CN102851218A - Production method of 11-O-acetylcyathatriol and 15-O-acetylcyathatriol, and special bacterial strain thereof - Google Patents
Production method of 11-O-acetylcyathatriol and 15-O-acetylcyathatriol, and special bacterial strain thereof Download PDFInfo
- Publication number
- CN102851218A CN102851218A CN2012103527737A CN201210352773A CN102851218A CN 102851218 A CN102851218 A CN 102851218A CN 2012103527737 A CN2012103527737 A CN 2012103527737A CN 201210352773 A CN201210352773 A CN 201210352773A CN 102851218 A CN102851218 A CN 102851218A
- Authority
- CN
- China
- Prior art keywords
- africanus
- formula
- compound shown
- cyathus
- formula iii
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 230000001580 bacterial effect Effects 0.000 title abstract description 13
- 238000004519 manufacturing process Methods 0.000 title description 3
- 150000001875 compounds Chemical class 0.000 claims abstract description 73
- 241000480243 Cyathus africanus Species 0.000 claims abstract description 49
- 239000007787 solid Substances 0.000 claims abstract description 27
- 238000000855 fermentation Methods 0.000 claims abstract description 26
- 230000004151 fermentation Effects 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 23
- 239000000287 crude extract Substances 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 244000005700 microbiome Species 0.000 claims abstract description 9
- 239000003960 organic solvent Substances 0.000 claims abstract description 9
- 238000000605 extraction Methods 0.000 claims abstract description 7
- 238000004821 distillation Methods 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 24
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 11
- 239000002904 solvent Substances 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 240000007594 Oryza sativa Species 0.000 claims description 7
- 235000007164 Oryza sativa Nutrition 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- 235000009566 rice Nutrition 0.000 claims description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 238000002425 crystallisation Methods 0.000 claims description 6
- 230000008025 crystallization Effects 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 238000010898 silica gel chromatography Methods 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 4
- 235000005822 corn Nutrition 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 230000003068 static effect Effects 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 244000077995 Coix lacryma jobi Species 0.000 claims description 3
- 235000007354 Coix lacryma jobi Nutrition 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 241000209140 Triticum Species 0.000 claims description 3
- 235000021307 Triticum Nutrition 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 239000012156 elution solvent Substances 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 2
- 238000010828 elution Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 240000003370 Cucumis africanus Species 0.000 claims 4
- 244000268590 Euryale ferox Species 0.000 claims 1
- 235000006487 Euryale ferox Nutrition 0.000 claims 1
- 235000013399 edible fruits Nutrition 0.000 claims 1
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 230000002906 microbiologic effect Effects 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- 238000002791 soaking Methods 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 10
- 238000000926 separation method Methods 0.000 description 8
- 244000000626 Daucus carota Species 0.000 description 7
- 235000005770 birds nest Nutrition 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 235000005765 wild carrot Nutrition 0.000 description 7
- 206010028980 Neoplasm Diseases 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- 241000233866 Fungi Species 0.000 description 5
- 150000001335 aliphatic alkanes Chemical class 0.000 description 5
- 229930004069 diterpene Natural products 0.000 description 5
- 238000000638 solvent extraction Methods 0.000 description 5
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- XSTXAVWGXDQKEL-UHFFFAOYSA-N Trichloroethylene Chemical compound ClC=C(Cl)Cl XSTXAVWGXDQKEL-UHFFFAOYSA-N 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 150000004141 diterpene derivatives Chemical class 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 229960001866 silicon dioxide Drugs 0.000 description 4
- 241000157727 Cyathus earlei Species 0.000 description 3
- -1 alkane triols Chemical class 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- OAIVIYSBZFEOIU-UHFFFAOYSA-N chloroform;propan-2-one Chemical compound CC(C)=O.ClC(Cl)Cl OAIVIYSBZFEOIU-UHFFFAOYSA-N 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000002207 metabolite Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000959617 Cyathus Species 0.000 description 2
- 241000123222 Hericium Species 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 230000000118 anti-neoplastic effect Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000012531 culture fluid Substances 0.000 description 2
- 239000002024 ethyl acetate extract Substances 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 241000480176 Cyathus helenae Species 0.000 description 1
- 241001147747 Cyathus sp. Species 0.000 description 1
- 241000959620 Cyathus striatus Species 0.000 description 1
- 241001232787 Epiphragma Species 0.000 description 1
- 240000007817 Olea europaea Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 241000778150 Sarcodon scabrosus Species 0.000 description 1
- ULLCSSTZNGODKE-KRNPDVCNSA-N [4-[4-[(5s,11ar)-3,11a-bis[(2s)-butan-2-yl]-2,4-dihydroxy-5-oxido-1-oxopyrazino[1,2-b][1,4,2]benzodioxazin-5-ium-9-yl]-2,3-diacetyloxy-5,6-dihydroxyphenyl]phenyl] acetate Chemical compound O([N@@+]1([O-])C(O)=C(N(C(=O)[C@@]1([C@@H](C)CC)OC1=C2)O)[C@@H](C)CC)C1=CC=C2C(C(=C1OC(C)=O)OC(C)=O)=C(O)C(O)=C1C1=CC=C(OC(C)=O)C=C1 ULLCSSTZNGODKE-KRNPDVCNSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 244000000007 bacterial human pathogen Species 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000012230 colorless oil Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 229930185448 cyathin Natural products 0.000 description 1
- 239000002254 cytotoxic agent Substances 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229930191277 erinacine Natural products 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 208000011645 metastatic carcinoma Diseases 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 229930190558 sarcodonin Natural products 0.000 description 1
- 229930188740 scabronine Natural products 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000005292 vacuum distillation Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparation of a compound shown in formula II and/or a compound shown in formula III by microbial fermentation, and a special bacterial strain thereof. The bacterial strain is Cyathus africanus L38, which is preserved in the General microbiological center of China Committee for Culture Collection of Microorganisms and has a preservation number of CGMCC NO.6259. The method disclosed in the invention comprises the steps of: 1) inoculating the fermentation Cyathus africanus L38 into a solid medium to conduct solid fermentation culture; and 2) adding an organic solvent into the fermented system and carrying out soaking extraction so as to obtain an extracted solution, which is then subjected to pressure reduced distillation, thus obtaining a crude extract containing the compound shown in formula II and the compound shown in formula III. In the invention, Cyathus africanus L38 is utilized to prepare the compounds shown in formula II and formula III, and is cultured by a variety of conditions. The optimal obtaining period of the compounds is detected, and high yields of the compounds can be achieved. (formula II) (formula III).
