CN117838742A - Preparation method of ganoderma lucidum triterpene crude extract with acetylcholine inhibition activity - Google Patents
Preparation method of ganoderma lucidum triterpene crude extract with acetylcholine inhibition activity Download PDFInfo
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a preparation method of ganoderma lucidum triterpene crude extract with acetylcholinesterase inhibition activity, which comprises the following steps: performing tissue separation on fresh ganoderma lucidum to obtain mother seeds, activating, inoculating the mother seeds into a seed culture medium for culture, and inoculating the mother seeds into a liquid fermentation culture medium which takes corn meal as a carbon source and peptone as a nitrogen source for fermentation culture; vacuum concentrating and evaporating fermentation liquor, adding ethanol solution according to feed-liquid ratio of 25-45%, ultrasonic extracting at 80-100deg.C for 1-2 hr, centrifuging at 4000rpm/min for 5min, collecting supernatant, and drying at 70deg.C to obtain Ganoderma triterpene crude extract. The invention can obviously improve the content and the extraction rate of the active ingredients of the ganoderma lucidum triterpene in the fermentation liquor, and simultaneously, the crude extract of the ganoderma lucidum triterpene has better acetylcholinesterase inhibition activity, and can be used for developing medicaments or health-care foods for preventing and treating or improving neurodegenerative diseases caused by neurotransmitter choline deficiency, such as senile dementia.
Description
Technical Field
The invention relates to a preparation method and efficacy application of a fungus fermentation product, in particular to a preparation method of a ganoderma lucidum triterpene crude extract with acetylcholinesterase inhibition activity.
Background
Senile dementia, which is a cerebral degenerative disorder characterized by progressive neurodegeneration occurring in the elderly and in the early stages of the elderly, has become one of the major diseases that seriously threatens the life health of the elderly following cardiovascular diseases and tumors. Senile dementia has multiple causative factors and complex pathogenesis, single component and single target point are difficult to work, and the multi-angle and multi-strategy attack disease system can overcome the limitation of a plurality of single-target point medicaments. In recent years, the natural extract components with homology of medicine and food have the characteristics of multiple components, multiple targets, synergistic effect, high safety, small toxic and side effects and the like, and have great advantages and development and application prospects in the prevention and treatment of complex pathogenesis and chronic diseases. Ganoderma lucidum is used as the first choice food for preventing and treating tumor, diabetes, hypertension and other chronic diseases by dietotherapy in traditional Chinese medicine from ancient times, and researches report that ganoderma lucidum triterpene has good anti-inflammatory, anti-tumor, liver-protecting, antioxidant and other biological activities and pharmacological effects. AChE, also known as true cholinesterase, is a key enzyme in biological nerve conduction that specifically degrades neurotransmitter-acetylcholine, causing it to hydrolyze into acetic acid and choline, thereby deleting acetylcholine, terminating the excitation of the neurotransmitter to postsynaptic membranes, and causing failure of nerve signaling, thereby causing degeneration of the brain. At present, AChE inhibitors are considered as a relatively effective drug treatment for senile dementia. Therefore, the AChE inhibitor with neuroprotective activity can be searched from ganoderma lucidum, and related health-care food or medicine is developed, which has the advantages of being suitable for long-term administration of patients, small in toxic and side effect, high in safety and the like compared with chemical synthesis inhibitors, and is particularly suitable for long-term administration of senile dementia.
Disclosure of Invention
The invention aims to: the invention aims to provide a preparation method of a ganoderma lucidum triterpene crude extract with acetylcholinergic inhibitory activity, which realizes the short-time obtaining of high-content ganoderma lucidum triterpene, and simultaneously searches for an AChE inhibitor with neuroprotective activity, thereby providing a basis for developing health-care foods or medicines related to senile dementia prevention and treatment.
The technical scheme is as follows: a preparation method of Ganoderma triterpene liquid fermented coarse body with neuroprotective effect comprises the following steps:
(1) Preparation of culture medium
Preparing a slant culture medium and a seed culture medium: corn meal glucose medium: 40g of corn flour, 1000mL of water is added, boiling is carried out, the mixture is kept for 30 minutes, stirring is carried out continuously, filtering is carried out by gauze, and filtrate is taken. Adding 20g of agar, adding glucose, stirring to dissolve completely, adding water to 1000mL, placing into test tube and conical flask, and sterilizing with high pressure steam for 25min.
