CN1629179A - Ganoderma lucidum sterol extract and its preparation process and application - Google Patents
Ganoderma lucidum sterol extract and its preparation process and application Download PDFInfo
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Abstract
The invention provides a medicinal preparation containing effective organic compositions, in particular a lucid ganoderma Sterols extract which is extracted from lucid ganoderma, its principal ingredient is white tabular crystal, its name is 24-methyl-cholest-5, 22-diolefin-3beta alcohol, its molecular formula is C28H46O, then invention also discloses the process for preparation, and its use in preparing cerebral ischemia caused by various etiology.
Description
Technical field
The present invention relates to a kind of pharmaceutical product that contains organic effective ingredient, particularly a kind of glossy ganoderma sterol (Sterols) class extract and preparation method thereof and application.
Background technology
Glossy ganoderma has stronger pharmaceutical use, and traditional instructions of taking is decoction or cooks that the active pharmaceutical ingredients that is extracted is less, generally has only the polysaccharide composition to be extracted, and promptly the utilization ratio of pharmaceutically active substance is low.
The practice that separation and Extraction medicine effective ingredient from glossy ganoderma is also arranged in the prior art, for example Chinese patent 93101870.6 (May 17 nineteen ninety-five Granted publication day) discloses a kind of compound glossy ganoderma nutrient solution and preparation method thereof, but this patented technology just combines the crude extract of glossy ganoderma and is a kind of medicine as effective extract of a kind of component and Tuber Fleeceflower Root and gynostemma pentaphylla, be used to improve immunizing power and delay senility, this patented technology this of glossy ganoderma is extracted composition and pharmacological effect is done further further investigation.
Kind surplus the sterol that separation and Extraction obtains from glossy ganoderma has nearly 20, wherein be divided into two types of ergosterol and cholesterol, retrieve the pharmacologically active report of the sterols extract of finding not see separation and Extraction from glossy ganoderma and obtaining and be applied to clinical because of skeleton is different.
Summary of the invention
The active substance glossy ganoderma sterols extract that the object of the present invention is to provide a kind of from glossy ganoderma, accurately the extraction and obtain;
This preparation method of extract is provided;
And this extract has application in the medicine of the cerebral ischemia effect that the anti-various causes of disease cause in preparation.
Purpose of the present invention can realize through following scheme.
Glossy ganoderma sterols extract, its main points are that it is extracted by glossy ganoderma and obtains, and its main component is the white plates crystallization, and its name is called 24-methyl-courage steroid-5,22-diene-3 β alcohol, molecular formula C
28H
46O, its chemical structural formula is as follows:
This extraction from glossy ganoderma and the material that obtains is a kind of phytosterin compound, this compound exists 28 carbon, wherein 6 CH
38 CH
2, 11 CH, and 3 quaternary carbons.Utilize one dimension (
1H,
13C, DEPT) and the two dimensional NMR technology [
1H-
1H,
1H-
13The relevant spectrum of C,
1H-
13C heteronuclear volume relevant spectrum of son (HMBC) and NOESY spectrum] compound has been carried out the structure evaluation, determine that its structure is 24-methyl-courage steroid-5,22-diene-3 β alcohol.
Adopting this glossy ganoderma sterols extract that the cortex neurone anoxic of rat is reoxidized damage (being called for short H/R) treats, be specially and adopt external former being commissioned to train to support SD rat cerebral cortex cell, reoxidize (24h) damage by anoxic (12h) and set up the cerebral ischemia re-pouring external model, observe and find glossy ganoderma sterols extract (consumption group 0.01; 0.1; 1ug/ml) damaged cell all there is obvious provide protection.
Identify neuronic life state with PI and the two calibration methods of Hoechst33258, under laser confocal microscope, observe the mark situation.The two cells that dye of PI and Hoechst33258 are dead cell, and Hoechst33258 singly dyes and is viable cell.The two dead cell showed increased of dying of PI and Hoechst33258 in the visible H/R model group of the result cell, and use the ratio (seeing Fig. 1 for details) that can obviously increase viable cell behind the glossy ganoderma sterols extract.
Use mtt assay and measure the survival rate and the cellular metabolism situation of cell.H/R obviously descends neuronal survival as a result, and glossy ganoderma sterols extract has significant provide protection to damaged cell.
Use free radical in the fluorescent probe DCF-DA specific marker cell, under laser confocal microscope, measure free-radical contents in the cell.As seen H/R can significantly induce the generation of free radical, and the visible fluorescence intensity of probe obviously strengthens under the mirror, and glossy ganoderma sterols extract for treating group fluorescence intensity then obviously reduces, and just glossy ganoderma sterols extract can significantly suppress the free radical level that increases in the cell.
Prepare cell lysate with lysate, identify protein content with the bradford method.Use Nanjing and build up test kit that bio-engineering research is produced, colorimetric method for determining lipid peroxidation product glutaraldehyde (MDA) level.The result as seen, glossy ganoderma sterols extract also can reduce the unusual MDA that raises in the injured neurons, illustrates that antioxygenation may be a glossy ganoderma sterols extract to one of mechanism of neurocyte protection effect.
Simultaneously, use Nanjing and build up test kit that bio-engineering research is produced, we have also detected total superoxide-dismutase (SOD) and Mn-SOD activity in the neurone.We find that glossy ganoderma sterols extract can obviously increase SOD and Mn-SOD activity in the neurone, and to the active not obviously effect of Cu-ZnSOD, this proof Mn-SOD may be the key enzyme that glossy ganoderma sterols extract is brought into play its antioxygenation, strengthens the resistance of oxidation (seeing Fig. 2 for details) of neurocyte by the activity that increases Mn-SOD.
