 adsorbed, after the distillation water elution of 3 times of bed volumes, successively with 10%,
30%, each 4 times of bed volumes of 50%, 70%, 90% and 100% ethanol solution set constant-temperature table 110rpm, 25 DEG C, the condition of 2h
Lower carry out gradient elution, color is shallow by being deep to, be washed till respectively it is colourless, collect eluent, be concentrated into cream.Condensed cream dilutes certain
Multiple surveys the bacteriostatic activity to H.pylori.
(2) dynamic desorption
Hole resin fills pillar (blade diameter length ratio 1：8), after the distillation water elution of 3 times of bed volumes, successively with 10%, 30%,
50%, each 4 times of bed volumes of 70%, 90% and 100% ethanol solution carry out gradient elution, Hericium erinaceus aqueous extract sample concentration
For 0.1kg/L, resin applied sample amount is 1kg/1L, and flow velocity is washed till colourless, Fractional Collections eluent respectively for 1.5mL/min, and dense
Shorten cream into.Bacteriostatic activity of certain multiple measurement to H.pylori is diluted with condensed cream.
(3) experimental result
The result shows that two kinds of elution requirements are identical to the effect of bacteriostatic activity, it is all higher than using MIC when distillation water elution
10mg/mL, and use 10%, 30%, 50% ethanol elution when static state elution and dynamic desorption liquid MIC 5-10mg/mL it
Between；The poor MIC of eluent bacteriostatic activity obtained using HPD-100 and 10%-50% ethanol elutions is all higher than 2.5mg/mL, and
70%, the MIC of eluent obtained after 90% and 100% elution is respectively 0.25-0.5mg/mL, 125-250mg/mL,
0.25mg/mL, wherein the extract after 90% ethanol elution inhibits the minimum 125- of minimum inhibitory concentration of H.pylori ATCC
250mg/mL.As a result show that the active constituent for inhibiting helicobacter pylori is concentrated mainly on 70%, 90%, 100% ethanol elution
In liquid, in the ethyl alcohol stripping liquid of 70%, 90%, 100% 3 kind of volume fraction, 90% ethyl alcohol to H.pylori bacteriostatic activities most
It is good, and the eluent fungistatic effect difference obtained after 90% ethyl alcohol, 100% ethanol elution is little.
Embodiment 5
It is external to inhibit helicobacter pylori confirmatory experiment
The hedgehogt fungus crude extract medicinal extract that embodiment 1 is prepared is configured to the water extract solution of 100mg/ml, then
Carry out following method extraction.
(1) HPD-100 resins extract：It is added in the pretreated macroreticular resin HPD-100 to lysate of 5L and adsorbs for 24 hours,
Period need to stir frequently, remove supernatant, the macroreticular resin after being adsorbed, and the macroreticular resin after absorption is filled pillar (blade diameter length ratio 1：
8) gradient desorption, is carried out with distilled water, each 4 times of bed volumes of 50% ethanol solution successively, flow velocity is that 1.5mL/min is washed till respectively
It is colourless, then with 95% ethanol elution and it is condensed into cream.
(2) experimental result
The five kinds of pylorus spirals of Hericium erinaceus ethanol extract pair extracted using HPD-100 macroreticular resins and 95% ethyl alcohol
The minimum inhibitory concentration of bacillus is respectively 0.5-1mg/mL (Helicobacter pylori ATCC), 0.125-0.25mg/mL
(Helicobacter pylori SS1)、0.5-1mg/mL(Helicobacter pylori W2504)、1mg/mL
(Helicobacter pylori DXF) and 0.5mg/mL (Helicobacter pylori 78)；And five kinds of water extract pair
The minimum inhibitory concentration of helicobacter pylori is respectively 2.5-5mg/mL (Helicobacter pylori ATCC), 5mg/mL
(Helicobacter pylori SS1)、5mg/mL(Helicobacter pylori W2504)、2.5-5mg/mL
(Helicobacter pylori DXF) and 5mg/mL (Helicobacter pylori 78).Comprehensive analysis uses HPD-
100 and 95% the obtained activity of Hericium erinaceus ethanol extract of ethanol elution improve 20 times.
