CN103205365A - Aspergillus tubingensis and application thereof to preparation of ginsenoside Rh4 and aglycone of ginsenoside Rh4 - Google Patents
Aspergillus tubingensis and application thereof to preparation of ginsenoside Rh4 and aglycone of ginsenoside Rh4 Download PDFInfo
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- CN103205365A CN103205365A CN2013101016534A CN201310101653A CN103205365A CN 103205365 A CN103205365 A CN 103205365A CN 2013101016534 A CN2013101016534 A CN 2013101016534A CN 201310101653 A CN201310101653 A CN 201310101653A CN 103205365 A CN103205365 A CN 103205365A
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Abstract
The invention discloses Aspergillus tubingensis and application thereof to preparation of ginsenoside Rh4 and aglycone of the ginsenoside Rh4. A strain Aspergillus tubingensis with hydrolyzed ginsenoside activity is selected, an aerobic fermentation biotransformation technology is adopted for transforming total ginsenoside so as to generate the ginsenoside Rh4 and the aglycone thereof, and the Rh4 and the aglycone are extracted and purified. The selected strain with the hydrolyzed ginsenoside activity is the Aspergillus tubingensis which is derived from Phuket, Thailand in June, 2007, and is preserved in China General Microbiological Culture Collection Center of China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC6992, the laboratory preservation number is SYP-F-2612, and the colony of the Aspergillus tubingensis on a PDA (potato dextrose agar) solid culture medium is black. The Aspergillus tubingensis and the application thereof are advanced in production process, low in cost of raw material, suitable for mass industrial production, free of emission of harmful substances during production, free of pollution to surroundings and free of public nuisances, and meet the requirement for environment friendliness.
Description
Technical field:
The present invention relates to field of pharmaceutical technology, relate to a kind of Tabin aspergillus (
Aspergillus tubingensis.) and the application in preparation Ginsenoside Rh4 and aglycon thereof.
Background technology:
Genseng (
Panax ginseng C. A. Meyer) be Araliaceae Panax perennial herb, be the famous Chinese medicinal materials of China, at China's medicinal history about 4,000 years, first of " northeast Triratna ", have won fame both at home and abroad, obey body-building for a long time and prolong life, have very big pharmaceutical use.Garriques at first began in 1854 genseng and homologue thereof are carried out The Chemical Constituents, and from then on, people just begin genseng is carried out a large amount of chemistry, biological chemistry and pharmacological research.Up to the present, the chemical ingredients of therefrom finding is mainly compositions such as triterpenoid saponin, sesquiterpene, flavones, polysaccharide, dencichine, polyene alkynes and polyamines.
Chinese scholars shows the structure activity study of ginsenoside anti-tumor activity: saponin(e and the aglycon of low sugar chain have stronger antitumor action, and its rule is as follows: aglycon〉monoglycosides〉bioside〉three glucosides〉the tetrose glycosides.As Rg
1And Rh
1The effect that all has anticancer propagation, but Rh
1C-20 lacks a part on the position
β-D-glucose, the ability of its anticancer propagation is about Rg
115 times.As seen, the glycosyl side chain in the ginsenoside molecule has remarkable influence for its biological activity.Therefore, change the structure of original ginsenoside by certain means, obtain active stronger rare ginsenoside, become the important topic of a lot of scholar's research.
At present, dark people's research has been carried out in the prevention of its main activeconstituents ginsenoside and the effect of inhibition tumour both at home and abroad, found that, Radix Ginseng total saponins and each monomeric compound are to promoting apoptosis of tumor cells, impel the tumour cell differentiation, improve tumour cell aspects such as the susceptibility of chemotherapeutics and the antineoplastic immunizing power of raising body are had important effect.And rare ginsenoside Rh4 and aglycon thereof carry out the detection of biologic activity through mtt assay, find that it has anti-tumor activity in various degree, therefore find the method tool for preparing rare ginsenoside Rh4 and aglycon thereof to have very important significance.