With anti-p65 labeling of monoclonal antibodies intracellular nucleic transcription factor NF-KB, under laser confocal microscope, NF-κ B is positioned observation with the method for immunofluorescence.As seen NF-κ B basic fixed position under normal circumstances is in cytoplasm, and at 12 hours reoxygenations of anoxic in the time of 45 minutes, then transposition in nucleus in a large number of NF-κ B, use glossy ganoderma sterols extract then obviously antagonism activation and the nuclear translocation of NF-κ B.
Prepare cell pyrolysis liquid, detect the expression level of NF-κ B specificity inhibition I-κ B with the method for Western blot.Specificity one anti-application concentration is 1: 100, and the two anti-concentration of using were with 1: 1000.Experiment finds that H (12h)/R (45min) can increase the degraded of I-κ B, and glossy ganoderma sterols extract can suppress this variation.Therefore we think that glossy ganoderma sterols extract is by degraded that suppresses I-κ B and then activation and the nuclear translocation that has suppressed NF-κ B.
The preparation cell pyrolysis liquid, and application ELISA test kit, detect inflammatory cytokine TNF α and IL-1 β at intracellular expression level according to the test kit illustration method, find can significantly the raise level of TNF α and IL-1 β of H/R, and glossy ganoderma sterols extract can suppress this variation.
Because NF-κ B is the crucial regulatory factor of various kinds of cell factor expression, so we infer that glossy ganoderma sterols extract may influence the expression of TNF α and IL-1 β by the activation that suppresses NF-κ B, thereby have suppressed inflammatory cytokine to neuronic immunologic injury.Experimental results show that because of having SOD can suppress the activation of NF-κ B again,, and then damage from two mechanism antagonism of anti-oxidant and anti-immunity H/R so we infer that Mn-SOD may be the keying action site of glossy ganoderma sterols extract to H/R damaging cells performance provide protection.
Claim that medically cerebral anoxia causes because of cerebral blood supply insufficiency, thereby Ben Lingzhi sterols extract has provide protection to pallium neurone anoxic, so equally cerebral ischemia is had provide protection.
A series of research all shows, the new sterols extract that the contriver extracts from glossy ganoderma has outstanding beneficial effect to the cerebral ischemia (particularly to pallium neurone anoxic) that treatment is caused by the various causes of disease, and make its mechanism of action of curing the disease comparatively clear and definite because of its molecular structural formula accurately that has, pharmacological action is outstanding, the utilization ratio height of pharmaceutically active substance.
Glossy ganoderma sterols extract, its main points are, also comprise submember white crystals courage steroid-5,7-diene-3 β alcohol, and its chemical structural formula is as follows:
This compound be equally by one dimension (
1H,
13C, DEPT) and the two dimensional NMR technology [
1H-
1H,
1H-
13The relevant spectrum of C,
1H-
13Relevant spectrum of C heteronuclear volume son (HMBC) and NOESY spectrum] extract is identified and obtained.
Another object of the present invention: provide a kind of
Glossy ganoderma sterols preparation method of extract, its main points are, comprise following step of carrying out in regular turn:
(1) provide a kind of glossy ganoderma as the raw material parent;
(2) broken this raw material parent and broken thing;
(3) with ethanol to broken thing carry out refluxing extraction 4～10h, centrifugal and extracting solution;
(4) extracting solution is concentrated into contains crude drug amount 0.5～1.5g/ml in the concentrated solution, leave standstill more than 10 hours, 4～5 ℃ of temperature are collected crude extract;
(5) crude extract is dissolved with hot ethanol, extract lysate and leave standstill more than 10 hours 4～5 ℃ of temperature, collection coarse crystallization;
(6) coarse crystallization was handled 16～48 hours with the ethanol dilute alkaline soln, is collected insolubles,
(7) with insolubles with the concentrated alkali solution 24h that alkalizes, filter, collect filtrate;
(8) filtrate is extracted and collects ether layer with the equivalent ether;
(9) ether layer is washed with water to neutrality, carry out drying, treat that the complete back of ether volatilization gained solids is glossy ganoderma sterols extract.
Can make description below to processing step:
As the glossy ganoderma of raw material parent can be cultivating in bag and the Ganoderma sporophore that obtains, also can be the sporophore of segment wood cultivated glossy ganoderma, even can also be to obtain Ganoderma mycelium through microbial fermentation.
Adopt shredder assembly commonly used that glossy ganoderma raw material parent is carried out fragmentation, the glossy ganoderma state after the fragmentation can be sheet or pipe tobacco shape, can obtain the pulverized parent of cell walls as long as satisfy.
Because the glossy ganoderma phytosterin compound that desire is extracted is dissolved in ethanol, thus adopt ethanol that broken thing is carried out refluxing extraction, and obtain pure clear extracting solution by centrifugal removing slag.The time of refluxing extraction is long more, and extraction yield is high more, but length consuming time, the cost height, experience shows that adopting the Best Times of alcohol reflux sterol compound is 5～6 hours.And the weight ratio of ethanol and broken thing can be 10: 1.
