CN103798293B - Preparation and applications of salvia miltiorrhiza endophytic fungi hypha water-soluble extract and parts - Google Patents
Preparation and applications of salvia miltiorrhiza endophytic fungi hypha water-soluble extract and parts Download PDFInfo
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Abstract
The invention discloses a salvia miltiorrhiza endophytic fungi hypha water-soluble extract (WSE). The salvia miltiorrhiza endophytic fungi hypha water-soluble extract (WSE) is prepared by following steps: salvia miltiorrhiza endophytic fungi hyphostoma obtained via 9 to 14 days of fermentation is collected; distilled water is added for resuspending; an obtained suspension liquid is sterilized for 30 to 60min at a temperature of 110 to 130 DEG C, and then is subjected to vacuum pumping filtration so as to obtain a filtrate. The invention also discloses polysaccharide protein fractions (PPF) of the salvia miltiorrhiza endophytic fungi hypha water-soluble extract, and polysaccharide fractions (PSF) of the salvia miltiorrhiza endophytic fungi hypha water-soluble extract. The invention further discloses applications of SWE, PPF, and PSF in promoting growth of tissue culture object salvia miltiorrhiza hairy roots of host salvia miltiorrhiza, and increasing tanshinone content of salvia miltiorrhiza hairy roots.
Description
Technical field
The present invention relates to a kind of water-soluble extractive of red sage root Endophytic Fungal Hyphae and GL-PP position thereof and polysaccharide position, in addition, the invention still further relates to the purposes at this red sage root Endophytic Fungal Hyphae water-soluble extractive and GL-PP position and polysaccharide position, grow for promoting Hairy Root Cultures of Salvia miltiorrhiza and improve tanshinone component content.
Background technology
The red sage root (Salvia miltiorrhiza Bge.) is Lamiaceae Salvia platymiscium, dry root and rhizome is of the same name is used as medicine for it, begin to be loaded in Shennong's Herbal, being classified as top grade, is that China commonly uses traditional Chinese medicine, bitter, cold nature, the thoughts of returning home, Liver Channel, have stasis-dispelling and pain-killing, activating blood to promote menstruation, clear away heart-fire the effects such as relieving restlessness.Clinically being widely used in cardio-cerebrovascular disorder, is one of focus kind of domestic and international modernization traditional Chinese medicine research.Modern study shows, the chemical composition of the red sage root is mainly divided into two large classes: one is take danshensu as water soluble ingredient---the phenolic acid compound of basic structure, comprises protocatechualdehyde, danshensu, caffeic acid, Rosmarinic acid, danshinolic acid etc.; Two is lipid soluble ingredients---diterpene-kind compound, comprises salvia miltiorrhiza bge I, tanshinone IIA, II B, Cryptotanshinone I, dihydrotanshinone Ⅰ etc.Clinical research confirmation, the number of chemical composition that the red sage root contains has biologically active widely, and its traditional pharmacology is to expand blood vessel, promoting blood circulation and removing blood stasis, and it is main for improving microcirculation.Modern pharmacological research shows, the red sage root still has antitumor, anti-inflammation, antiandrogen, suppresses hyperplasia of sebacous glands, anti-cirrhosis, anti-fibrosis, promotes the multiple pharmacologically active such as tissue repair and regeneration.The market demand of the red sage root is large, and wild resource reduces day by day, and cultivar degenerates serious.There is the impact of introducing a fine variety the objective factors such as difficulty and planting environment restriction in artificial introducing and planting, the content of active secondary metabolism composition is unstable, and quality of medicinal material can not be guaranteed.
Utilize animal nutrition to obtain red sage root active chemical and in succession become study hotspot, Hairy Root Cultures of Salvia miltiorrhiza culture technique is exactly one of them.It utilizes agrobacterium rhizogenes Agrobacterium rhizogenes to infect host injury cell and produces and transforms adventive root in a large number, also known as hair shape root.Compared with traditional cell culture technology, hair shape root have growth rapidly, the feature such as stabilization characteristics of genetics and Hormone autotrophy.Current Hairy Root Cultures of Salvia miltiorrhiza culture technique is quite ripe, but active constituent content is lower greatly constrains a mao application for shape root.Adopt biology and abiotic elicitor to stimulate, effectively can promote the growth of Hairy Root Cultures of Salvia miltiorrhiza and improve the content of secondary metabolite in Hairy Root Cultures of Salvia miltiorrhiza.