(3) it discusses
The present embodiment takes antibacterial component of the method that activity tracks to separation hedgehog hydnum solid fermentation mycelia to H.pylori
Be enriched with, in four kinds of macroreticular resins of selection, HPD-100 macroreticular resins to the activated adoption ability of hedgehog hydnum solid mycelia most
It is good, and it is 0.5-1mg/mL, activity to be determined that active material is concentrated mainly in 50-95% ethanol eluates minimum bacteriostatic activity
20 times are improved than water extract.This is adsorbed with effect active material to studying HPD-100 macroreticular resins later to hedgehog hydnum solid mycelia
Technical study in play basic research, and there is macroporous absorbent resin adsorption separation method consumption of organic solvent can return less
Many advantages, such as receiving, can be used repeatedly, regenerating simply, reduce cost, is a kind of efficient separation purifying technique, can use
The concentration and separation of active ingredient and big industrial production in Hericium erinaceus.
Claims (6)
1. a kind of preparation method of Hericium erinaceus ethanol extract, including：
(1) it takes Hericium erinaceus to be ground into particle, is then dissolved in 1~3h of boiling stirring, standing in water and is cooled to room temperature laggard
Row ultrasonic extraction, is then centrifuged, and supernatant is taken after standing, obtains Hericium erinaceus aqueous extract；Wherein Hericium and water
Weight ratio is 1:5~10；
(2) Hericium erinaceus aqueous extract is added to pretreated macroreticular resin HPD-100, is adsorbed at room temperature, then uses 70%
~100% ethyl alcohol is eluted, and Hericium erinaceus ethanol extract is obtained after dry；Wherein, macroreticular resin HPD-100 pre-treatment steps
It is as follows：90% ethyl alcohol being added in resin column higher than 10 centimetres of resin layer impregnates 4 hours, releases immersion liquid, is washed with distilled water
It is diluted with water until not muddy and eluent uv scan must not detect absorption peak to efflux, then uses in test tube
Water washing to ethanol content is less than 1%, you can.
2. a kind of preparation method of Hericium erinaceus ethanol extract according to claim 1, it is characterised in that：The step
(1) centrifugal speed in is 8000rpm, centrifugation time 10min.
3. a kind of preparation method of Hericium erinaceus ethanol extract according to claim 1, it is characterised in that：The step
(2) it is adsorbed as Static Adsorption in, is as follows：A concentration of 100mg/ml of Hericium erinaceus aqueous extract, solution and macropore tree
The volume ratio of fat is 10：1, shaking table 110rpm adsorb 12h.
4. a kind of preparation method of Hericium erinaceus ethanol extract according to claim 1, it is characterised in that：The step
(2) elution in is static elution or dynamic desorption.
5. a kind of preparation method of Hericium erinaceus ethanol extract according to claim 4, it is characterised in that：The static state is washed
It is de- to be specially：The macroreticular resin HPD-100 that will have been adsorbed, after the distillation water elution of 3 times of bed volumes, with 95% ethanol solution
4 times of bed volumes set constant-temperature table 110rpm, 25 DEG C, eluted under conditions of 2h, color is shallow by being deep to, and is washed till colourless；It is dynamic
State elutes：Column is filled by the way of wet method or dry column-packing, chromatographic column specification is blade diameter length ratio 1：8, the extraction of Hericium erinaceus water
Liquid sample concentration is 0.1kg/L, and resin applied sample amount is 1kg/1L, and flow velocity 1.5mL/min is washed with 95% ethanol solution
It is de-.
6. a kind of preparation method of Hericium erinaceus ethanol extract according to claim 1, it is characterised in that：The step
(2) drying in is freeze-drying.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102731365A (en) * | 2011-04-06 | 2012-10-17 | 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 the hericium erinaceum biological micro-molecules in treatment of digestive tract diseases |
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| KR20040066344A (en) * | 2003-01-17 | 2004-07-27 | 학교법인 영광학원 | Hericium erinaceus exo-biopolymer having a hypolipidemic effect, method for isolation and the use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102731365A (en) * | 2011-04-06 | 2012-10-17 | 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 the hericium erinaceum biological micro-molecules in treatment of digestive tract diseases |
Non-Patent Citations (1)
| Title |
|---|
| 猴头菌提取物抗氧化活性研究;潘伟等;《食用菌学报》;20121231;第19卷(第2期);第95-99页，尤其是第95页右栏1.3.1水提物 * |
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