Summary of the invention
The invention provides a kind of bio-transformation bacterial classification and implementation method, change the method that the ginsenoside glycosyl prepares rare ginsenoside Rh4 and aglycon thereof.Select a kind of fungi (Tabin aspergillus) bacterial classification for use, adopt a kind of or two kinds of methods in aerobic fermentation conversion technology, the immobilized cell technology enzyme immobilization technology to be used in conjunction, directly with total ginsenoside as substrate, transform preparation Ginsenoside Rh4 and aglycon thereof.
This method comprises: the activation of original strain, the various steps that shake-flask seed is cultivated, bio-transformation is cultivated and extract the back.Its technological process of production is as follows:
Original strain → slant activation → shake-flask seed → bio-transformation bottle that skimmed milk freeze-drying pipe is preserved drops into the ginsenoside substrate and carries out bio-transformation → termination conversion → extraction purifying → acquisition Rh4 and aglycon thereof.
Concrete processing step is as follows:
(1) the medium slant substratum is the PDA substratum, fills a prescription to be: potato 200g glucose 10g agar 20g pH 7.2-7.4; 30min is boiled in the peeling potatoes stripping and slicing, and four layers of filtered through gauze are supplied water and are settled to 1L then, 121 ℃ * 30min of sterilization.Nutrition in seed culture medium and the microbial transformation substratum comprises all kinds of carbon sources and nitrogenous source and the inorganic salt that the professional and technical personnel often adopts in the fermenting process.Can be as the carbon source that contains in the 1L substratum: 1 ~ 30g glucose, 1 ~ 100g dextrin; 1 ~ 50g sucrose; 1 ~ 100g starch; 1 ~ 50g maltose; Nitrogenous source can be in the 1L substratum: 1 ~ 20g peptone; 1 ~ 50g groundnut meal; 1 ~ 20g yeast powder; 1 ~ 50g soybean cake powder; 1 ~ 50g cottonseed meal; 1 ~ 10g urea; 1 ~ 10g ammonium chloride.Need to add inorganic salt in addition, as calcium chloride; Potassium primary phosphate; Sal epsom; Zinc sulfate; Sodium-chlor; Ammonium sulfate; KH
2PO
4, MgSO
47H
2O, ZnSO
4, (NH
4)
2SO
4
(2) bio-conversion process: the original strain that skimmed milk freeze-drying pipe is preserved is received on the slant medium, under 25 ~ 28 ℃ of environment, cultivated 3 ~ 7 days, treat that the lawn growth is fine and close.Inclined-plane after the activation is dug piece to be inoculated in the seed bottle, after 25 ~ 28 ℃ of isothermal vibrations (200r/min) on the automatic rotation bottle swingging machine are cultivated 48h, transferred species amount transferred species by 10% is in biotransformation medium, simultaneously according to 0.5%(w/v) ratio add Radix Ginseng total saponins, in 28 ℃, 200r/min concussion incubation time 144h transforms and finishes.
(3) extraction process: three extractions of isopyknic water-saturated n-butanol suction filtration liquid, merge the n-butanol extraction part, be evaporated to dried.The concentrated solution dissolve with methanol, silica gel mixed sample, carry out silica gel (200-300 order) column chromatography, adopt the chloroform-methanol gradient (wash-out of 15:1 → 12:1 → 10:1 → 8:1 → 5:1), every 100ml is a cut, analyze according to TLC, merge the close part of polarity, obtain Section A and Section B two portions.Respectively A, B two portions are prepared liquid phase and are further purified, obtain 2 monomeric compounds, purification of samples detects through carbon-13 nmr spectra, identifies that its converted product is rare ginsenoside Rh4 and aglycon thereof.
Shake-flask seed of the present invention is cultivated and is referred to: adopt the needed carbon source of strain growth, nitrogenous source, inorganic salt etc., after sterilization, take an amount of thalline inclined-plane, transfer to seed and shake in the bottle, at rotation bottle swingging machine concussion cultivation automatically, 200rpm, 28 ℃, cultivate and obtained shake-flask seed in 48 hours.
Microbial transformation of the present invention refers to: adopt the needed carbon source of strain growth, nitrogenous source, inorganic salt etc., after sterilization, transferred species amount transferred species by 10% is in fermention medium, simultaneously according to 0.5%(w/v) ratio add substrate (Radix Ginseng total saponins), temperature is 28 ℃, 200r/min incubation time 144h.