Summary of the invention
According to embodiments of the invention, wish to utilize endophytic fungus resources of plants, biology and abiotic elicitor is adopted to stimulate, effectively can promote the growth of Hairy Root Cultures of Salvia miltiorrhiza and improve the content of secondary metabolite in Hairy Root Cultures of Salvia miltiorrhiza, and a kind of red sage root Endophytic Fungal Hyphae water-soluble extractive (WSE) is provided, red sage root Endophytic Fungal Hyphae GL-PP position (PPF) and red sage root Endophytic Fungal Hyphae polysaccharide position (PSF), and then red sage root Endophytic Fungal Hyphae water-soluble extractive (WSE) is provided, red sage root Endophytic Fungal Hyphae GL-PP position (PPF), the application of tanshinone component content is improved at red sage root Endophytic Fungal Hyphae polysaccharide position (PSF) in Hairy Root Cultures of Salvia miltiorrhiza, for the exploitation of novel elicitor and the correlation between discussion endogenetic fungus and host provide foundation.
Disclosed in the red sage root endogenetic fungus that the present invention relates to and Chinese patent CN102676392A, red sage root endogenetic fungus is identical, its called after Trichoderma atroviride Trichoderma atroviride, bacterial strain code name is D16, and it is CGMCCNo.4712 at the preservation registration number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
1. the preparation of red sage root Endophytic Fungal Hyphae water-soluble extractive (WSE).
Collect the red sage root endophytic fungal hypha of fermented and cultured 9-14 days, it is resuspended to add appropriate distilled water, pressure cooker 110-130 DEG C of sterilizing 30-60min.Treat that nature cools, the filtrate obtained after decompress filter, be red sage root endogenetic fungus water-soluble extractive.Sulfuric acid-phynol method is adopted to measure total sugar concentration.
2. the preparation of red sage root Endophytic Fungal Hyphae water-soluble extractive GL-PP position (PPF).
Reduced pressure concentration red sage root endogenetic fungus water-soluble extractive WSE is to proper volume, add the ethanol 2-8 DEG C of standing 24-72 hour of 4-7 times of volume 70-95%, within 10-15 minute, white precipitate is obtained with the rotating speed centrifugation of 2000-10000 rev/min, collecting precipitation after absolute ethyl alcohol washes 3-5 time, vacuum cooling drying gained white powder is red sage root Endophytic Fungal Hyphae water-soluble extractive GL-PP position PPF.
3. the preparation of red sage root Endophytic Fungal Hyphae water-soluble extractive polysaccharide position (PSF).
Red sage root Endophytic Fungal Hyphae water-soluble extractive GL-PP position PPF is dissolved in appropriate distilled water again, adopt Sevag method except deproteinized, add the ethanol 2-8 DEG C of standing 24-72 hour of the 70-95% of 4-7 times of volume, within 10-15 minute, white precipitate is obtained, collecting precipitation after absolute ethyl alcohol washes 3-5 time with the rotating speed centrifugation of 2000-10000 rev/min.The appropriate distilled water of precipitation is dissolved, room temperature 24-72h(molecular cut off of dialysing in distilled water is 1000-10000Da), then obtain supernatant with the rotating speed centrifugation of 2000-10000 rev/min, vacuum cooling drying gained white powder is red sage root Endophytic Fungal Hyphae water-soluble extractive polysaccharide position PSF.
4. Hairy Root Cultures of Salvia miltiorrhiza suspension cultural characteristic.
Medium: 1/2B5 medium (NaH
2pO
4h
2o:75mg/L, CaCl
2: 56.62mg/L, KNO
3: 1250mg/L, (NH
4)
2sO
4: 67mg/L, MgSO
4: 61.05mg/L, disodium ethylene diamine tetraacetate: 18.65mg/L, FeSO
47H
2o:13.9mg/L, MnSO
42H
2o:5.0mg/L, ZnSO
47H
2o:1.0mg/L, boric acid: 1.5mg/L, Na
2moSO
4: 0.125mg/L, CuSO
45H
2o:0.0125mg/L, CoCl
25H
2o:0.0125mg/L, KI:0.375mg/L, vitamin B1: 5.0mg/L, vitamin B5: 0.5mg/L, nicotinic acid: 0.5mg/L, inositol: 50mg/L, sucrose: 20000mg/L, PH(25 DEG C): 5.5), after 15 minutes, cooling is stand-by naturally in 121 DEG C of sterilizings.