The strain activation culture medium is a conventional PDA culture medium.
Preparing a fermentation medium: corn flour as carbon source, peptone as nitrogen source, 0.3%, inorganic salt MgSO4 0.05%, KH 2 PO4 is added in an amount of 0.1%, glucose is added and stirred until all the materials are dissolved, water is added to 1000mL, the materials are put into a test tube and a conical flask, and the materials are sterilized in an autoclave for 25min.
(2) Culturing ganoderma lucidum strains: the whole process is operated on a sterile operating table. Selecting a Ganoderma with good shape, cutting off upper epidermis and lower epidermis at the junction of its stipe and pileus, and cutting fungus meat into mung bean size (knife is sharp to prevent damage to surface strain). Soaking the cut strain in alcohol for half an hour to kill the mixed strain. Placing sterilized Ganoderma strain into sterilized slant culture medium, and culturing in incubator (27deg.C without light) for 5 days. The middle part is used for observing the growth vigor of the strain, whether the strain grows into mixed bacteria and whether the strain grows into blank. After five days, a small piece of mycelium is cut out, and the mycelium is put into a culture dish for secondary culture, and is cultivated again for five days to observe the growth condition of the strain, and finally the mother strain is obtained, and the strain number is 2021GL01.
(3) Activating strains: inoculating Ganoderma strain onto PDA plate, and culturing at 27deg.C in dark for 7d.
(4) Preparing seed liquid: under aseptic condition, taking activated Ganoderma strain, inoculating into 500mL conical flask containing 100mL seed solution culture medium, and culturing at 27deg.C and 50-200r/min for 7d.
(5) Liquid fermentation: inoculating the cultured seed culture solution into a fermentation culture medium according to the inoculum size of 5-15%, and culturing for 3-10 days at the temperature of 27 ℃ and at the speed of 50-200r/min after inoculation.
(6) Preparing a ganoderma lucidum triterpene crude body: vacuum concentrating and evaporating the ganoderma lucidum fermentation liquid obtained in the step (5), adding ethanol solution according to the feed liquid ratio of 25-45%, extracting by ultrasonic wave at 80-100 ℃ for 1-2h, centrifuging at 4000rpm/min for 5min, and drying the supernatant at 70 ℃ to obtain the product.
The beneficial effects are that: compared with the traditional technology for obtaining the ganoderma triterpene through the ganoderma lucidum fruiting body, the invention shortens the preparation time and the cost, simultaneously obviously improves the content and the extraction rate of the ganoderma triterpene, and simultaneously has better neuroprotective activity, can develop related health-care foods or medicaments, and has wide application prospect for preventing and treating senile dementia.
Drawings
FIG. 1 is a single factor experimental result of liquid fermentation medium optimization;
FIG. 2 shows the result of a single factor experiment for optimizing the triterpene extraction process
FIG. 3 is a graph showing the change of triterpene extraction rate due to the influence of extraction time and extraction temperature;
FIG. 4 is a contour plot of the effect of extraction time and extraction temperature on triterpene extraction rate;
FIG. 5 is a graph showing the effect of the extraction time on the triterpene extraction rate and the specific concentration of the feed liquid;
FIG. 6 is a contour plot of the effect of extraction time and specific feed concentration on extraction rate;
FIG. 7 is a graph showing the effect of specific concentration of triterpene extract on extraction temperature;
FIG. 8 is a contour plot of the effect of specific feed concentration and extraction temperature on extraction rate;
Detailed Description
1. Preparation of culture medium
Preparing a slant culture medium and a seed culture medium: corn meal glucose medium: 40g of corn flour, 1000mL of water is added, boiled, kept for 30 minutes, stirred continuously, filtered by dense gauze, and the filtrate is taken. Adding 20g of agar, adding glucose, stirring to dissolve completely, adding water to 1000mL, placing into test tube and conical flask, and sterilizing with high pressure steam for 25min.
The strain activation culture medium is a conventional PDA culture medium.