The extraction purge process of bioconversion product of the present invention-rare ginsenoside Rh4 and aglycon thereof is: with fungi conversion fluid suction filtration, collect filtrate, divide three extractions with water-saturated n-butanol, merge the n-butanol extraction part, be evaporated to dried.Concentrated solution dissolves with MeOH, and silica gel mixed sample carries out silica gel (200-300 order) column chromatography, adopt chloroform-methanol gradient (15:1--5:1) wash-out, every 100ml is a cut, analyzes according to TLC, merge the close part of polarity, obtain Section A and Section B two portions.Respectively A, B two portions are prepared liquid phase and are further purified, obtain monomeric compound Rh4 and aglycon thereof.
The Tabin aspergillus bacterial strain that the present invention uses has been delivered to China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation.Preservation date on December 14th, 2012, deposit number is CGMCC No.6992, classification called after Tabin aspergillus
Aspergillus tubingensis.
Description of drawings
The chemical structural formula of Fig. 1 Ginsenoside Rh4 and aglycon thereof
Fig. 2 bacterial strain SYP-F-2612 colonial morphology
Fig. 3 bacterial strain SYP2612 sporocyst and spore shape
Fig. 4 bacterial strain SYP2612 is according to the systematic evolution tree of ITS-5.8S rDNA sequence.
Embodiment
Below in conjunction with preferred embodiment the present invention is done further narration, but protection domain is not limited by the examples.
Embodiment 1: the cultural characteristic of fungi SYP-A-2612 of the present invention on different substratum
The growth conditions of above-mentioned bacterial strains is asked for an interview accompanying drawing 1.
The feature of bacterial strain under the opticmicroscope after the growth of PDA substratum:
Mycelia and spore shape: the conidial head sphere is to radiation shape; The conidiophore wall is smooth, top capsule sphere, conidial fructification bilayer, conidium sphere, the coarse or tool verruca of wall.Referring to accompanying drawing 2.
With molecular biology method, extract above-mentioned fungal gene group DNA, with ITS1, ITS4(ITS1:5 '-TCC GTC GGT GAA CCT GCG G-3 '; ITS4:5 '-TCC TCC GCT TAT TGA TAT GC-3 ') for primer carries out pcr amplification, order-checking, and with the rDNA homologous sequence comparison among check order row and the Genbank, choose the bacterial strain of the higher homology of 11 strains, and with
Sphingobacterium siyangensisBe reference, utilize software Clustal X and Mega3.1 to make up the bacterial classification evolutionary tree.Bacterial strain SYP2612 with
Aspergillus tubingensisStrain CBS 103.12 homologys the highest (100%).Combining form is learned qualification result again, and this bacterial strain is accredited as Tabin aspergillus
Aspergillus tubingensisReferring to accompanying drawing 3.
Embodiment 2: the aerobic fermentation method transforms the method for producing rare ginsenoside Rh4 and aglycon thereof
One, substratum
(1) PDA substratum
Potato 200g glucose 10g agar 20g pH 7.2-7.4
30min is boiled in the peeling potatoes stripping and slicing, and four layers of filtered through gauze are supplied water and are settled to 1L then, 121 ℃ * 30min of sterilization
(2) seed culture medium (g/L)
(3) biotransformation medium (g/L)
Two, aerobic fermentation microbial transformation
1, the slant activation of bacterial classification: open bacterial classification skimmed milk freeze-drying pipe, a small amount of powder of picking is even to be coated on the PDA inclined-plane after the sterilization, cultivates 5-7 days under 28 ℃ of temperature, and the lawn growth is fine and close, be covered with the inclined-plane, do not have assorted bacterium, 4 ℃ of preservations of refrigerator.
2, shake-flask seed is cultivated: will activate good PDA slant strains, and dig piece 1*1cm
2Be inoculated in the seed culture bottle, isothermal vibration is cultivated under 28 ℃ of temperature, and 200rpm cultivates 448h, and the microscopy mycelial growth is unfolded, dyeed deeply, do not have assorted bacterium and gets final product.