Cultivation temperature: 25 ± 2 DEG C.
Incubation time: 20-50 days are not etc.
Shaking speed: 180rpm.
Cultural characteristic: Hairy Root Cultures of Salvia miltiorrhiza is polymerized to group and grows to surrounding.
5. red sage root Endophytic Fungal Hyphae water-soluble extractive (WSE) is on Hairy Root Cultures of Salvia miltiorrhiza growth and the biosynthetic impact of tanshinone.
At Hairy Root Cultures of Salvia miltiorrhiza suspension cultivation 16-24 days, add the red sage root Endophytic Fungal Hyphae water-soluble extractive WSE that total sugar concentration is 30-400mg/L, continue to suspend and cultivate 12-30 days, hair shape root biomass improves 30%-80%, the content of dihydrotanshinone Ⅰ improves 15-40 doubly, the content of salvia miltiorrhiza bge I improves 1-3 doubly, the content of Cryptotanshinone improves 50-90 doubly, the content of tanshinone IIA improves 2-5 doubly, and the content (i.e. dihydrotanshinone Ⅰ, salvia miltiorrhiza bge I, Cryptotanshinone and tanshinone IIA content sum) of total-tanshinone improves 10-25 doubly.Red sage root Endophytic Fungal Hyphae water-soluble extractive mycelia WSE can improve the biomass of Hairy Root Cultures of Salvia miltiorrhiza effectively, promotes the biosynthesis of tanshinone component.
6. red sage root Endophytic Fungal Hyphae water-soluble extractive GL-PP position (PPF) and polysaccharide position (PSF) is on Hairy Root Cultures of Salvia miltiorrhiza growth and the biosynthetic impact of tanshinone.
At Hairy Root Cultures of Salvia miltiorrhiza suspension cultivation 16-24 days, adding concentration is 30-400mg/L red sage root Endophytic Fungal Hyphae water-soluble extractive GL-PP position (PPF) or red sage root Endophytic Fungal Hyphae water-soluble extractive polysaccharide position (PSF), continue to suspend and cultivate 12-30 days, hair shape root dry weight all improves 10%-70%, the content of dihydrotanshinone Ⅰ all improves 10-40 doubly, the content of salvia miltiorrhiza bge I all improves 1-8 doubly, the content of Cryptotanshinone all improves 20-50 doubly, the content of tanshinone IIA all improves 5-15 doubly, content (the i.e. dihydrotanshinone Ⅰ of total-tanshinone, salvia miltiorrhiza bge I, Cryptotanshinone and tanshinone IIA content sum) all improve 10-25 doubly.PPF and PSF all can improve the biomass of Hairy Root Cultures of Salvia miltiorrhiza effectively, promotes the biosynthesis of tanshinone component.
The present invention has prepared water-soluble extractive (WSE), GL-PP position (PPF) and polysaccharide position (PSF) first from red sage root Endophytic Fungal Hyphae, find that WSE, PPF and PSF all can effectively promote that Hairy Root Cultures of Salvia miltiorrhiza grows, significance ground improves the content of tanshinone component in hair shape root simultaneously, thus effectively improves the output of tanshinone component in Hairy Root Cultures of Salvia miltiorrhiza.
Accompanying drawing explanation
Fig. 1 is the aspect graph of red sage root endogenetic fungus on 1/2B5 plating medium.
Fig. 2 is the aspect graph of red sage root endogenetic fungus in 1/2B5 liquid nutrient medium.
Fig. 3 is Hairy Root Cultures of Salvia miltiorrhiza liquid suspension culture aspect graph.
Fig. 4 is red sage root Endophytic Fungal Hyphae water-soluble extractive and GL-PP position thereof and the polysaccharide position effect Hairy Root Cultures of Salvia miltiorrhiza aspect graph of 12 days (A is contrast, and B is the process of mycelia water-soluble extractive, and C is the process of GL-PP position, and D is the process of polysaccharide position).
Embodiment
Below in conjunction with the drawings and specific embodiments, set forth the present invention further.These embodiments are interpreted as only being not used in for illustration of the present invention limiting the scope of the invention.After the content of having read the present invention's record, those skilled in the art can make various changes or modifications the present invention, and these equivalence changes and modification fall into the scope of the claims in the present invention equally.
