CN109825540B - Preparation method and application of inonotus obliquus non-saccharide extract - Google Patents
Preparation method and application of inonotus obliquus non-saccharide extract Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a non-saccharide extract of inonotus obliquus. The preparation method is simple and short in period, the main component of the prepared non-saccharide extract is the s-triazine derivative, the non-saccharide extract has excellent bacteriostatic effect on bacteria and fungi, belongs to a biological preparation, has little pollution to the environment, can be applied to preparation of environment-friendly bactericides and bacteriostatic drugs, has a good application prospect, fully develops the application value of the inonotus obliquus, and improves the utilization rate of the medicinal fungi.
Description
Technical Field
The invention relates to a medicinal fungus extract, in particular to a preparation method and application of a non-saccharide extract of inonotus obliquus.
Background
Currently, Inonotus obliquus, also known as Chaba, is a high-value medicinal fungus belonging to the Basidiomycotina (Basidiomycotina), Hymenomycetes (Hymenomycetes), Aphyllophorales (Aphyllophorales), and Hymenochaetaceae (Hymenohaeaeeae). The cold-loving wood rot fungi are distributed in cold zone, and are grown under bark of living standing trees such as white birch, silver birch, elm, and alder or on withered trunk of felled trees, and can cause white rot of white birch, silver poplar, elm, and alder. The method is mainly distributed in regions with north latitude of 45-50 degrees in northern hemisphere, such as North America, Finland, Poland, Russia, Heilongjiang, Changbai mountain region of Jilin province, and North Hai Dai of Japan.
In foreign countries, the application of inonotus obliquus appears in russia and Polish early, and is used for treating various difficult and complicated diseases, such as cancer, diabetes and the like, at present, numerous reports on the extraction and application research of chemical components of inonotus obliquus exist, the research mainly focuses on the research of antitumor, blood sugar reducing and blood fat reducing of polysaccharide substances of inonotus obliquus, and because the active components in the inonotus obliquus are various and have high application value, the research in the field does not fully utilize and develop the value of non-saccharide active components of the inonotus obliquus, and a corresponding extraction preparation method of the non-saccharide active components is also lacked.
Therefore, the prior art has yet to be developed.
Disclosure of Invention
In order to solve the technical problems, the embodiment of the invention provides a preparation method and application of a non-saccharide extract of inonotus obliquus.
The invention provides the following technical scheme:
the invention provides a preparation method of a non-saccharide extract of inonotus obliquus, which comprises the following steps:
s1, inoculating inonotus obliquus thalli into a liquid culture medium, performing shake culture at 22-27 ℃ for 1-2d to obtain a seed solution, inoculating the seed solution into the liquid culture medium according to the inoculum size of 5-10%, performing shake culture for 5-7d, and obtaining a culture solution at the rotating speed of a shaking table of 130-;
s2, centrifuging the culture solution, and respectively storing the separated thalli and fermentation liquor in an environment of 0-8 ℃ for cold induction for 20-40 days; the step is to induce the inonotus obliquus to express specific active substances under adverse environment.
S3, mixing the thalli subjected to cold induction treatment with fermentation liquor, performing vacuum freeze drying, and grinding the freeze-dried mixture into powder to obtain freeze-dried powder;
s4, performing chloroform extraction on the freeze-dried powder to obtain a primary extracting solution, and performing filter paper filtration on the primary extracting solution to remove impurities;
s5, dialyzing the filtered primary extracting solution, and removing macromolecules above 500Da to obtain a secondary extracting solution; this step removes polysaccharides or other macromolecular interferents from the primary extract in large quantities by dialysis.
And S6, carrying out HPLC chromatographic purification, and separating to obtain a refined extract. The refined extract is non-saccharide extract of Inonotus obliquus.
In the preparation method, in step S1, the adopted liquid culture medium comprises the following components in percentage by mass: birch wood powder 5%, glucose 10%, peptone 1.5%, KH 0.2%2PO4、0.2％MgSO4And the balance of water.
In the preparation method, in step S3, the vacuum freeze-drying conditions are as follows: and (3) freezing the mixture of the thalli and the fermentation liquor at-20 ℃ for 2-3h, freezing at-80 ℃ for 4-8h, taking out the mixture, and drying in a vacuum freeze dryer until a freeze-dried mixture is obtained.