3, bio-transformation: biotransformation medium as above, by 10% transferred species amount transferred species in biotransformation medium, simultaneously according to 0.5%(w/v) ratio add and transform with substrate-Radix Ginseng total saponins, 28 ℃, 200r/min behind the incubation time 144h, transforms and finishes.
Three, the evaluation of rare ginsenoside Rh4 and aglycon thereof and purifying
1, with bioconversion product liquid under the room temperature normal pressure, remove mycelium with the decompress filter method, collect supernatant liquor and adopt TLC, HPLC method, carry out the qualitative and quantitative analysis of Ginsenoside Rh4 and aglycon thereof.
2, TLC testing conditions: silica GF254; Developping agent: chloroform: methyl alcohol=8:1(v/v).Test sample is the sample after Ginsenoside Rh4's standard substance, Ginsenoside Rh4's aglycon, the bio-transformation.Behind the thin-layer chromatography, volatilize developping agent, spray 10%(v/v) H
2SO
4-EtOH is in 105 ℃ of baking 10min, spot displaing amaranth.The Rf value of standard of comparison product and sample (Rf), can see Tabin aspergillus can with ginsenoside in various degree change into Ginsenoside Rh4 and aglycon thereof.
3, the HPLC testing conditions is as follows.When dropping into 0.5% ginsenoside in bio-transformation liquid, Tabin aspergillus can be converted into the ginsenoside more than 90% rare ginsenoside rh4 and aglycon thereof.
4, the separation and purification of converted product
Three extractions of isopyknic water-saturated n-butanol suction filtration liquid merges the n-butanol extraction part, is evaporated to dried.The concentrated solution dissolve with methanol, silica gel mixed sample, carry out silica gel (200-300 order) column chromatography, adopt the chloroform-methanol gradient (wash-out of 15:1 → 12:1 → 10:1 → 8:1 → 5:1), every 100ml is a cut, analyze according to TLC, merge the close part of polarity, obtain Section A and Section B two portions.Respectively A, B two portions are prepared liquid phase and are further purified, obtain 2 monomeric compounds, purification of samples detects through carbon-13 nmr spectra, identifies that its converted product is rare ginsenoside Rh4 and aglycon thereof.Be correlated with
13C-NMR structure elucidation data see Table 1.
5, converted product rare ginsenoside Rh
4And the result of study of the biologic activity of aglycon
Adopt mtt assay, research converted product rare ginsenoside Rh
4And aglycon is to the inhibited proliferation of gastric carcinoma cells (SGC-7901), human desmocyte sarcoma cell (HT-1080), human oral cancer cells (KB-A-1), inhibiting rate and corresponding IC
50See Table 2 and table 3 shown in.
Table 2 ginsenoside Rh
4And aglycon is to the investigation of three kinds of inhibitory rate of cell growth
Inhibiting rate<20% background is bright grey; Inhibiting rate background between 20-90% is Dark grey; Inhibiting rate〉90% background is black.
Can be found rare ginsenoside Rh by table 2
4Above-mentioned three kinds of cells are all had restraining effect, and along with the increase of concentration, restraining effect increases gradually, and when concentration reached 156.99 μ M, inhibiting rate all reached more than 90%, and to the inhibiting rate of SGC apparently higher than HT and KB.Equally, rare ginsenoside Rh
4The concentration of aglycon increases, and restraining effect increases gradually, and when concentration reached 218.34 μ M, three kinds of cell inhibiting rates were all greater than 90%.By table 3 as seen, compare rare ginsenoside Rh with the positive control drug Dx
4And aglycon IC
50Be worth bigger.
Claims (6)
- Tabin aspergillus ( Aspergillus tubingensis), it is characterized in that: its deposit number is: CGMCC No.6992.
- 2. the application of the described Tabin aspergillus of claim 1 in preparation rare ginsenoside Rh4 and aglycon.
- 3. application according to claim 2 is characterized in that, adopts the aerobic fermentation conversion technology, withTotal ginsenoside generates Ginsenoside Rh4 and aglycon thereof as the substrate conversion Radix Ginseng total saponins, extracts purifying.