Embodiment 1: the fermentation of interior raw Trichoderma atroviride bacterium D16
Red sage root endogenetic fungus D16 is seeded to incubated at room temperature 3-5 days (see figure 1)s on 1/2B5 solid culture medium; Then the mode of card punch punching is adopted by the D16 mycelium inoculation on solid culture medium in 250ml triangular flask (including 100ml 1/2B5 culture fluid), 26 DEG C, 180rpm cultivates namely to obtain for 3-5 days and cultivate seed liquor (see figure 2); Finally cultivation seed liquor is seeded in 5L triangular flask (including 4L 1/2B5 culture fluid), 26 DEG C, 160rpm cultivates 10 days.
Embodiment 2: the preparation of red sage root Endophytic Fungal Hyphae water-soluble extractive (WSE)
Collect the cultivation D16 mycelium of 10 days by decompress filter, and wash 3 times with distilled water; Then appropriate distilled water is added by resuspended for D16 mycelium, 121 DEG C of high temperature high pressure process 40 minutes.Treat that nature cools, decompress filter gained filtrate is suitably concentrated is interior raw Trichoderma atroviride bacterium D16 mycelia WSE.
Sulfuric acid-phynol method is adopted to measure total sugar concentration in interior raw Trichoderma atroviride bacterium D16 mycelia WSE.With glucose drawing standard curve, obtain equation of linear regression: Y=0.0136X-0.0151, coefficient R
2=0.9989, glucose concentration range 10-50ug/ml.
Embodiment 3: the preparation of red sage root Endophytic Fungal Hyphae water-soluble extractive GL-PP position (PPF)
In reduced pressure concentration, raw Trichoderma atroviride bacterium D16 mycelia WSE solution (30L) is to proper volume, the ethanol 4 DEG C adding 5 times of volumes 95% leaves standstill 48 hours, within 10 minutes, white precipitate is obtained with the rotating speed centrifugation of 5000 revs/min, collecting precipitation after absolute ethyl alcohol washes 3 times, vacuum cooling drying gained white powder is interior raw Trichoderma atroviride bacterium D16 mycelia PPF.
Embodiment 4: the preparation of red sage root Endophytic Fungal Hyphae water-soluble extractive polysaccharide position (PSF)
Interior raw Trichoderma atroviride bacterium D16 mycelia PPF is dissolved in appropriate distilled water again, adopt Sevag method except deproteinized, the ethanol 4 DEG C adding 95% of 5 times of volumes leaves standstill 48 hours, within 10 minutes, obtains white precipitate, collecting precipitation after absolute ethyl alcohol washes 3 times with the rotating speed centrifugation of 5000 revs/min.The appropriate distilled water of precipitation is dissolved, room temperature dialyses 48 hours (molecular cut off is 2000Da) in distilled water, then obtain supernatant by the rotating speed centrifugation of 5000 revs/min, vacuum cooling drying gained white powder is interior raw Trichoderma atroviride bacterium D16 mycelia PSF.
Embodiment 5: Hairy Root Cultures of Salvia miltiorrhiza suspension cultural characteristic
What the present invention adopted is that shaking table is cultivated, 1/2B5 culture fluid 100ml is contained in the triangular flask of 250ml volume, Hairy Root Cultures of Salvia miltiorrhiza good for liquid culture is in advance seeded in sterilized medium, every bottle graft kind 1g, 25 DEG C, light culture 20 days-50 days not etc., does not change a culture fluid (see figure 3) weekly under 180rpm.During Hairy Root Cultures of Salvia miltiorrhiza results, outwell culture fluid, after distillation washing 3 times, filter paper blots residual moisture.Dry to constant weight for 50 DEG C, measure the content of tanshinone component by describing method in embodiment 6.
Embodiment 6: the mensuration of tanshinone component content in Hairy Root Cultures of Salvia miltiorrhiza
Adopt high performance liquid chromatography (HPLC) to analyze the content of tanshinone component in Hairy Root Cultures of Salvia miltiorrhiza, mainly quantitatively detect the content of dihydrotanshinone Ⅰ, salvia miltiorrhiza bge I, Cryptotanshinone and tanshinone IIA.Concrete steps are as follows:
The preparation of Hairy Root Cultures of Salvia miltiorrhiza HPLC sample introduction sample: precision weighing dries the hair shape root to constant weight, pulverize after record dry weight.Precision takes a mao shape root powder 0.15g, adds the ultrasonic extraction of 5ml methyl alcohol 3 times, each 40 minutes.Gained extraction solution is crossed 0.45um miillpore filter and is namely obtained HPLC sample introduction sample solution.
