The ganoderma spove powder and application of a kind of method for breaking trachytectum of glossy ganoderma and its preparation
Technical field
The invention belongs to breaking trachytectum of glossy ganoderma technical field, and in particular to a kind of method for breaking trachytectum of glossy ganoderma and its be prepared into
The ganoderma spove powder arrived and application.
Background technology
Ganoderma lucidum is a kind of material containing abundant physiologically active, including GL-B class, triterpenes, ucleosides, sterol
Class, alkaloids, furan derivatives, amino acid polypeptide class, mineral element, aliphatic acid etc..Its pharmacological action, which has mainly, anti-swollen
Knurl, immunological regulation, radioresistance, calmness, stable, cardiac stimulant, decompression, antibechic, relieving asthma, it is hypoglycemic etc..The active component of ganoderma lucidum is most
It is present in Reishi sporule, therefore, wants to make full use of the active component of Reishi sporule, it is necessary to which Reishi sporule is carried out at broken wall
Reason, could so discharge more active materials.But Reishi sporule outer wall is thicker, therefore breaking trachytectum of glossy ganoderma technology
The key just utilized into Reishi sporule.
At present, the wall breaking technology for Reishi sporule includes a variety of, such as ultramicro grinding broken wall method, and homogenate wall-cracking surpasses
Sound broken wall method, sprouts broken wall method, biology enzyme broken wall method, CO2Overcritical broken wall method, high-energy ball milling impingement method etc..But Ultramicro-powder
Broken broken wall method, homogenate wall-cracking, ultrasonication method, high-energy ball milling impingement method sporoderm-broken rate are all than relatively low, and ultramicro grinding broken wall
Method, high-energy ball milling impingement method and ultrasonication method are larger to the destructive power of active material, active material is gone bad.And sprout
Broken wall method broken wall process is longer, cumbersome, is unsuitable for mass disposal, and broken ratio is also than relatively low.Biology enzyme broken wall method
Influence factor it is too many, wherein enzyme dosage, temperature, pH value, time etc. can all influence the efficiency of broken wall, therefore practicality is not strong.
CO2Overcritical broken wall method broken wall process is carried out under superelevation pressure condition, CO2Can be by significant quantities of fat under the conditions of subcritical liquid state
Acids active material is discharged, and causes the loss of active material.
Therefore, exploitation is a kind of can be sufficiently reserved the active material of lucidum spore powder, and can realize higher sporoderm-broken rate
Method will be the key that makes lucidum spore powder effect obtain optimal performance.
The content of the invention
In order to solve the deficiencies in the prior art, it is an object of the invention to provide a kind of method for breaking trachytectum of glossy ganoderma and the party
Ganoderma spove powder and application that method is prepared.
Inventor is had found, lucidum spore powder is mixed and pre-processed with fullerene powder, is made in fullerene powder
Clustered particles are ground into micron and/or nano particle, into Reishi sporule shell wall and inside;Then from external energy source
Mode irradiates fullerene, is allowed to produce phase transformation, occurs volumetric expansion, Reishi sporule outer wall is strutted, Reishi sporule is realized
Broken wall.The fullerene phase transition process that the external energy source is triggered does not produce high heat, and without high pressure, can keep Reishi sporule
The stability of interior active component, while the sporoderm-broken rate of Reishi sporule at least 98% can be realized.Meanwhile, the fullerene used can
To be reclaimed and be reused, the angle utilized from raw material greatlys save production cost.Based on such thinking, complete
The present invention.
The present invention seeks to what is be achieved through the following technical solutions:
A kind of method for breaking trachytectum of glossy ganoderma, the wall-breaking method comprises the following steps:
1) fullerene powder is mixed and pre-processed with lucidum spore powder, so that fullerene powder enters ganoderma lucidum spore
Son;
2) pretreated mixed-powder is irradiated.