2. Cultivation of ganoderma lucidum strain
The whole process is operated on a sterile operating table. Selecting a Ganoderma with good shape, cutting off upper epidermis and lower epidermis at the junction of its stipe and pileus, and cutting fungus meat into mung bean size (knife is sharp to prevent damage to surface strain). Soaking the cut strain in alcohol for half an hour to kill the mixed strain. Placing sterilized Ganoderma strain into sterilized slant culture medium, and culturing in incubator (27deg.C without light) for 5 days. The middle part is used for observing the growth vigor of the strain, whether the strain grows into mixed bacteria and whether the strain grows into blank. After five days, a small piece of mycelium is cut, and the mycelium is put into a culture dish for secondary culture, and is cultured again for five days to observe the growth condition of the strain, and finally the mother strain is obtained, and the strain number is 2021GL01.
3. Activating the bacterial species
Inoculating Ganoderma strain onto PDA plate, and culturing at 27deg.C in dark for 7d.
4. Preparation of seed liquid
Under aseptic condition, taking activated Ganoderma strain, inoculating into 500mL conical flask containing 100mL seed solution culture medium, and culturing at 27deg.C and 50-200r/min for 7d.
5. Liquid fermentation
Inoculating the cultured seed culture solution into a fermentation culture medium according to the inoculum size of 5-15%, and culturing for 3-10 days at the temperature of 27 ℃ and at the speed of 50-200r/min after inoculation.
6. Glossy ganoderma triterpene content determination
Vacuum concentrating and evaporating the Ganoderma fermentation broth obtained in step 5, adding ethanol solution according to the ratio of 25-45%, ultrasonic extracting at 80-100deg.C for 1-2 hr, centrifuging at 4000rpm/min for 5min, collecting supernatant, and drying at 70deg.C to obtain Ganoderma triterpene crude extract. The detection of Ganoderma triterpene adopts vanillin-glacial acetic acid method. Adding a proper amount of sample solution into a test tube, adding distilled water to a volume of 2.0mL, evaporating to dryness in a water bath at 60 ℃, adding 0.4mL of 5% vanillin-glacial acetic acid and 1mL of perchloric acid which are used for preparation, shaking, standing in the water bath, and standing at room temperature. Absorbance was measured at 546nm, and absolute ethanol was used as a blank.
7. Optimization of liquid fermentation media
7.1 Single factor experiment
7.1.1 determination of carbon Source and amount to be added
Yeast powder with fixed nitrogen source of 0.5% and KH with inorganic salt of 0.2% 2 PO 4 And 0.1% MgSO 4 Natural pH. Corn flour and potato were modified to have a carbon source of 3% and fermented for 5 days to determine the optimal carbon source as corn flour, see table 1.
After the optimal carbon source scheme is determined, the addition amount of the carbon source is changed to be 0%, 1%, 2%, 3% and 4%, the content of the ganoderma lucidum triterpene is measured, and the optimal carbon source addition amount is determined.
According to FIG. 1, it is known that the extraction rate of Ganoderma triterpene is highest when the optimal addition amount of corn flour is 3%.
TABLE 1 influence of carbon sources on mycelium growth and extracellular triterpene production
The "+" is used to qualitatively indicate how many cells are, and the more "+" indicates the more cells are. Table 2 is the same.
7.1.2 determination of Nitrogen Source and addition amount thereof
Glucose with 3% fixed carbon source and KH with 0.2% inorganic salt 2 PO 4 And 0.1% MgSO 4 Natural pH. Changing nitrogen source, dividingThe other is 0.5% peptone, yeast powder and bran, and the fermentation is carried out for 5 days, and the optimal nitrogen source is determined to be peptone after comparison, and the peptone is shown in Table 2.
TABLE 2 influence of Nitrogen Source on mycelium growth and extracellular triterpene production
After determining the optimal nitrogen source scheme, changing the adding amount of the nitrogen source to be 0%, 0.1%, 0.2%, 0.3% and 0.4% respectively, measuring the content of the ganoderma lucidum triterpene, and determining the optimal adding amount of the nitrogen source.