HPLC chromatographiccondition: chromatographic column: ZORBAX-C18 reversed-phase column (4.6 × 250mm × 5 μm); Mobile phase: H
2o (+0.5%HCOOH)-HCN, gradient elution: [time (min): D (HCN)]=[0.0: 20%]-[20.0: 40%]-[21.0: 80%]-[40.0: 90%]-[45.0: 20%]; Column temperature: 30 DEG C; Flow velocity: 0.8ml/min; Determined wavelength 280nm.
The determination of calibration curve: (1) accurately takes dihydrotanshinone Ⅰ 2mg and is dissolved in 10ml methyl alcohol, accurate dilution obtain concentration be 2.5,5,10,20, the dihydrotanshinone Ⅰ standard items HPLC sample introduction solution of 40ng/ml.(2) accurately taking salvia miltiorrhiza bge I 1mg is dissolved in 10ml methyl alcohol, accurate dilution obtain concentration be 2,4,6,8, the salvia miltiorrhiza bge I standard items HPLC sample introduction solution of 10ng/ml.(3) accurately taking Cryptotanshinone 1mg is dissolved in 10ml methyl alcohol, accurate dilution obtain concentration be 1,5,25,50, the Cryptotanshinone standard items HPLC sample introduction solution of 100ng/ml.(4) accurately taking tanshinone IIA 1mg is dissolved in 10ml methyl alcohol, accurate dilution obtain concentration be 0.5,1.0,2.0,4.0, the tanshinone IIA standard items HPLC sample introduction solution of 8.0ng/ml.All standard solutions are analyzed by above-mentioned HPLC chromatographic condition sample introduction.With the peak area of HPLC for Y, with sample introduction standard solution concentration (mg/ml) for X, obtain 4 kinds of tanshinone equations of linear regression (see table 1).
The assay of tanshinone in Hairy Root Cultures of Salvia miltiorrhiza sample: the sample solution 20ul sample introduction getting above-mentioned preparation, carries out the analysis of tanshinone component, quantitatively calculates according to regression equation by above-mentioned H PLC chromatographic condition.
The equation of linear regression of table 1 four kinds of tanshinones
Embodiment 7: red sage root Endophytic Fungal Hyphae water-soluble extractive (WSE) is on Hairy Root Cultures of Salvia miltiorrhiza growth and the biosynthetic impact of tanshinone
Interior raw Trichoderma atroviride bacterium D16 mycelia WSE solution is prepared according to embodiment 2.After the total sugar concentration measured by sulfuric acid-phynol method, add the WSE solution preparation of certain volume become total sugar concentration to be 40,150, the 1/2B5 induction broth of 300mg/L, 121 DEG C of sterilizings cool stand-by naturally after 15 minutes.The culture fluid of the Hairy Root Cultures of Salvia miltiorrhiza of normally cultivating 21 days is replaced by induction broth, continues cultivation and gather in the crops (see figure 4) after 18 days.Hairy Root Cultures of Salvia miltiorrhiza dry weight and four kinds of tanshinone content are in table 2.As can be seen from the table, the WSE of variable concentrations all can improve to significance the biomass of Hairy Root Cultures of Salvia miltiorrhiza; When induced concentration is 300mg/L, effect is best, and the biomass of hair shape root improves 46.4%.The WSE of variable concentrations also all can improve to significance the content of tanshinone component in Hairy Root Cultures of Salvia miltiorrhiza simultaneously: when induced concentration is 300mg/L, the content of dihydrotanshinone Ⅰ is the highest improves 33.3 times, and the content of salvia miltiorrhiza bge I is the highest improves 1.7 times; When induced concentration is 150mg/L, the content of Cryptotanshinone is the highest improves 82.7 times; When induced concentration is 40mg/L, the content of tanshinone IIA improves 4.1 times; When induced concentration is 150mg/L, the content (i.e. dihydrotanshinone Ⅰ, salvia miltiorrhiza bge I, Cryptotanshinone and tanshinone IIA content sum) of total-tanshinone is the highest improves 21.1 times.Interior raw Trichoderma atroviride bacterium D16 mycelia WSE can improve the biomass of Hairy Root Cultures of Salvia miltiorrhiza effectively, promotes the biosynthesis of tanshinone component.