Extracting solution concentrated obtain the higher concentrated solution of medicament active composition concentration, also reclaim ethanol simultaneously,, thereby adopt the heating evaporation mode to reclaim ethanol usually because ethanol is volatile.The concentration that contains the crude drug amount in the concentrated solution can produce certain influence to ensuing purifying process, but can not produce too much influence to the effect of whole extraction process, thus only need reasonable control just can, be 1g/ml the best but be controlled at concentration as far as possible.Because phytosterin compound can crystallization be separated out under the low temperature, thereby adopt and leave standstill (or claiming standing over night) more than 10 hours, 4～5 ℃ of temperature can be collected the crude extract of comparatively coarse sterols extract, and the mode of collection can be centrifuging.
Crude extract is dissolved with hot ethanol, carry out the purifying of sterols extract, the alcoholic acid temperature may be controlled to and is higher than normal temperature but is not higher than the alcoholic acid volatilization temperature, concrete temperature is set those skilled in the art of the present technique and can be provided with accurately according to local real air temperature and the knowledge of being grasped, abandon alcohol-insoluble substance, and collect the lysate of hot ethanol solute and leave standstill more than 10 hours 4～5 ℃ of temperature, the coarse crystallization that collection is further purified.
Coarse crystallization was handled 16～48 hours with the ethanol dilute alkaline soln, and collection is insoluble to the neutral substance sterols extract of this ethanol dilute alkaline soln and removes unwanted acidic substance.
Insolubles with the concentrated alkali solution 24h that alkalizes, is filtered, and this moment, required sterols extract was dissolved in this concentrated alkali solution, collected filtrate.The alkaline solution at this place can be 12%KOH.
Adopt extraction equipment commonly used that filtrate is extracted, extraction agent is and the ether of filtrate equivalent that the ether layer that is dissolved with phytosterin compound is collected in liquid layering to be mixed.
Ether layer is carried out drying, remove ether, the gained solids is glossy ganoderma sterols extract.
The glossy ganoderma sterols extract that is obtained includes multiple components, wherein includes major ingredient 24-methyl-courage steroid-5, the pure and mild less important composition courage steroid-5 of 22-diene-3 β, 7-diene-3 β alcohol.The medicinal effect institute of this steroidal extract is preceding as stating.
Concentration of ethanol in order to the dissolving sterol compounds in the above-mentioned technology can adopt typical concentrations of the prior art, for example can adopt 95% ethanol.Certainly, ethanol or use chloroform in actually operating, or use methyl alcohol, or replace with organic solvents such as benzene.
The present invention further also is
Extracting solution is carried out spissated step is:
(1) provide a kind of Rotary Evaporators,
(2) extracting solution is imported in the Rotary Evaporators, rotating speed 98r/min,
(3) contain crude drug amount 0.5～1.5g/ml until concentrated solution, leave standstill more than 10 hours, 4～5 ℃ of temperature are collected crude extract.
And to crude extract can also be refined as with hot ethanol dissolved step:
(1) dissolve crude extract with hot ethanol,
(2) collect lysate and insolubles respectively,
(3) insolubles is dissolved with hot ethanol,
(4) repeat (2)～(3) step repeatedly,
(5) collect all lysates and leaving standstill more than 10 hours, 4～5 ℃ of temperature are collected coarse crystallization.
Above-mentioned steps can increase the extraction yield of phytosterin compound, has avoided the waste of pharmaceutically active substance.
Providing of ethanol dilute alkaline soln can be for adding 1%KOH or NaOH solution in ethanolic soln.
To ether layer exsiccant concrete steps can for:
(1) ether layer is washed with water to neutrality,
(2) use anhydrous Na
2SO
4Get solids after moisture content being blotted and makes ether volatilization fully.
Below the glossy ganoderma sterols extract that is obtained is further purified, to obtain more accurate material.
Concrete steps are as follows:
(1) provide a kind of chromatography column, stationary phase silica gel, moving phase sherwood oil and ethyl acetate mixed solution,
(2) with sherwood oil silica gel is inserted chromatography column earlier,
(3) lysate is added a small amount of silica gel and mixes, normal temperature remove solvent and solids,
(4) solids is packed into chromatography capital drips sherwood oil and with ethyl acetate mixed solution gradient elution each component is separated,
(5) with automatic segmentation collector Fractional Collections parting liquid,
(6) various parting liquids are carried out tlc analysis, follow the tracks of to detect, merge similarly, the weight ratio of used developping agent sherwood oil and ethyl acetate is 7: 3, developer 1% 4-hydroxyl-3-methoxylbenxaldehyde-sulfuric acid;
(7) similar parting liquid is concentrated;
(8) provide a kind of ether: the mixed solution of Glacial acetic acid=1: 1;
(9) in concentrated solution, add equivalent mixed liquor, leave standstill, collect the white plates crystallization of separating out and be respectively major ingredient glossy ganoderma sterols purification thing 24-methyl-courage steroid-5, the pure and mild less important composition courage steroid-5 of 22-diene-3 β, 7-diene-3 β alcohol.
Adopt chromatography column that lysate is carried out different compounds in the separate dissolved liquid, automatic segmentation collector Fractional Collections this different compound and concentrated respectively.Chromatography column can the extremely proximate material of separating property, just has been widely used in fields such as chemistry, chemical industry, biochemistry as far back as decades ago.
When column chromatography was analyzed, stationary phase was that the surface has certain active sorbent material, and the effect of moving phase is sample (solids) to be carried out wash-out separate.
This separate mode is based on solid adsorbent (stationary phase) to carry out the difference of the adsorptive power of each component in the solids.