According to the present invention, it will be understood by those skilled in the art that various fullerenes can be used.It is preferred that the fullerene is
C60, C70, C76In one or more of mixtures, more preferably C60, C70In one kind or its mixture, most preferably C60。
, according to the invention it is preferred to the particle diameter of the fullerene powder is 10nm~50 μm, more preferably 10nm~5000nm,
Most preferably 100nm~1000nm;The particle diameter of fullerene powder is 50nm-500nm, most preferably 100nm- after preferred pretreatment
200nm。
According to the present invention, it will be understood by those skilled in the art that the lucidum spore powder is not limited, using prior art
In it is any.
, according to the invention it is preferred to which the mass ratio of the fullerene powder and lucidum spore powder is 1:5~10000, more preferably
For 1:10~1000, most preferably 1:100~500.
According to the present invention, in step 1) in, temperature≤50 DEG C of preferably described pretreatment.
According to the present invention, in step 1) in, preferably described pretreatment includes but is not limited to low temperature high-energy ball milling, high-pressure homogeneous
Processing, high speed homogenization processing etc.;Most preferably, the pretreatment is low temperature high-energy ball milling.
, according to the invention it is preferred to the ball milling temperature of the low temperature high-energy ball milling is 20~40 DEG C, more preferably 25~35 DEG C,
Most preferably 30 DEG C;It is preferred that Ball-milling Time is 1~20h, more preferably more preferably 2~16h, 4~12h, it is most preferably
8h;It is preferred that ball milling frequency be 200~2000r/min, more preferably 300~1500r/min, more preferably 500~
1000r/min, most preferably 700r/min.
In the present invention, by pretreatment (being, for example, low temperature high-energy ball milling, high-pressure homogeneous processing, high speed homogenization processing etc.),
On the one hand the clustered particles in fullerene powder can be ground into micron and/or nano particle, while can be in Reishi sporule outer wall
Certain crack is produced, can so cause fullerenic particles preferably to enter Reishi sporule shell wall and inside.
According to the present invention, the irradiation is realized by external energy source, can be magnetic field, radio frequency, one in ray
Plant or a variety of;Preferably radio frequency.
, according to the invention it is preferred to which the energy in the magnetic field is 0.1-10T;It is preferred that the time of the magnetic field radiation is 1-12h.
, according to the invention it is preferred to which the energy of the radio frequency is 100~1000MHz, most preferably 300~500MHz;It is preferred that
The time of the radio frequency irradiation is 0.5~20h, most preferably more preferably 1~12h, 3~9h.
, according to the invention it is preferred to which the energy of the ray is 1-1000eV;It is preferred that the time of the x ray irradiation x is 0.5-
12h。
In the present invention, the fullerene in Reishi sporule is produced phase transformation by irradiation, occur volumetric expansion, by epispore
Strut, it is achieved thereby that to the broken wall treatment of Reishi sporule.
The present invention also provides a kind of preparation method of ganoderma spove powder, and methods described comprises the following steps:
1) fullerene powder is mixed and pre-processed with lucidum spore powder, so that fullerene powder enters ganoderma lucidum spore
Son;
2) pretreated mixed-powder is irradiated, obtains wall-broken ganoderma spore fullerene mixed-powder;
3) to above-mentioned steps 2) add water in obtained wall-broken ganoderma spore fullerene mixed-powder, stirring, centrifugation, remove
Precipitation, takes supernatant to be dried, that is, prepares ganoderma spove powder.
, according to the invention it is preferred to which the temperature of the water is 50~100 DEG C, most preferably 60~80 DEG C.
, according to the invention it is preferred to which the mass volume ratio of the wall-broken ganoderma spore fullerene mixed-powder and water is 1-
1000mg/mL;Most preferably 50-500mg/mL.
, according to the invention it is preferred to which the addition water, stirring and centrifugation are repeated 3~5 times.
According to the present invention, it will be understood by those skilled in the art that various dried forms can be used.It is preferred that, the drying
It is that vacuum drying or normal pressure are air-dried;It is preferred that the temperature of the drying is 20-50 DEG C, more preferably 30-45 DEG C, most preferably 40
℃;It is preferred that the time of the drying is 1-48h, most preferably more preferably 10-24h, 12h.