According to FIG. 1, it is found that the extraction rate of Ganoderma lucidum triterpene is highest when the optimum addition amount of peptone is 0.3%.
7.1.3 determination of the amount of inorganic salt to be added
Yeast powder with fixed nitrogen source of 0.5% and glucose with carbon source of 3% to change inorganic salt KH 2 PO 4 The addition amounts of (a) are 0%, 0.05%, 0.1%, 0.15%, 0.2%, respectively, and the inorganic salt MgSO 4 The addition amount of (2) was 0.05%, and KH was measured 2 PO 4 Effect on triterpene extraction rate, KH was determined 2 PO 4 Optimum addition ratio.
Yeast powder with fixed nitrogen source of 0.5% and glucose with carbon source of 3% and inorganic salt KH 2 PO 4 The addition amount of (2) was 0.1%, and the inorganic salt was MgSO 4 The amounts added were 0%, 0.025%, 0.05%, 0.075%, 0.1%, respectively, and MgSO was measured 4 Influence on the extraction yield of triterpenes, mgSO was determined 4 Optimum addition ratio.
From FIG. 1, KH is known 2 PO 4 And MgSO 4 The optimal addition amounts of the ganoderma lucidum triterpene are respectively 0.15 percent and 0.075 percent, and the extraction rate of the ganoderma lucidum triterpene is the highest.
7.2 orthogonal Experimental design
According to the single factor experimental result, for carbon source, nitrogen source and inorganic salt KH 2 PO 4 、MgSO 4 Four factors influence the triterpene content of ganoderma lucidum orthogonal experiments were designed and are shown in Table 3.
From Table 4, it is determined that the factors are specific to Ganoderma lucidumThe effect of triterpene extraction rate, and R value comparison shows that the effect sequence of triterpene on ganoderma lucidum is A>D>C>B, the most obvious effect is the addition amount of the corn flour as a carbon source, and the lowest effect is the addition amount of the peptone. Comparing k values, the optimal extraction rate combination of the ganoderma triterpenes is A 2 B 2 C 2 D 2 Namely, the corn meal addition amount is 3%, the peptone addition amount is 0.3%, and the inorganic salt MgSO 4 Additive amount and KH 2 PO 4 The addition amounts are 0.075% and 0.15% respectively, and the culture medium prepared by the ratio can obtain more ganoderma lucidum triterpenes. Experiments prove that the content of the ganoderma lucidum triterpene can reach 0.656 percent
TABLE 3 culture medium optimization orthogonal experiment design table
TABLE 4 results of culture medium optimization orthogonal experiments
8. Optimization of ganoderma lucidum triterpene extraction process
8.1 Single factor experiment
8.1.1 Effect of specific feed concentration on triterpene extraction yield
Controlling the time to be 2 hours, measuring the triterpene content by precisely measuring 0.4mL, 0.5mL, 0.6mL, 0.7mL and 0.8mL of bacterial liquid at the temperature of 80 ℃. As can be seen from FIG. 2, the optimal specific concentration of the liquid for extracting the ganoderma lucidum triterpenes is 30%.
8.1.2 Effect of extraction time on triterpene extraction yield
Controlling the specific concentration of the feed liquid to be 30%, extracting at 80 ℃ and heating in water bath for 0.5h, 1h, 1.5h, 2h and 2.5h respectively, and determining the triterpene content. As can be seen from FIG. 2, the optimal time for extracting Ganoderma triterpene is 2h.
8.1.3 influence of extraction temperature on triterpene extraction yield
Controlling the specific concentration of the feed liquid to be 30%, extracting for 2h, heating in water bath at 60deg.C, 70deg.C, 80deg.C, 90deg.C and 100deg.C, and measuring triterpene content. As can be seen from FIG. 2, the optimal temperature for extracting Ganoderma triterpene is 80deg.C.
8.2 response surface experiments with Ganoderma triterpene
According to the single factor experimental result, a response surface experimental scheme is established by design expert software, and then a response surface curve is drawn according to data obtained by the given scheme experiment, so that the technological parameter with the highest triterpene extraction rate is determined.