Its sepn process is such: moving phase is carried solids and is entered chromatography column, when contacting with stationary phase, solids be fixed very soon phased soln or absorption, continuous feeding along with moving phase, dissolved or adsorbed components elutes from stationary phase again, the component that elutes be fixed once more during along with the moving forward of moving phase phased soln or absorption.Along with constantly pouring in of moving phase, the process of dissolving-wash-out is carried out repeatedly.Obviously, because the difference of constitutive property, stationary phase is to the ability difference of their dissolving or absorption, the time that stops in chromatography column is also different, thereby, through the component 24-methyl-courage steroid-5 of certain time interval (certain column length) back different in kind, the pure and mild courage steroid-5 of 22-diene-3 β, 7-diene-3 β alcohol or other composition are just separated from one another.
The collection mouth of automatic segmentation collector is connected with the chromatography column discharge channel.
A kind of ether is provided: the mixed solution of Glacial acetic acid=1: 1, in concentrated solution, add equivalent mixed liquor and make sterols purification thing recrystallization in the concentrated solution, leave standstill for some time after, collect the white plates crystallization of separating out and be glossy ganoderma sterols purification thing.
One of purpose of the present invention: provide the sterols extract to have the cerebral ischemia effect that the anti-various causes of disease cause in preparation, the medicine that particularly has Chinese People's Anti-Japanese Military and Political College's cortex neurone anoxia functions can be realized in the following way.
Medicine with treating cerebral ischemia, it comprises the glossy ganoderma sterols extract of effective dose.
The present invention also is, comprises the glossy ganoderma sterols purification thing 24-methyl-courage steroid-5 of effective dose in this medicine, 22-diene-3 β alcohol.
The present invention advances-goes on foot also to be, comprises the glossy ganoderma sterols purification thing courage steroid-5 of effective dose in this medicine, 7-diene-3 β alcohol.
Or this medicine comprises the glossy ganoderma sterols purification thing 24-methyl-courage steroid-5 of effective dose, the pure and mild courage steroid-5 of 22-diene-3 β, the mixture of 7-diene-3 β alcohol simultaneously.
Glossy ganoderma sterols extract, glossy ganoderma sterols purification thing 24-methyl-courage steroid-5, the result of treatment of the cerebral ischemia that 22-diene-3 β alcohol and courage steroid-5,7-diene-3 β alcohol cause the various causes of disease is with the embodiment 3 that above reaches in the following embodiment.Then decide as for the definition of effective dose according to the dosage of changing acquisition according to the experiment dose in the existing field of medicaments.
And the formulation of this medicine can be of the prior art commonly used, routine injection, tablet, oral liquid or the like.
In sum, the present invention has following advantage compared to prior art: the cerebral ischemia that the new phytosterin compound that the present invention extracts from glossy ganoderma causes the various causes of disease has outstanding curative effect, and make its mechanism of action of curing the disease comparatively clear and definite because of its molecular structural formula accurately that has, drug effect is outstanding, the utilization ratio height of pharmaceutically active substance.
Description of drawings
Fig. 1 is the provide protection of glossy ganoderma sterols extract (1 μ g/ml) to the cortical neuron of H/R damage.Use viable cell and dead cell in the two mark technology difference of fluorescent probe Hoechst dye 33258 and the PI culture system, all viable cell karyons of Hoechst dye 33258 (blueness) mark wherein, PI (redness) mark dead cell karyon.
A. normal control group cell b.H (12h)/R (24h) damaging cells c.GS (1 μ g/ml) handles cell
Fig. 2 is that glossy ganoderma sterols extract is to total SOD, Mn-SOD and the active influence of Cu-Zn SOD in the neurone of H/R damage.Data represented mean+SD.
*P＜0.05,
*P＜0.01 and the contrast of H/R model group.
Fig. 3 adopts mtt assay to detect sterol purification thing 24-methyl-courage steroid-5, and 22-diene-3 β alcohol is to the provide protection of H/R damage tegumental cell, data represented mean+SD.
*P＜0.01 and the contrast of H/R model group.
Fig. 4 is glossy ganoderma sterols purification thing 24-methyl-courage steroid-5, and 22-diene-3 β alcohol (1 μ g/ml) is to the provide protection of the cortical neuron of H/R damage.
A. normal control group cell B.H (12h)/R (24h) damaging cells C.GSP (1 μ g/ml) handles cell
Embodiment
Below in conjunction with accompanying drawing the present invention is described in more detail.
Embodiment 1:
Glossy ganoderma sterols preparation method of extract comprises following step of carrying out in regular turn:
1, provide the cultivating in bag Ganoderma sporophore as the raw material parent, and with the cleaning of raw material parent, drying;
2, the employing crusher carries out fragmentation with this raw material parent and gets broken thing;
3, with 95% ethanol broken thing is carried out refluxing extraction 5～6h, the weight ratio of ethanol and broken thing is 10: 1, and 3000r/min15 minute is centrifugal, waste and extracting solution;
4, extracting solution is concentrated, step is:
(1) provide a kind of film Rotary Evaporators,
(2) extracting solution is imported in the film Rotary Evaporators, rotating speed 98r/min,
(3) contain the about 1g/ml of crude drug amount until concentrated solution, leave standstill more than 10 hours, 4～5 ℃ of temperature are collected crude extract.