In the preparation method, the chloroform extraction method comprises the following steps: dissolving and extracting the lyophilized powder by Soxhlet extraction with chloroform as solvent, soaking for 10-12h, and refluxing for 6-8h to obtain primary extractive solution.
In the preparation method, in the step of S6, the adopted chromatographic column is a Hypersil GOLD C18 column, and the column temperature is 28-32 ℃.
In the preparation method, in the step of S6, an acetonitrile-water elution system is adopted, and the volume ratio of water to acetonitrile in the eluent is as follows: 1: (6-10), the flow rate of the secondary extract was 0.2 ml/min.
The invention also provides a non-saccharide extract of inonotus obliquus prepared by the preparation method.
Mass spectrum detection shows that the main component of the non-saccharide extract is the s-triazine derivative, which is found by extracting the s-triazine compound in the inonotus obliquus for the first time, and the extract has excellent bacteriostatic effect and wide application prospect.
The invention also provides application of the non-saccharide extract of the inonotus obliquus in preparing bactericides and bacteriostatic medicaments.
The invention has the following beneficial effects:
the invention provides a novel preparation method of a non-saccharide extract in inonotus obliquus, which is designed aiming at the property of the non-saccharide extract, is different from the complicated steps of the prior inonotus obliquus extract, and has the advantages of simple and easy operation, less types of adopted extraction solvents, short extraction step time consumption and good extraction effect; the main component of the non-saccharide extract obtained by the method is the s-triazine derivative, which is extracted from microorganisms (inonotus obliquus) for the first time in the field, and the s-triazine compound is mainly obtained by a chemical synthesis mode. And the non-saccharide extract is found to have an excellent bacteriostatic function and bacteriostatic effects on pathogenic bacteria escherichia coli, staphylococcus aureus, candida albicans and the like, can be applied to preparation of bactericides and bacteriostatic drugs, has a good application prospect, fully develops the application value of the inonotus obliquus, and improves the utilization rate of the medicinal fungus.
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FIG. 1 is a high performance liquid chromatography chromatogram of a chloroform refined extract of Inonotus obliquus;
FIG. 2 is a mass spectrum of chloroform extract of Inonotus obliquus.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1 preparation of non-saccharide extract of Inonotus obliquus
1. Culture and fermentation of inonotus obliquus
The formula of the culture medium is as follows: birch wood powder 5%, glucose 10%, peptone 1.5%, KH 0.2%2PO4、0.2％MgSO4And the balance of water. The prepared liquid medium was autoclaved.
Inoculating Fuscoporia obliqua mycelium into 50ml liquid culture medium, shake culturing at 25 deg.C and 150rpm/min for 1-2d to obtain seed solution, inoculating the seed solution into 300ml culture medium according to inoculum size of 5-10%, shake culturing at 25 deg.C and 150rpm/min for 5-7d to obtain culture solution.
2. Cold induction of Inonotus obliquus
Placing the culture solution of Inonotus obliquus in refrigerator at 4 deg.C for cold induction for 20-40 d. Preferably, the cold induction time is 30 days. This step helps the Inonotus obliquus to induce the expression of the target product under extreme environment.
3. Preparation of lyophilized powder
And (3) carrying out vacuum freeze drying on the fermentation liquor (including mycelium pellets and the fermentation liquor) subjected to the cold induction treatment, and grinding the freeze-dried mixture into powder to obtain a primary treatment product. In order to fully break the wall, can add 150U/ml of cell lysate into the mixture of thallus and fermentation broth for preliminary enzymolysis, and vacuum freeze-dry after 20min of treatment. In other embodiments, the mixed solution of the cells and the fermentation solution may be directly subjected to freeze-drying treatment.
The method for vacuum freeze drying comprises the following steps: freezing the mixed solution of the thalli and the fermentation liquor at-20 ℃ for 2h, freezing at-80 ℃ for 8h, taking out, and drying in a vacuum freeze dryer until a freeze-dried mixture is obtained; grinding the freeze-dried mixture into powder to obtain the freeze-dried powder.