TABLE 5 regression equation analysis of the triterpene extraction yield results
As can be seen from fig. 3 and fig. 4, the contour plot of the extraction temperature and the extraction time shows a curve instead of a straight line, which illustrates that the effect of the interaction of the extraction temperature and the extraction time on the extraction rate of the ganoderma triterpene is remarkable. The extraction rate increases with increasing extraction temperature and extraction time, and reaches peak value when the temperature reaches 80deg.C and the time is 2 hr.
As can be seen from fig. 5 and fig. 6, the contour plot of the specific concentration of the feed liquid and the extraction temperature shows a curve instead of a straight line, which illustrates that the effect of the interaction of the specific concentration of the feed liquid and the extraction temperature on the extraction rate of the ganoderma triterpenes is remarkable. The extraction rate is increased along with the increase of the extraction temperature and the specific concentration of the feed liquid, and the extraction rate reaches a peak value when the temperature reaches 80 ℃ and the specific concentration of the feed liquid is 30%.
From fig. 7 and fig. 8, it can be seen that the contour plot of the specific concentration of the feed liquid and the extraction time shows a curve instead of a straight line, which illustrates that the effect of the interaction of the specific concentration of the feed liquid and the extraction time on the extraction rate of the ganoderma lucidum triterpenes is remarkable. When the specific concentration of the feed liquid is 30%, the extraction rate reaches the peak value when the time is 2 hours. The triterpene extraction efficiency is correspondingly highest at the moment.
And (3) predicting an experimental model: the optimal extraction process of the ganoderma triterpene is that the specific concentration of the feed liquid is 30 percent, the extraction temperature is 80 ℃, and the highest extraction rate at the time is 0.675 percent when the extraction time is 2 hours. And (3 times of repetition) carrying out verification experiments on the prediction results, wherein the yield of the ganoderma lucidum triterpene is 0.674%, and the model is very close to the prediction value of the model, so that the model has feasibility for optimizing the triterpene extraction process.
9. Measurement of acetylcholinesterase inhibitory Activity
Acetylcholinesterase (AChE) inhibition activity was assayed using the modified Ellman method. The principle is as follows: the substrate analogue of the thioacetylcholine iodide is catalyzed by AChE to generate acetic acid and thiocholine, and the two compounds react with a color reagent DTNB to generate yellow substances with specific light absorption value at 405 nm. And mixing the compound to be tested with an acetylcholinesterase solution, wherein if the degradation amount of the thiocholine iodide is reduced, and meanwhile, the yellow compound generated with the color developing agent is reduced, the specific light absorption value at 405nm is reduced, the compound to be tested has the inhibition activity on the acetylcholinesterase, and otherwise, the compound to be tested has no inhibition effect. The experimental method is as follows:
(1) Na is mixed with 2 HPO 4 (94.7 mL, 0.1M) and NaH2PO4 (5.3 mL, 0.1M) were mixed to prepare a phosphate buffer (pH 8.0). DMSO was diluted to 2% with phosphate buffer, AChE was diluted to 0.1U/mL working solution, and DTNB and thiocholine iodide were prepared as 6.25mM working solutions, respectively. The crude extract of ganoderma triterpene is diluted with 2% DMSO to 1mM working solution. The positive control was tacrine (final concentration 0.333. Mu.M) and the negative control (NC group) was 2% DMSO solvent.
(2) To the reaction system, 110. Mu.L of phosphate buffer (pH 8.0) -10. Mu.L of test compound (1 mM) -AChE enzyme (0.1U/mL) 40. Mu.L were sequentially added, incubated in a microplate reader for 20min, measured for 2 background values at 405nm, and then 40. Mu.L of an equal volume of a mixture of DTNB (6.25 mM) and thioacetylcholine iodide (6.25 mM) was added, each sample was repeated 3 times.
(3) After adding the color developing agent and the substrate, detecting the light absorption value of 405nm every 30 seconds;
(4) Sample absorbance at NC (average) of about 1 was selected, and the average of compound absorbance (compound measured value-background value) was calculated according to the formula: (NC-mean absorbance of compound)/NC 100%, the compound AChE inhibition was calculated.
The experimental determination results are shown in Table 6, and the crude extract of ganoderma lucidum triterpene has 53.7 percent of acetylcholinesterase inhibition activity and has better development prospect.