5, crude extract is done following processing:
(1) with 95% hot ethanol dissolving crude extract,
(2) collect lysate and insolubles respectively,
(3) insolubles is dissolved with 95% hot ethanol,
(4) repeat (2)～(3) step repeatedly,
(5) abandon the hot insolubles of ethanol, collect all lysates and leave standstill more than 10 hours 4～5 ℃ of temperature, collection coarse crystallization.
6, coarse crystallization was handled 16～24 hours with the 1%KOH ethanolic soln, is collected insolubles,
7, insolubles is used 12%KOH alkaline solution alkalization 24h, filtered, collect filtrate;
8, use the ether with filtrate equivalent filtrate to be extracted and collects ether layer;
9, ether layer is washed with water to neutrality, use anhydrous Na
2SO
4Get solids glossy ganoderma steroid class extract after moisture content being blotted and makes ether volatilization fully.
Glossy ganoderma sterols extract is further purified, to obtain more accurate material.
Concrete steps are as follows:
(1) provide a kind of chromatography column, stationary phase silica gel, moving phase sherwood oil and ethyl acetate mixed solution,
(2) with sherwood oil silica gel is inserted chromatography column earlier,
(3) lysate is added a small amount of silica gel and mixes, normal temperature remove solvent and solids,
(4) solids is packed into chromatography capital drips sherwood oil and with ethyl acetate mixed solution gradient elution each component is separated,
(5) with automatic segmentation collector Fractional Collections parting liquid,
(6) various parting liquids are carried out tlc analysis, follow the tracks of to detect, merge similarly, the weight ratio of used developping agent sherwood oil and ethyl acetate is 7: 3, developer 1% 4-hydroxyl-3-methoxylbenxaldehyde-sulfuric acid;
(7) similar parting liquid is concentrated;
(8) provide a kind of ether: the mixed solution of Glacial acetic acid=1: 1;
(9) add equivalent mixed liquor in concentrated solution, leave standstill, collect the white plates crystallization of separating out and be respectively major ingredient glossy ganoderma sterols purification thing 24-methyl-courage steroid-5, the pure and mild less important composition of 22-diene-3 β is a courage steroid-5,7-diene-3 β alcohol.
Embodiment 2:
The maximum sterols purification thing of ratio that provides embodiment 1 to obtain, promptly major ingredient carries out following experiment:
Experiment condition
The about 20mg of compound is dissolved in the deuterochloroform, pack into then in the 5mm nuclear-magnetism pipe, carry out the detection of chloroform one peacekeeping two dimensional NMR spectrum at Bruker DRX-400 type superconduction nuclear magnetic resonance spectrometer, probe temperature is 298k, marks in the chemical shift of TMS as hydrogen spectrum and carbon spectrum.
Experimental result
By
13CNMR and DEPT spectrum can determine to exist in the molecule 28 carbon, wherein 6 CH
3, 8 CH
2, 11 CH, and 3 quaternary carbons.Utilize one dimension (
1H,
13C, DEPT) and the two dimensional NMR technology
1H-
1H,
1H-
13The relevant spectrum of C,
1H-
13Relevant spectrum of C heteronuclear volume son (HMBC) and NOESY spectrum can be determined the chemical shift (see Table 1 and table 2) of the hydrogen atom and the carbon atom of compounds X
Table 1: compound
1H NMR data
| The hydrogen atom numbering | Chemical shift (δ ppm) | The hydrogen atom numbering | Chemical shift (δ ppm) | The hydrogen atom numbering | Chemical shift (δ ppm) |
| 1α | 1.065 | ?9α | 1.613 | ?18 | ?0.534 |
| 1β | 1.799 | ?11α | 1.419 | ?19 | ?0.776 |
| 2α | 1.374 | ?11β | 1.588 | ?20 | ?1.837 |
| 2β | 1.770 | ?12α | 1.237 | ?21 | ?0.897 |
| 3α | 3.573 | ?12β | 1.681 | ?22 | ?5.138 |
| 4α | 1.220 | ?14α | 1.225 | ?23 | ?5.184 |
| 4β | 1.987 | ?15α | 1.338 | ?24 | ?1.995 |
| 6 | 5.139 | ?15β | 1.470 | ?25 | ?1.440 |
| 7α | 1.234 | ?16α | 1.265 | ?26 | ?0.801 |
| 7β | 1.742 | ?16β | 1.695 | ?27 | ?0.812 |
| 8α | 1.337 | ?17α | 1.784 | ?28 | ?0.995 |
Table 2: compound
13C NMR data
| Carbon atoms numbered | Chemical shift (δ ppm) | The hydrogen atom numbering | Chemical shift (δ ppm) | The hydrogen atom numbering | Chemical shift (δ ppm) |
| ????1 | ????37.2 | ????11 | ????21.6 | ????21 | ????17.6 |
| ????2 | ????31.5 | ????12 | ????28.1 | ????22 | ????135.7 |
| ????3 | ????71.1 | ????13 | ????43.3 | ????23 | ????131.9 |
| ????4 | ????39.5 | ????14 | ????56.0 | ????24 | ????49.5 |
| ????5 | ????139.5 | ????15 | ????22.9 | ????25 | ????33.1 |
| ????6 | ????117.5 | ????16 | ????37.9 | ????26 | ????19.7 |
| ????7 | ????29.6 | ????17 | ????55.1 | ????27 | ????20.0 |
| ????8 | ????40.5 | ????18 | ????12.1 | ????28 | ????21.1 |
| ????9 | ????40.3 | ????19 | ????13.0 | ||
| ????10 | ????34.2 | ????20 | ????42.8 |
The structural formula of this main compound is
The less important composition that in like manner obtains this glossy ganoderma sterols extract is to reach courage steroid-5,7-diene-3 β alcohol, and its structural formula is
Embodiment 3:
Glossy ganoderma sterols extract and purification thing thereof reoxidize the provide protection of damage to rat cerebral cortex neurone anoxic
Observe glossy ganoderma sterols extract (Ganoderma Sterols; GS) and the purification thing (be main component 24-methyl-courage steroid-5; 22-diene-3 β alcohol; purification ofGanoderma Sterols; GSP) rat cerebral cortex neurone anoxic is reoxidized the provide protection of damage (H/R), for the prevention protection that the glossy ganoderma steroidal extract is applied to cerebral ischemia re-pouring provides experimental basis.