4. Extraction of active ingredients
Dissolving and extracting the freeze-dried powder by using chloroform as a solvent by using a Soxhlet extraction method, soaking for 12h, refluxing for 6h to obtain a primary extracting solution, and filtering by using filter paper to remove impurities.
5. Dialysis treatment of primary extract
Taking a section of dialysis bag, intercepting molecular weight of 500Da, tying one end with cotton rope, adding 5ml of filtered primary extract from the other end, sealing, suspending the dialysis bag in a beaker filled with distilled water, and stirring with a magnetic stirrer to promote solution exchange until dialysis balance. And (3) removing polysaccharide molecules in the primary extracting solution after the dialysis treatment to obtain a secondary extracting solution.
6. Purification treatment
The secondary extract was purified by HPLC using a Hypersil GOLD C18 column (2.1X 100mm,5 μm; Thermo Fisher scientific, Waltham, USA) at a column temperature of 30 ℃ in a two-dimensional nano-liquid system at a flow rate of 0.2 ml/min. In the elution process, water is adopted as eluent A, an acetonitrile elution system is adopted as eluent B, and the volume ratio of the two eluents is as follows: under the condition of elution at a ratio of 1:8, the chromatographic retention time is 1.3min, and a purified product, namely a chloroform refined extract of the inonotus obliquus is collected, and the chromatogram is shown in figure 1.
7. Mass spectrometric analysis
Performing mass spectrometry on the chloroform refined extract, performing positive ion and negative ion full scanning in a nuclear-to-mass ratio range of 50-1500m/z by electrospray mass spectrometry, and taking nitrogen as atomizing gas at the flow rate of 6L/min; the temperature is 180 ℃; the pressure was 1.0Bar and the data acquisition and processing used LC/MS data analysis software supplied with the instrument (version 4.0). The nuclear to proton ratio data corresponding to a particular elemental composition is calculated using formula prediction software provided with the instrument. Requiring an error between the measured nuclear-to-cytoplasmic ratio and a standard nuclear-to-cytoplasmic ratio for the substanceNot exceeding 5 ppm. The mass spectrum result is shown in FIG. 2, and the analysis of the mass spectrum shows that the maximum peak is the molecular ion peak, the M/Z value is 203, and the molecular formula of the main active ingredient is C9H7N4O2Molecular weight 203, combined with analysis of other fragment ion peaks, ultimately yielding: the compound C9H7N4O2The compound contains a 1, 3, 5-s-triazine ring, and a 4-nitro group and a benzene ring are connected on the ring, so that the compound is supposed to be an s-triazine derivative. The s-triazine derivative is separated from the inonotus obliquus for the first time.
In parallel with the above experiment, the following control groups were also set: the extraction materials and the steps are the same, only the cold induction treatment step is omitted, and the chloroform refined extract of the inonotus obliquus extracted from the control group is detected to find that: compound C is not found in the refined extract9H7N4O2This explains that: without cold induction treatment, the inonotus obliquus does not express compound C9H7N4O2。
Example 2 bacteriostasis test of non-saccharide extract of Inonotus obliquus
1. Culturing indicator bacteria:
respectively inoculating escherichia coli, staphylococcus aureus, salmonella typhimurium and shigella dysenteriae to a beef extract peptone medium, and performing shake culture at 37 ℃; inoculating candida albicans into a PDA culture medium, and performing shake culture at 28 ℃ to respectively obtain bacterial solutions.
2. Sample set setting:
experimental and control groups were set up separately as follows, with at least 3 replicates per group:
experimental group 1: preparing a chloroform refined extract of inonotus obliquus as a sample according to the complete experimental steps; experimental group 2: carrying out fermentation, cold induction treatment and low-temperature concentration on fermentation supernatant obtained by filtering and removing inonotus obliquus thalli to obtain a sample; experimental group 3: basically the same procedure as in experimental group 1, only the cold induction treatment step was omitted and the extract was collected as a sample. Positive control group: streptomycin was used as a sample. The samples of each experimental group were dissolved in sterile distilled water to prepare 10mg/ml solution, and filtered through a 0.22 μm sterile filter membrane.