In the present invention, because fullerene is water insoluble, the lucidum spore powder of broken wall or non-broken wall is all insoluble at room temperature
Water, and the lucidum spore powder of broken wall or non-broken wall is soluble in water under 50~100 DEG C (being preferably 60~80 DEG C), utilizes ganoderma lucidum spore
The dissolubility of sub- powder, by it, separation and Extraction comes out from fullerene powder;And the fullerene can be removed it by centrifugation.
The present invention also provides a kind of ganoderma spove powder, and the ganoderma spove powder is prepared into by the above method
Arrive.
The present invention also provides a kind of preparation method of ganoderma lucidum spore oil, and methods described comprises the following steps:
1) fullerene powder is mixed and pre-processed with lucidum spore powder, so that fullerene powder enters ganoderma lucidum spore
Son;
2) pretreated mixed-powder is irradiated, obtains wall-broken ganoderma spore fullerene mixed-powder;
3) to above-mentioned steps 2) add water in obtained wall-broken ganoderma spore fullerene mixed-powder, stirring, centrifugation, remove
Precipitation, takes supernatant to be dried, prepares ganoderma spove powder;
4) by above-mentioned steps 3) obtained ganoderma spove powder extracts ganoderma lucidum spore oil.
It will be understood by those skilled in the art that can be using various methods known in the art in ganoderma spove powder
Extract ganoderma lucidum spore oil.
The present invention also provides a kind of ganoderma lucidum spore oil, and the ganoderma lucidum spore oil is prepared by the above method.
Invention further provides the present invention ganoderma spove powder or ganoderma lucidum spore oil prepare be used for prevent and/
Or the purposes in the medicine for the treatment of tumour or the medicine in preparation for preventing and/or treating caused by chemotherapeutic medicines hepar damnification
In purposes.
Beneficial effects of the present invention:
1. the invention provides a kind of method for breaking trachytectum of glossy ganoderma, methods described is by lucidum spore powder and fullerene powder
Mix and pre-processed, the clustered particles in fullerene powder is ground into micron and/or nano particle, into ganoderma lucidum spore
Sub- shell wall and inside;Then the mode from external energy source irradiates fullerene, is allowed to produce phase transformation, occurs volumetric expansion, will
Reishi sporule outer wall is strutted, and realizes the broken wall of Reishi sporule, it is possible to achieve the sporoderm-broken rate of Reishi sporule at least 98%.
2. present invention also offers a kind of preparation method of ganoderma spove powder and its wall-breaking lucidum spore prepared
Sub- powder;Methods described is not related to HTHP, farthest remains the active component content and activity of Reishi sporule, tumor suppression
Rate is high, while it has significant protective effect to caused by chemotherapeutic medicines hepar damnification.
3. it is described present invention also offers a kind of preparation method of ganoderma lucidum spore oil and its ganoderma lucidum spore oil prepared
Ganoderma lucidum spore oil is extracted from preparation-obtained ganoderma spove powder of the invention and obtained, and its recovery rate is high.
Brief description of the drawings
Fig. 1 is different group liver cell situations in the embodiment of the present invention 9.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.Furthermore, it is to be understood that after described content of the invention has been read, this area skill
Art personnel can make various changes or modifications to the present invention, and these equivalent form of values equally fall within limited range of the present invention.
Ball milling in embodiment of the present invention is carried out in XQM-2L type ball mills, and described ball milling operation is ball
It is 30 DEG C to grind temperature;Ball-milling Time is 8h;Ball milling frequency is 700r/min.
In the present invention, the computational methods of described sporoderm-broken rate:
1) lucidum spore powder that 2.0mg broken walls at room temperature, are weighed with assay balance (precision 0.lmg) is put into 10mL examinations
Guan Zhong, then 2mL distilled water is added with accurate hand-held dropper in each test tube, 5min or so is swayed, because lucidum spore powder exists
At room temperature, it is insoluble in water, thus prepares the suspension of lucidum spore powder;
2) the μ L of suspension 6 are drawn with micro syringe (10 μ L), disposably dripped to suspension in counting scale, and cover one
Sheet glass, stands 2min~3min, suspension is evenly distributed in counting scale, is counted after lucidum spore powder sinks and no longer moved
Number.By light microscope differentiate spore broken wall whether, the conidial cell wall of non-broken wall is smooth complete, plentiful, and double-walled construction is clear,
Individual morphology is consistent;After broken wall, conidial cell wall is imperfect, content is in irregular block;
3) calculating of wall breaking rate of ganoderma lucidum spores:
The calculation formula of wall breaking rate of ganoderma lucidum spores:Sporoderm-broken rate=[(A-B)/A] × 100%;
Wherein, A is intact spore number before broken wall;B is intact spore number after broken wall;Intact spore number is after broken wall before broken wall
Imperfect spore count sum after intact spore number and broken wall.