(1) glossy ganoderma sterols extract (GS) reoxidizes the provide protection of damage to rat cerebral cortex neurone anoxic
At first we have studied the provide protection of glossy ganoderma sterols extract (GS) to the cerebral ischemia re-pouring external model, and its mechanism has been carried out preliminary discussion.
External former being commissioned to train supported SD rat cerebral cortex cell, reoxidizes (24h) damage by anoxic (12h) and sets up the cerebral ischemia re-pouring external model.Cultivate vessel and handle with the 12mg/l poly-lysine in advance, dry standby naturally.Take out the SD rat that gave birth to 1 day, aseptic separation suckling mouse pallium, 0.25% tryptic digestion, the preparation cell suspension is with the DMEM dilution that contains 10% foetal calf serum, 10% horse serum, with 1 * 10
6/ ml cell density is inoculated in to be cultivated in the vessel, places incubator (CO
2Concentration is 5%), hatch in 37 ℃.Vitro culture added 10 μ mol/L cytosine arabinosides in 72 hours and suppresses non-neuronal growth.(NSE) checks neuronic purity as the neuronal specificity mark with Hydratase, phosphoenolpyruvate, and the result proves that the neurone proportion is 83-92% in our culture system.
The neurone vitro culture is removed former DMEM nutrient solution after 8 days, change with low sugar, serum-free medium, puts into 37 ℃, 5%CO
2, 95%N
2The anoxic jar in, behind anoxic 12h, cell is moved to 37 ℃ at once, 5%CO
2, reoxygenation 24h in the 95% air incubator, taking-up experimentizes.
Identify neuronic life state with PI and the two calibration methods of Hoechst33258, under laser confocal microscope, observe the mark situation.The two cells that dye of PI and Hoechst33258 are dead cell, and Hoechst33258 singly dyes and is viable cell.The two dead cell showed increased of dying of PI and Hoechst33258 in the visible H/R model group of the result cell, and use the ratio (seeing Fig. 1 for details) that can obviously increase viable cell behind the GS.
Use mtt assay and measure the survival rate and the cellular metabolism situation of cell.4h adds MTT (final concentration is 0.5mg/m1) in every hole of 96 orifice plates before stopping cultivation, sucking-off nutrient solution behind the cultivation 4h, and every hole adds the inferior maple (DMSO) of 100 μ l dimethyl, and crystallization is fully dissolved, and reads the A value of wavelength 570nm on the spectrophotometer.H/R obviously descends neuronal survival as a result, and GS has significant provide protection to damaged cell.
Use free radical in the fluorescent probe DCF-DA specific marker cell, DCF-DA is by H in cell
2O
2Be oxidized to dichlorofluorescein (DCF), DCF produces green fluorescence, by measuring the fluorescence intensity of DCF in the cell, and can the interior H of relative quantification cell
2O
2Level.DCF-DA (10 μ mol/L) reacted 30 minutes under 37 ℃ of conditions with cell, measured free-radical contents in the cell under laser confocal microscope.As seen H/R can significantly induce the generation of free radical, and the visible fluorescence intensity of probe obviously strengthens under the mirror, and GS treatment group fluorescence intensity then obviously reduces, and just GS can significantly suppress the free radical level that increases in the cell.
Prepare cell lysate with lysate, identify protein content with the bradford method.Use Nanjing and build up test kit that bio-engineering research is produced, colorimetric method for determining lipid peroxidation product glutaraldehyde (MDA) level.The result as seen, GS also can reduce the unusual MDA that raises in the injured neurons, illustrates that antioxygenation may be GS to one of mechanism of neurocyte protection effect.
Simultaneously, use Nanjing and build up test kit that bio-engineering research is produced, we have also detected total superoxide-dismutase (SOD) and Mn-SOD activity in the neurone.We find that GS can obviously increase SOD and Mn-SOD activity in the neurone, and to the active not obviously effect of Cu-Zn SOD, this proof Mn-SOD may be the key enzyme that GS brings into play its antioxygenation, strengthens the resistance of oxidation (seeing Fig. 2 for details) of neurocyte by the activity that increases Mn-SOD.
With anti-p65 labeling of monoclonal antibodies intracellular nucleic transcription factor NF-KB, under laser confocal microscope, NF-κ B is positioned observation with the method for immunofluorescence.As seen NF-κ B basic fixed position under normal circumstances is in cytoplasm, and at 12 hours reoxygenations of anoxic in the time of 45 minutes, then transposition in nucleus in a large number of NF-κ B, use GS then obviously antagonism activation and the nuclear translocation of NF-κ B.