3. Determination of bacteriostatic Activity
Adding 200uL of the bacterial suspension on a solid culture medium plate, uniformly coating to prepare bacteria-containing plates, respectively soaking sterilized filter paper sheets in sterile solution of the extract for 30min in two experimental groups, clamping the filter paper sheets by using sterilization tweezers, and sticking the filter paper sheets on each bacteria-containing plate; in the positive control group, the bacterial-containing plates are treated by using filter paper sheets soaked in streptomycin for 30min, each group is repeated for three times, the plates are placed in a constant-temperature incubator and are placed in an inverted manner for 24h, then the plates are taken out, the diameter of the antibacterial zone of the plates of the experimental group and the plates of the control group are measured by using a cross method respectively, the numerical average value is obtained, and the test results are shown in the following table 1.
The calculation method of the bacteriostatic rate comprises the following steps:
the bacteriostasis rate is [ (the diameter of the bacteriostasis zone of the test reagent-the diameter of the filter paper sheet)/(the diameter of the bacteriostasis zone of the positive reagent-the diameter of the filter paper sheet) ] × 100%
TABLE 1 results of the bacteriostatic ratio test
From the bacteriostasis test results, the inonotus obliquus chloroform refined extract of the experimental group 1 has excellent bacteriostasis activity on bacteria (escherichia coli, staphylococcus aureus and shigella dysenteriae) and fungi (candida albicans); the experiment group 3 without cold induction has far inferior bacteriostatic test result to the experiment group 1, which shows that the bacteriostatic active component of the chloroform refined extract is expressed under the cold induction condition, and the cold induction is an important experiment step; comparing the experimental group 2 with the experimental group 1, the method for extracting the chloroform refined extract has the advantages of good extraction effect, high extraction rate of bacteriostatic components and high bacteriostatic activity.
It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.
Claims (6)
1. A preparation method of a non-saccharide extract of inonotus obliquus is characterized by comprising the following steps:
s1, inoculating inonotus obliquus thalli into a liquid culture medium, performing shake culture at 22-27 ℃ for 1-2d to obtain a seed solution, inoculating the seed solution into the liquid culture medium according to the inoculum size of 5-10%, performing shake culture for 5-7d, and obtaining a culture solution at the rotating speed of a shaking table of 130-;
s2, centrifuging the culture solution, and respectively storing the separated thalli and fermentation liquor in an environment of 0-8 ℃ for cold induction for 20-40 days;
s3, mixing the thalli subjected to cold induction treatment with fermentation liquor, performing vacuum freeze drying, and grinding the freeze-dried mixture into powder to obtain freeze-dried powder;
s4, performing chloroform extraction on the freeze-dried powder to obtain a primary extracting solution, and filtering the primary extracting solution by using filter paper to remove impurities;
s5, dialyzing the filtered primary extracting solution, and removing macromolecules above 500Da to obtain a secondary extracting solution;
s6, carrying out HPLC chromatographic purification on the secondary extracting solution, and separating to obtain a refined extract, namely a non-saccharide extract.
2. The method according to claim 1, wherein in step S1, the liquid medium used comprises the following components in percentage by mass: birch wood powder 5%, glucose 10%, peptone 1.5%, KH 0.2%2PO4、0.2％MgSO4And the balance of water.
3. The method according to claim 1, wherein in step S3, the vacuum freeze-drying conditions are as follows: and (3) freezing the mixture of the thalli and the fermentation liquor at-20 ℃ for 2-3h, freezing at-80 ℃ for 4-8h, taking out the mixture, and freeze-drying in a vacuum freeze dryer until a freeze-dried mixture is obtained.
4. The method according to claim 1, wherein the chloroform extraction method comprises: dissolving and extracting the lyophilized powder by Soxhlet extraction with chloroform as solvent, soaking for 10-12h, and refluxing for 6-8h to obtain primary extractive solution.
5. The method according to claim 1, wherein the step of S6, the chromatographic column used is Hypersil GOLD C18 column, and the column temperature is 28-32 ℃.
6. The preparation method according to claim 5, wherein in the step S6, an acetonitrile-water elution system is adopted, and the volume ratio of water to acetonitrile is as follows: 1: (6-10), the flow rate of the secondary extract was 0.2 ml/min.
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