TABLE 6 acetylcholinesterase inhibitory Activity of crude extract of Ganoderma lucidum triterpene (%)
Note that: TA, tacrine reaction final concentration 0.333. Mu.M, compound reaction final concentration 100. Mu.M;
"+" indicates that the inhibition rate is between 10% and 20%; "++" indicates that the inhibition rate is between 20% and 60%, and it is recommended not to make IC 50 ；
"+". ++'s representation of inhibition rate>60%, propose to further test IC 50 。
Claims (9)
1. A preparation method of ganoderma lucidum triterpene crude extract with acetylcholinesterase inhibition activity is characterized in that: the method comprises the following steps: performing tissue separation on fresh ganoderma lucidum to obtain mother seeds, activating, inoculating the mother seeds into a seed culture medium for culture, and inoculating the mother seeds into a liquid fermentation culture medium which takes corn meal as a carbon source and peptone as a nitrogen source for fermentation culture; vacuum concentrating and evaporating fermentation liquor, adding ethanol solution according to the ratio of 25-45%, ultrasonic extracting at 80-100deg.C for 1-2 hr, centrifuging at 4000rpm/min for 5min, collecting supernatant, and drying at 70deg.C to obtain the final product.
2. The method according to claim 1, characterized in that: preparing a slant culture medium and a seed culture medium: 40g of corn flour, 1000mL of water is added, boiling is carried out, the mixture is kept for 30 minutes, stirring is carried out continuously, filtering is carried out by gauze, and filtrate is taken. Adding 20g of agar, adding glucose, stirring to dissolve completely, adding water to 1000mL, placing into test tube and conical flask, and sterilizing with high pressure steam for 25min.
3. The method according to claim 1, characterized in that: culturing ganoderma lucidum strains: the whole process is operated on a sterile operating table. Selecting a Ganoderma with good shape, cutting off upper epidermis and lower epidermis at the junction of its stipe and pileus, and cutting fungus meat into mung bean size (knife is sharp to prevent damage to surface strain). Soaking the cut strain in alcohol for half an hour to kill the mixed strain. Placing sterilized Ganoderma strain into sterilized slant culture medium, and culturing in incubator (27deg.C without light) for 5 days. After five days, a small piece of mycelium is cut out, and the mycelium is put into a culture dish for secondary culture, and is cultivated again for five days to observe the growth condition of the strain, and finally the mother strain is obtained, and the strain number is 2021GL01.
4. The method according to claim 1, characterized in that: activating strains: the ganoderma lucidum strain is transferred onto a PDA plate and cultured for 7 days under the dark condition at the temperature of 27 ℃.
5. The method according to claim 1, characterized in that: preparing seed liquid: under aseptic condition, taking activated Ganoderma strain, inoculating into 500mL conical flask containing 100mL seed solution culture medium, and culturing at 27deg.C and 50-200r/min for 7d.
6. The method according to claim 1, characterized in that: the formula of the liquid fermentation medium comprises the following components: corn meal addition amount is 3%, peptone addition amount is 0.3%, mgSO 4 With KH 2 PO 4 The addition amounts are 0.075% and 0.15%, respectively, glucose is added and stirred until all are dissolved, and water is added to 1000mL.
7. The method according to claim 1, characterized in that: liquid fermentation: inoculating the cultured seed culture solution into a fermentation culture medium according to the inoculum size of 5-15%, and culturing for 3-10 days at the temperature of 27 ℃ and at the speed of 50-200r/min after inoculation.
8. The method according to claim 1, characterized in that: vacuum concentrating and evaporating to dryness, adding ethanol solution according to a feed liquid ratio of 30%, ultrasonic extracting at 80deg.C for 2 hr, centrifuging at 4000rpm/min for 5min, collecting supernatant, and drying at 70deg.C to obtain crude extract of Ganoderma triterpene with a content of 0.675%.
9. The method according to claim 1, characterized in that: the ganoderma triterpene crude extract has good acetylcholinesterase inhibiting activity, and can be used for preventing and treating or improving neurodegenerative diseases caused by neurotransmitter choline deficiency, such as senile dementia, or health food.
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