Prepare cell pyrolysis liquid, detect the expression level of NF-κ B specificity inhibition I-κ B with the method for Western blot.Specificity one anti-application concentration is 1: 100, and the two anti-concentration of using were with 1: 1000.Experiment finds that H (12h)/R (45min) can increase the degraded of I-κ B, and GS can suppress this variation.Therefore we think that GS is by degraded that suppresses I-κ B and then activation and the nuclear translocation that has suppressed NF-κ B.
Preparation cell pyrolysis liquid, and use the ELISA test kit detects inflammatory cytokine TNF α and IL-1 β at intracellular expression level according to the test kit illustration method, find can significantly the raise level of TNF α and IL-1 β of H/R, and GS can suppress this variation.
Because NF-κ B is the crucial regulatory factor of various kinds of cell factor expression,, thereby suppressed inflammatory cytokine to neuronic immunologic injury so we infer that GS may influence the expression of TNF α and IL-1 β by the activation that suppresses NF-κ B.Experimental results show that because of having SOD can suppress the activation of NF-κ B again,, and then damage from two mechanism antagonism of anti-oxidant and anti-immunity H/R so we infer that Mn-SOD may be the keying action site of GS to H/R damaging cells performance provide protection.
(2) glossy ganoderma sterol purification thing (24-methyl-courage steroid-5,22-diene-3 β alcohol, GSP)
Rat cerebral cortex neurone anoxic is reoxidized the provide protection of damage
(24-methyl-courage steroid-5,22-diene-3 β alcohol GSP) replace the glossy ganoderma steroidal extract in above-mentioned (one) experiment to test to the sterols purification thing that employing is further purified, and draw following result.
Use the cerebral ischemia re-pouring external model that the H/R damage is set up, we have carried out preliminary discussion to the pharmacodynamics and the pharmacology of this purification thing.
Use mtt assay and measure the survival rate and the cellular metabolism situation (seeing Fig. 3 for details) of cell, can find that GSP has obvious provide protection to damaged cell, can obviously increase neuronal survival (seeing Fig. 4 for details).Use free radical in the fluorescent probe DCF-DA specific marker cell; under laser confocal microscope, measure free-radical contents in the cell; as seen GSP can significantly suppress the free radical level that increases in the H/R model group cell, proves that antioxygenation may be GSP to one of mechanism of neurocyte protection effect.
We find to compare with the GS of equal in quality, and GSP all has more significant effect to neuronic protective effect and the effect of inhibition free-radical generating. The method of explanation purifying GSP from GS has been protected the composition that wherein has neuroprotection effectively, and can therefore reduce dosage and possible side effect.
Claims (16)
1, glossy ganoderma sterols extract is characterized in that, it is extracted by glossy ganoderma and obtains, and its main component is white plates crystallization 24-methyl-courage steroid-5,22-diene-3 β alcohol, molecular formula C
28H
46O, its chemical structural formula is as follows:
2, glossy ganoderma sterols extract according to claim 1 is characterized in that, also comprises submember white crystals courage steroid-5,7-diene-3 β alcohol, and its chemical structural formula is as follows:
3, glossy ganoderma sterols preparation method of extract is characterized in that, comprises following step of carrying out in regular turn:
(1) provide a kind of glossy ganoderma as the raw material parent;
(2) broken this raw material parent and broken thing;
(3) with ethanol to broken thing carry out refluxing extraction 4～10h, centrifugal and extracting solution;
(4) extracting solution is concentrated into contains crude drug amount 0.5～1.5g/ml in the concentrated solution, leave standstill more than 10 hours, 4～5 ℃ of temperature are collected crude extract;
(5) crude extract is dissolved with hot ethanol, extract lysate and leave standstill more than 10 hours 4～5 ℃ of temperature, collection coarse crystallization;
(6) coarse crystallization was handled 16～48 hours with the ethanol dilute alkaline soln, is collected insolubles,
(7) with insolubles with the concentrated alkali solution 24h that alkalizes, filter, collect filtrate;
(8) filtrate is extracted and collects ether layer with the equivalent ether;
(9) ether layer is washed with water to neutrality, carry out drying, treat that the complete back of ether volatilization gained solids is the sterols extract.
4, glossy ganoderma sterols preparation method of extract according to claim 3 is characterized in that, ethanol is 5～6 hours to the Best Times that broken thing carries out the refluxing extraction sterol compound.
5, glossy ganoderma sterols preparation method of extract according to claim 3 is characterized in that, the weight ratio of ethanol and broken thing is 10: 1.
6, glossy ganoderma sterols preparation method of extract according to claim 3 is characterized in that, being provided as of ethanol basic solution adds 1%KOH or NaOH solution in ethanolic soln.
7, glossy ganoderma sterols preparation method of extract according to claim 3 is characterized in that, alkaline solution can be 12%KOH.
8, glossy ganoderma sterols preparation method of extract according to claim 3 is characterized in that, ethanol or use chloroform, or use methyl alcohol, or replace with organic solvents such as benzene.
9, glossy ganoderma sterols preparation method of extract according to claim 3 is characterized in that, extracting solution is carried out spissated step be:
(1) provide a kind of Rotary Evaporators,
(2) extracting solution is imported in the Rotary Evaporators, rotating speed 98r/min,
(3) contain crude drug amount 0.5～1.5g/ml until concentrated solution, leave standstill more than 10 hours temperature
4～5 ℃, collect crude extract.