In the present invention, the extracting method of described ganoderma lucidum spore oil is supercritical CO2Extracting process;Described Reishi sporule
The recovery rate computational methods of oil are to judge the recovery rate of ganoderma lucidum spore oil by detecting the content of aliphatic acid in ganoderma lucidum spore oil,
Specially:
Spore oil obtained by 20mg is weighed in l0mL in tool plug test tube, addition 0.4mol/L KOH- methanol solution 2mL, in
70 DEG C of heating water bath 30min, after completion of the reaction, add distilled water 2mL, then enriching hydrochloric acid is neutralized to pH value in neutrality.Then plus
Enter 2mL n-hexane extractions, take supernatant, cyclic washing 2~3 times.Merge supernatant and be concentrated under reduced pressure into dry, then the sulphur for Jia 1%
Sour methanol solutions 2mL, in 70 DEG C of water-bath 30min, after completion of the reaction, n-hexane extraction, then with distilled water, fully vibration is stood
Supernatant is taken after layering, cyclic washing 2~3 times merges supernatant and uses anhydrous sodium sulfate drying, finally takes the μ L of supernatant 1 to enter
Row GC-MS is analyzed, and the carboxylate obtained by analysis calculates content of fatty acid in ganoderma lucidum spore oil, and then determines recovery rate.
Embodiment 1
By 1g fullerenes C60Carry out in ball mill being sufficiently mixed ball milling with 100g lucidum spore powders, then by mixed-powder
It is positioned in a vial, vial is linked by coil with radiofrequency generator, opens radiofrequency generator, regulation power is 300MHz, is continued
Irradiate after 2h, close radiofrequency generator.80 DEG C of water are added after mixed-powder is taken out to be sufficiently stirred for, and carry out centrifugally operated, repeat 3-5
It is secondary;Because fullerene is water insoluble, bottom can be deposited on, DDGS --- supernatant liquor is collected, is dried and (is dried at 40 DEG C
12h) prepare the ganoderma spove powder afterwards.
Embodiment 2
By 1g fullerenes C60Carry out in ball mill being sufficiently mixed ball milling with 500g lucidum spore powders, then by mixed-powder
It is positioned in a vial, vial is linked by coil with radiofrequency generator, opens radiofrequency generator, regulation power is 400MHz, is continued
Irradiate after 4h, close radiofrequency generator.80 DEG C of water are added after mixed-powder is taken out to be sufficiently stirred for, and carry out centrifugally operated, repeat 3-5
It is secondary;Because fullerene is water insoluble, bottom can be deposited on, DDGS --- supernatant liquor is collected, is dried and (is dried at 40 DEG C
12h) prepare the ganoderma spove powder afterwards.
Embodiment 3
By 1g fullerenes C60Carry out in ball mill being sufficiently mixed ball milling with 300g lucidum spore powders, then by mixed-powder
It is positioned in a vial, vial is linked by coil with radiofrequency generator, opens radiofrequency generator, regulation power is 300MHz, is continued
Irradiate after 3h, close radiofrequency generator.80 DEG C of water are added after mixed-powder is taken out to be sufficiently stirred for, and carry out centrifugally operated, repeat 3-5
It is secondary;Because fullerene is water insoluble, bottom can be deposited on, DDGS --- supernatant liquor is collected, is dried and (is dried at 40 DEG C
12h) prepare the ganoderma spove powder afterwards.