- 4. application according to claim 3 is characterized in that: its biotransformation step is as follows: the aftertreatment of the activation of original strain, the preparation of shake-flask seed, microbial fermentation, fermented liquid; Concrete technical process is as follows: original strain → slant activation → shake-flask seed → bio-transformation bottle that skimmed milk freeze-drying pipe is preserved, drop into the ginsenoside substrate and carry out bio-transformation → termination conversion → extraction purifying → acquisition Rh4 and aglycon thereof(1) carbon source and nitrogenous source are adopted in the nutrition in substratum seed culture medium and the fermentation culture medium for microbe, wherein carbon source is glucose, dextrin, sucrose, starch or maltose, nitrogenous source is peptone, hot rolling groundnut meal, yeast powder or soybean cake powder, also needs to add NH 4Cl, KH 2PO 4, MgSO 47H 2O, ZnSO 4, (NH 4) 2SO 4(2) microorganism fermentation process: obtain Ginsenoside Rh4 and Rh4 aglycon by shaking bottle second order fermentation, cultural method comprises: the preparation of shake-flask seed: substratum keeps nature pH before the sterilization, after digging piece inoculation production bacterial strain inclined-plane lawn after the sterilization, on automatic rotation bottle swingging machine, revolution 200rpm, temperature is 28 ℃, after 200r/min cultivates 48h, transferred species amount transferred species by 10% is in fermention medium, simultaneously according to 0.5%(w/v) ratio add substrate, 28 ℃, 200r/min, incubation time 144h transforms and finishes.
- 5. application according to claim 3, it is characterized in that: its purification step is as follows: with fungi conversion fluid suction filtration, collect filtrate, divide three extractions with isopyknic water-saturated n-butanol, merge the n-butanol extraction part, be evaporated to driedly, concentrated solution dissolves with MeOH, silica gel mixed sample, carry out silica gel column chromatography, adopt the chloroform-methanol gradient elution, every 100ml is a cut, analyzes according to TLC, merge the close part of polarity, obtain Section A and Section B two portions, respectively with A, B two portions are prepared liquid phase and are further purified, and obtain monomeric compound Rh4 and aglycon thereof.
- 6. application according to claim 5 is characterized in that, the gradient of described chloroform-methanol is 15:1-5:1.
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Cited By (5)
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CN103966105A (en) * | 2013-11-28 | 2014-08-06 | 河南科技学院 | Aspergillus oryzae for converting ginsenoside Rg3 to produce Rh2, production method and application |
CN104523790A (en) * | 2015-01-19 | 2015-04-22 | 成都天昕医药保健品有限公司 | Prepared pseudo-ginseng, as well as preparation method and application thereof |
CN106754422A (en) * | 2017-01-12 | 2017-05-31 | 华中科技大学 | A kind of Tabin aspergillus and its application in turmeric saponin is prepared |
CN109626598A (en) * | 2019-01-23 | 2019-04-16 | 南华大学 | A method of utilizing Tabin aspergillus and phytate in-situ immobilization hexavalent uranium polluted surface water |
CN110982869A (en) * | 2019-12-19 | 2020-04-10 | 昆明理工大学 | Preparation of 20(R) -ginsenoside Rh by biotransformation of panax notoginseng saponins1Method (2) |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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CN103966105A (en) * | 2013-11-28 | 2014-08-06 | 河南科技学院 | Aspergillus oryzae for converting ginsenoside Rg3 to produce Rh2, production method and application |
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CN106754422A (en) * | 2017-01-12 | 2017-05-31 | 华中科技大学 | A kind of Tabin aspergillus and its application in turmeric saponin is prepared |
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CN109626598A (en) * | 2019-01-23 | 2019-04-16 | 南华大学 | A method of utilizing Tabin aspergillus and phytate in-situ immobilization hexavalent uranium polluted surface water |
CN110982869A (en) * | 2019-12-19 | 2020-04-10 | 昆明理工大学 | Preparation of 20(R) -ginsenoside Rh by biotransformation of panax notoginseng saponins1Method (2) |
CN110982869B (en) * | 2019-12-19 | 2023-05-16 | 昆明理工大学 | Preparation of 20 (R) -ginsenoside Rh by bioconversion of Panax notoginseng saponins 1 Is a method of (2) |
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