10, glossy ganoderma sterols preparation method of extract according to claim 3 is characterized in that, crude extract is refined as with hot ethanol dissolved step:
(1) dissolve crude extract with hot ethanol,
(2) collect lysate and insolubles respectively,
(3) insolubles is dissolved with hot ethanol,
(4) repeat (2)～(3) step repeatedly,
(5) collect all lysates and leaving standstill more than 10 hours, 4～5 ℃ of temperature are collected coarse crystallization.
11, glossy ganoderma sterols preparation method of extract according to claim 3 is characterized in that, to ether layer exsiccant concrete steps is:
(1) ether layer is washed with water to neutrality,
(2) use anhydrous Na
2SO
4Get solids after moisture content being blotted and makes ether volatilization fully.
12, glossy ganoderma sterols preparation method of extract according to claim 3 is characterized in that, the concrete steps of the different components of sterols separated class extract are as follows:
(1) provide a kind of chromatography column, stationary phase silica gel, moving phase sherwood oil and ethyl acetate mixed solution,
(2) with sherwood oil silica gel is inserted chromatography column earlier,
(3) lysate is added a small amount of silica gel and mixes, normal temperature remove solvent and solids,
(4) solids is packed into chromatograph is analysed the top, and drip sherwood oil and each component is separated with ethyl acetate mixed solution gradient elution,
(5) with automatic segmentation collector Fractional Collections parting liquid,
(6) various parting liquids are carried out tlc analysis, follow the tracks of to detect, merge similarly, the weight ratio of used developping agent sherwood oil and ethyl acetate is 7: 3, developer 1% 4-hydroxyl-3-methoxylbenxaldehyde-sulfuric acid;
(7) similar parting liquid is concentrated.
(8) provide a kind of ether: the mixed solution of Glacial acetic acid=1: 1;
(9) in concentrated solution, add equivalent mixed liquor, leave standstill, collect the white plates crystallization of separating out and be respectively major ingredient glossy ganoderma sterols purification thing 24-methyl-courage steroid-5, the pure and mild less important composition courage steroid-5 of 22-diene-3 β, 7-diene-3 β alcohol.
13, have the medicine for the treatment of cerebral ischemia, it is characterized in that, it comprises the glossy ganoderma sterols extract of effective dose.
14, the medicine with treating cerebral ischemia according to claim 13 is characterized in that, it comprises the glossy ganoderma sterols purification thing 24-methyl-courage steroid-5 of effective dose, 22-diene-3 β alcohol.
15, the medicine with treating cerebral ischemia according to claim 13 is characterized in that, it comprises the glossy ganoderma sterols purification thing courage steroid-5 of effective dose, 7-diene-3 β alcohol.
16, the medicine with treating cerebral ischemia according to claim 13 is characterized in that, it comprises the glossy ganoderma sterols purification thing 24-methyl-courage steroid-5 of effective dose, the pure and mild courage steroid-5 of 22-diene-3 β, the mixture of 7-diene-3 β alcohol.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311475A (en) * | 2011-07-18 | 2012-01-11 | 福建医科大学 | New compound separated from Mythic Fungus, preparation method thereof and medicinal purpose thereof |
| CN104825463A (en) * | 2014-12-22 | 2015-08-12 | 中国科学院微生物研究所 | Metabolic-regulation and anti-hypoxia application of ganoderma leucocontextum extract |
| CN110632237A (en) * | 2019-09-26 | 2019-12-31 | 吕梁学院 | Method for evaluating oxidation resistance of phytosterol in red dates by applying TLC-CMS (thin layer chromatography-sodium carboxymethyl cellulose) technology |
| CN110860107A (en) * | 2019-11-29 | 2020-03-06 | 林东楷 | Microcapsule embedding phytosterol extraction element |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101461271B1 (en) * | 2011-02-25 | 2014-11-12 | 오노 야꾸힝 고교 가부시키가이샤 | Formulations for injection containing sivelestat sodium salt or hydrate thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1028410C (en) * | 1993-02-20 | 1995-05-17 | 李汝清 | Compound glossy ganoderma nutrient solution and its preparation method |
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2003
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311475A (en) * | 2011-07-18 | 2012-01-11 | 福建医科大学 | New compound separated from Mythic Fungus, preparation method thereof and medicinal purpose thereof |
| CN102311475B (en) * | 2011-07-18 | 2013-02-06 | 福建医科大学 | New compound separated from Ganoderma lucidum, preparation method thereof and medicinal purpose thereof |
| CN104825463A (en) * | 2014-12-22 | 2015-08-12 | 中国科学院微生物研究所 | Metabolic-regulation and anti-hypoxia application of ganoderma leucocontextum extract |
| CN104825463B (en) * | 2014-12-22 | 2017-09-15 | 中国科学院微生物研究所 | The Metabolism regulation of plain boiled pork Ganodenna Lucidum P.E and the purposes of anti anoxia |
| CN110632237A (en) * | 2019-09-26 | 2019-12-31 | 吕梁学院 | Method for evaluating oxidation resistance of phytosterol in red dates by applying TLC-CMS (thin layer chromatography-sodium carboxymethyl cellulose) technology |
| CN110632237B (en) * | 2019-09-26 | 2021-10-08 | 吕梁学院 | Method for evaluating oxidation resistance of phytosterol in red dates by applying TLC-CMS (thin layer chromatography-sodium carboxymethyl cellulose) technology |
| CN110860107A (en) * | 2019-11-29 | 2020-03-06 | 林东楷 | Microcapsule embedding phytosterol extraction element |
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