Embodiment 4
By 1g fullerenes C70Carry out in ball mill being sufficiently mixed ball milling with 100g lucidum spore powders, then by mixed-powder
It is positioned in a vial, vial is linked by coil with radiofrequency generator, opens radiofrequency generator, regulation power is 300MHz, is continued
Irradiate after 3h, close radiofrequency generator.80 DEG C of water are added after mixed-powder is taken out to be sufficiently stirred for, and carry out centrifugally operated, repeat 3-5
It is secondary;Because fullerene is water insoluble, bottom can be deposited on, DDGS --- supernatant liquor is collected, is dried and (is dried at 40 DEG C
12h) prepare the ganoderma spove powder afterwards.
Embodiment 5
By 1g fullerenes C76Carry out in ball mill being sufficiently mixed ball milling with 100g lucidum spore powders, then by mixed-powder
It is positioned in a vial, vial is linked by coil with radiofrequency generator, opens radiofrequency generator, regulation power is 300MHz, is continued
Irradiate after 3.5h, close radiofrequency generator.80 DEG C of water are added after mixed-powder is taken out to be sufficiently stirred for, and carry out centrifugally operated, are repeated
3-5 times;Because fullerene is water insoluble, bottom can be deposited on, DDGS --- supernatant liquor is collected, is dried (at 40 DEG C
Dry 12h) prepare the ganoderma spove powder afterwards.
Comparative example 1 --- high-energy ball milling impingement method
300g lucidum spore powders are taken to carry out being sufficiently mixed ball milling, 700 revs/min of the rotating speed of the ball mill in ball mill
Clock, Ball-milling Time is 10h.After ball milling terminates, powder in ball grinder is taken out, the lucidum spore powder after broken wall is obtained.
Comparative example 2 --- CO2Overcritical broken wall method
300g lucidum spore powders are taken in 1LCO2In supercritical extraction reactor, the temperature and pressure difference of extraction kettle and separating still
Be set to 35MPa, 40 DEG C, run 3h, then rapid release critical gas CO after static 12h under condition of high voltage2.Critical gas CO2
After release terminates, it is ganoderma spove powder to collect sample.
The sporoderm-broken rate pair for the ganoderma spove powder that embodiment 6 --- embodiment 3, comparative example 1, comparative example 2 are prepared
Than
Due in the preparation process in accordance with the present invention, when centrifugation removes fullerene, having minimal amount of non-wall-broken ganoderma spore
It is removed, in order to which more scientific progress is contrasted, the sediment of embodiment 3 is slightly handled together with fullerene:By in sediment
Excess toluene is added, supernatant is the purple solution for having dissolved fullerene, precipitation is cleaned and done afterwards several times by centrifugation repeatedly with ethanol
It is dry, dried powder is mixed with the ganoderma spove powder that embodiment 3 is obtained, mixed sample is used for broken wall
The comparative analysis of rate.
The ganoderma spove powder that comparative example 1 and comparative example 2 are obtained is without being handled.
Calculated by the computational methods of above-mentioned sporoderm-broken rate and obtain following result:
Case |
Sporoderm-broken rate |
Embodiment 3 |
98% |
Comparative example 1 |
75% |
Comparative example 2 |
80% |
Wall-breaking method of the present invention can greatly improve the sporoderm-broken rate of Reishi sporule it can be seen from upper table contrast.
Embodiment 7 --- spirit is extracted in the ganoderma spove powder prepared from embodiment 3, comparative example 1, comparative example 2
The recovery rate of Ganoderma lucidum spore oil
Calculated by said extracted method and the computational methods of recovery rate and obtain following result:
Case |
Ganoderma lucidum spore oil recovery rate |
Embodiment 3 |
45% |
Comparative example 1 |
23% |
Comparative example 2 |
22% |
Wall-breaking method of the present invention can greatly improve Reishi sporule active material it can be seen from upper table contrast
Utilization rate.
The anti-cancer properties for the ganoderma spove powder that embodiment 8 --- embodiment 3, comparative example 1, comparative example 2 are prepared
Contrast
The present embodiment has investigated growth inhibition effect of the ganoderma spove powder to rat liver cancer tumour, wherein,
Animal strains:Balb/c female mices, 5 weeks, body weight was between 16-20g;
Tumor model:The strain of rat liver cancer H22 knurls;
Administering mode:Orally;
Dosage:400mg/kg, is administered 10 times, is administered once daily altogether;
Experiment packet:Random packet, every group 6.