In table 3, raw Trichoderma atroviride bacterium D16 mycelia PPF and PSF is on Hairy Root Cultures of Salvia miltiorrhiza growth and the biosynthetic impact of tanshinone
Note: inoculum concentration is 10g/L, control group is the process not adding elicitor, and each process all in triplicate.The content of total-tanshinone is dihydrotanshinone I, Tanshinone I, Cryptotanshinone and tanshinone IIA four kinds of tanshinone content sums.*: P < 0.05, * *: P < 0.01, * * *: P < 0.001 VS control group.
Embodiment 8: red sage root Endophytic Fungal Hyphae water-soluble extractive GL-PP position (PPF) and polysaccharide position (PSF) are on Hairy Root Cultures of Salvia miltiorrhiza growth and the biosynthetic impact of tanshinone
Raw Trichoderma atroviride bacterium D16 mycelia PPF and PSF in preparing according to embodiment 3,4.Add in many PPF or PSF to 1/2B5 culture fluids, be made into the induction broth that concentration is 60mg/L PPF or PSF, after 15 minutes, cooling is stand-by naturally in 121 DEG C of sterilizings.The culture fluid of the Hairy Root Cultures of Salvia miltiorrhiza of normally cultivating 21 days is replaced by induction broth, continues cultivation and gather in the crops (see figure 4) after 24 days.Hairy Root Cultures of Salvia miltiorrhiza dry weight and four kinds of tanshinone content are in table 3.As can be seen from the table, concentration is the equal biomass that can improve to significance Hairy Root Cultures of Salvia miltiorrhiza of PPF and PSF of 60mg/L, and PPF improves 32%, PSF and improves 62%.Simultaneously concentration is the content that PPF and PSF of 60mg/L also all can improve to significance tanshinone component in Hairy Root Cultures of Salvia miltiorrhiza: the content PPF of dihydrotanshinone Ⅰ improves 27.7 times, and PSF improves 33.2 times; The content PPF of salvia miltiorrhiza bge I improves 1.9 times, and PSF improves 5.7 times; The content PPF of Cryptotanshinone improves 36.4 times, and PSF improves 43.5 times; The content PPF of tanshinone IIA improves 7.2 times, and PSF improves 10.6 times; Content (i.e. dihydrotanshinone Ⅰ, salvia miltiorrhiza bge I, Cryptotanshinone and the tanshinone IIA content sum) PPF of total-tanshinone improves 17.8 times, and PSF improves 22.8 times.Interior raw Trichoderma atroviride bacterium D16 mycelia PPF and PSF can improve the biomass of Hairy Root Cultures of Salvia miltiorrhiza effectively, promotes the biosynthesis of tanshinone component.
Claims (9)
1. a red sage root Endophytic Fungal Hyphae water-soluble extractive, is characterized in that, it collects the red sage root endophytic fungal hypha of fermented and cultured 9-14 days, and it is resuspended to add distilled water, 110-130 DEG C of sterilizing 30-60min, cooling, the filtrate obtained after decompress filter; The called after Trichoderma atroviride Trichoderma atroviride of described red sage root endogenetic fungus, bacterial strain code name is D16, and it is CGMCC No.4712 at the preservation registration number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. a red sage root Endophytic Fungal Hyphae water-soluble extractive GL-PP position, it is characterized in that, reduced pressure concentration red sage root Endophytic Fungal Hyphae according to claim 1 water-soluble extractive, adding 4-7 times of volume volumetric concentration is the ethanol of 70-95%, at 2-8 DEG C of standing 24-72 hour, within 10-15 minute, white precipitate is obtained with the rotating speed centrifugation of 2000-10000 rev/min, with absolute ethyl alcohol wash-out 3-5 rear collecting precipitation, vacuum cooling drying gained white powder is red sage root Endophytic Fungal Hyphae water-soluble extractive GL-PP position.
3. a red sage root Endophytic Fungal Hyphae water-soluble extractive polysaccharide position, it is characterized in that, red sage root Endophytic Fungal Hyphae water-soluble extractive GL-PP position according to claim 2 is dissolved in distilled water, adopt Sevag method except deproteinized, adding 4-7 times of volume volumetric concentration is the ethanol of 70-95%, at 2-8 DEG C of standing 24-72 hour, within 10-15 minute, obtain white precipitate with the rotating speed centrifugation of 2000-10000 rev/min, with absolute ethyl alcohol wash-out 3-5 rear collecting precipitation; Precipitation distilled water is dissolved, dialyse under room temperature 24-72h in distilled water, molecular cut off is 1000-10000Da, obtains supernatant with the rotating speed centrifugation of 2000-10000 rev/min, and vacuum cooling drying gained white powder is red sage root Endophytic Fungal Hyphae water-soluble extractive polysaccharide position.
4. red sage root Endophytic Fungal Hyphae water-soluble extractive is as the purposes promoting tanshinone component content in Hairy Root Cultures of Salvia miltiorrhiza growth and/or raising Hairy Root Cultures of Salvia miltiorrhiza, the called after Trichoderma atroviride Trichoderma atroviride of described red sage root endogenetic fungus, bacterial strain code name is D16, and it is CGMCC No.4712 at the preservation registration number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
5. purposes according to claim 4, is characterized in that, at Hairy Root Cultures of Salvia miltiorrhiza suspension cultivation 16-24 days, adds the red sage root Endophytic Fungal Hyphae water-soluble extractive that total sugar concentration is 30-400mg/L, continues to suspend and cultivate 12-30 days.
6. red sage root Endophytic Fungal Hyphae water-soluble extractive GL-PP position is as the purposes promoting tanshinone component content in Hairy Root Cultures of Salvia miltiorrhiza growth and/or raising Hairy Root Cultures of Salvia miltiorrhiza, the called after Trichoderma atroviride Trichoderma atroviride of described red sage root endogenetic fungus, bacterial strain code name is D16, and it is CGMCC No.4712 at the preservation registration number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
7. purposes according to claim 6, is characterized in that, at Hairy Root Cultures of Salvia miltiorrhiza suspension cultivation 16-24 days, adds the red sage root Endophytic Fungal Hyphae water-soluble extractive GL-PP position that total sugar concentration is 30-400mg/L, continues to suspend and cultivate 12-30 days.
8. red sage root Endophytic Fungal Hyphae water-soluble extractive polysaccharide position is as the purposes promoting tanshinone component content in Hairy Root Cultures of Salvia miltiorrhiza growth and/or raising Hairy Root Cultures of Salvia miltiorrhiza, the called after Trichoderma atroviride Trichoderma atroviride of described red sage root endogenetic fungus, bacterial strain code name is D16, and it is CGMCC No.4712 at the preservation registration number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
9. purposes according to claim 8, is characterized in that, at Hairy Root Cultures of Salvia miltiorrhiza suspension cultivation 16-24 days, adds the red sage root Endophytic Fungal Hyphae water-soluble extractive polysaccharide position that total sugar concentration is 30-400mg/L, continues to suspend and cultivate 12-30 days.
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| CN104745482A (en) * | 2014-09-12 | 2015-07-01 | 中国人民解放军第二军医大学 | Endophytic fungi of amebia enchroma and application of endophytic fungi |
| CN108030089B (en) * | 2017-10-30 | 2021-06-15 | 浙江科技学院 | Method for recycling salvia miltiorrhiza water extraction and alcohol precipitation herb residues |
| CN109797107B (en) * | 2019-01-29 | 2021-06-04 | 西北大学 | Preparation method of fungus mycelium fine powder |
| CN112369435B (en) * | 2020-12-07 | 2021-10-08 | 中南林业科技大学 | Salvia miltiorrhiza growth regulating bacteria suspension and preparation method and application thereof |
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| CN102676392A (en) * | 2011-04-08 | 2012-09-19 | 中国人民解放军第二军医大学 | Endophytic fungus in salvia miltiorrhiza bunge and application thereof |
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| CN102676392A (en) * | 2011-04-08 | 2012-09-19 | 中国人民解放军第二军医大学 | Endophytic fungus in salvia miltiorrhiza bunge and application thereof |
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| Promotion of Salvia miltiorrhiza hairy root growth and tanshinone production by polysaccharide–protein fractions of plant growth-promoting rhizobacterium Bacillus cereus;Jiang-Lin Zhao等;《Process Biochemistry》;20100930;第45卷(第9期);第1517–1522页 * |
| 沈双.诱导子对丹参毛状根生长和丹参酮含量的影响.《中国优秀硕士学位论文全文数据库(电子期刊)农业科技辑》.2012,第D047-370页. * |
| 诱导子对丹参毛状根酚酸类和丹参酮类成分积累的影响;张顺仓等;《中国中药杂志》;20110531;第36卷(第10期);第1269-1274页 * |
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