Medicine A groups:The ganoderma spove powder that embodiment 3 is prepared;
Medicine B groups:The ganoderma spove powder that comparative example 1 is prepared;
Medicine C groups:The ganoderma spove powder that comparative example 2 is prepared;
Control group:Physiological saline;
Experimental method:The μ L concentration of subcutaneous vaccination 100 is 5 × 107/ mL H22 liver cancer cells;Inoculation 24 hours after start to
Medicine, successive administration 10 times;Mouse weight is every other day weighed during experiment and tumour growth situation is observed, observation is to after being inoculated with 15
It terminates experiment, takes mouse tumor to weigh and measure volume, calculates tumour inhibiting rate.
The calculation formula of tumour inhibiting rate:
Tumour inhibiting rate=(the average knurl weight of the average knurl weight-treatment group of control group) average knurl weight × 100% of/control group
Calculated by above-mentioned anti-cancer methods and tumour inhibiting rate and obtain following result:
Case |
Average knurl weight (g) |
Tumour inhibiting rate |
Embodiment 3 |
0.52±0.13 |
65.5% |
Comparative example 1 |
0.78±0.08 |
47.9% |
Comparative example 2 |
0.67±0.15 |
55.3% |
Control group |
1.5±0.17 |
-- |
The ganoderma spove powder that wall-breaking method of the present invention is prepared it can be seen from upper table contrast has most
High tumour inhibiting rate.
Protective effect of embodiment 9 --- the ganoderma spove powder to hepar damnification caused by chemotherapeutics
Animal model:From the week old female mices of ICR 5,5 groups are randomly divided into, every group 6, control is corresponded to respectively
Group, model group and experimental group:3 groups of 1 group of comparative example, 2 groups of comparative example and embodiment.
Experimental group:Chemotherapeutics gives mouse by gavage mode, and specific dosage is:Endoxan 80mg/kg body weight/
It is secondary, busulfan 20mg/kg body weight/time;Ganoderma spove powder in embodiment 3, comparative example 1 and comparative example 2 passes through gavage side
Formula gives mouse, and specific dosage is:100 μ L/ times, concentration is 10mg/mL.
Control group:The chemotherapeutics and ganoderma spove powder of experimental group, other places are replaced with the physiological saline of same volume
Reason mode is consistent with experimental group.
Model group:Chemotherapeutics endoxan and busulfan give mouse by gavage mode, the same experimental group of its consumption, and
With the ganoderma spove powder in the physiological saline alternate embodiment 3, comparative example 1 and comparative example 2 of same volume, and pass through gavage side
Formula gives mouse, and other processing modes are consistent with experimental group.
First culture mouse 2~3 days, observes mouse state, it is in stable condition after, be used as the 1st day of experiment.Then respectively
2nd, 5, give mouse using gavage mode by chemotherapeutics or the physiological saline of same volume within 9 and 12 days, the 19th, 20,21,24 and
Ganoderma spove powder or the physiological saline of same volume in embodiment 3, comparative example 1 and comparative example 2 was used into gavage side in 25 days
Formula gives observing effect after mouse.
Model group is shown it can be seen from accompanying drawing 1, is given using gavage mode after chemotherapeutics, mouse liver has brighter
Aobvious damage;Experimental group shows that the ganoderma spove powder given using gavage mode in embodiment 3 is had to mouse liver damage
It is obviously improved, its liver cell situation is closer to control group, and is given using gavage mode broken in comparative example 1 and comparative example 2
The protecting effect that wall lucidum spore powder is damaged to mouse liver is weaker, and its liver cell situation is closer to model group.
Illustrate that the ganoderma spove powder that the present invention is prepared has well really to hepar damnification caused by chemotherapeutics
Protective effect.
More than, embodiments of the present invention are illustrated.But, the present invention is not limited to above-mentioned embodiment.It is all
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements done etc., should be included in the guarantor of the present invention
Within the scope of shield.