Description
Technical field
The present invention relates to method and the special strain therefore thereof of the production of cyathane class diterpene compound 11-oxygen acetyl Bird's Nest alkane triol (11-O-acetylcyathatriol) and 15-oxygen acetyl Bird's Nest alkane triol (15-O-acetylcyathatriol).
Background technology
In several centuries in past, the medicine that derives from microorganism is being brought into play huge effect aspect the disease of the serious threat human healths such as treatment tumour and cause pathogeny imcrobe infection.Fungi is one of important monoid of microorganism, and its meta-bolites abundant species, structure type uniqueness make it become the micromolecular main source of natural organic active; The active substance of originated from fungus accounts for microbe-derived more than 50% (Journal of Antibiotics 2005,58,1 – 26).Derive from the microbiotic (such as penicillin etc.) of fungi, disease-resistant fungal pathogens preparation, immunosuppressor and decreasing cholesterol preparation be used for decades in the past clinical, for human resist the disease, promote longevity and made huge contribution.The strain that the sixties in last century H.J.Brodie finds is named as the bird's-nest fungus novel species of Cyathus helenae Brodie, can be good at the growth (Masters Thesis.University of Alberta, Sept.1967) of anti-bacteria; So the people such as W.A.Ayer are to its further research, find that the mycelial crude extract of this bacterium has preferably antibiotic activity, and therefrom separate the microbiotic (this compounds has 5/,6/7 three ring skeleton) obtain several called afters " cyathin ", all has good anti-microbial activity, can anti-bacteria, growth (the Canadian Journal of Chemistry 1971 such as actinomycetes, some fungies and human pathogenic bacteria, 17,1401-1407).Afterwards, the method that the people such as W.A.Ayer utilize biological activity to follow the tracks of is separated again the diterpene that obtains this monoid in other Cyathus sp., the compound quantity of the type is constantly increased.Kawagishi etc. last century the nineties from Hericium ernaceum, separate and obtain erinacine; From Sarcodon scabrosus, separate and obtain scabronine and sarcodonin(Canadian Journal of Chemistry 1973,51,3842-3854; Tetrahedron Lett.1971,13,1917-1920.), this three compounds has identical 5/,6/7 three ring skeleton with caythin, and the distribution range of such diterpene is enlarged, and is all significant to biosynthesizing and the activity research thereof of studying such diterpene.
Compound 11-oxygen acetyl Bird's Nest alkane triol (11-O-acetylcyathatriol) (formula II) and 15-oxygen acetyl Bird's Nest alkane triol (15-O-acetylcyathatriol) (formula III) are (Canadian Journal of Chemistry 1979 found in Cyathus earlei Lloyd at first, 57 (24), 3332-3337), but pure compound productive rate lower (be pure compound and crude extract mass ratio, separate obtaining 132mg 11-O-acetylcyathatriol and 310mg 15-O-acetylcyathatriol in the 7.7g crude extract).Find also to have 11-O-acetylcyathatriol(Bioscience at Hericium ernaceum again afterwards, Biotechnology, and Biochemistry 2004,68 (8), 1786-1789.), the same pure compound productive rate with Cyathus earlei Lloyd lower (about 0.6%, separate obtain this compound of 2.8mg from the 461mg crude extract).
In document " Metabolites of bird ' s nest fungi.Part 11.Diterpenoid metabolites of Cyathusearlei.; Can.J.Chem.; 1979; 57; 3332-3337. ", the author has determined the structure of active compound 11-O-acetylcyathatriol and 15-O-acetylcyathatriol by methods such as nucleus magnetic resonance, and them have been tested to the anti-microbial activity of Staphyloccocus aureus, the result shows that these two compounds have certain inhibition active to institute's test strain.
Tumour be one of disease of well-known serious harm human health (CA-Cancer J.Clin.2011,61,69-90).In at present numerous methods for the treatment of, because chemotherapeutics can arrive each one of whole body with blood circulation, (Nat.Rev.Cancer 2005 so chemotherapy (comprising immunotherapy) is being brought into play the effect that can not replace in primary carcinoma, secondary metastatic carcinoma and leukemic treatment, 5,65-72).It is single-minded that desirable chemotherapeutics should have action target spot, body internal specific target tumor tissue, and (Science 2001,293,58-59) for the characteristics such as mechanism of action is clear and definite, and the normal tissue toxic side effect is little.Because toxicity, side effect and the chemical sproof continuous appearance of traditional chemotherapeutics so that the anti-tumor chemotherapeutic curative effect reduces or curative effect lost efficacy, therefore, are sought new type antineoplastic medicine, and be extremely urgent.The focus of at present antitumor drug research and development is mainly transferred to new type antineoplastic medicine for the tumor signal Signal Transduction Pathways from traditional cytotoxic drug.
In the patent literature, all do not relate to the anti-tumor activity report of 11-O-acetylcyathatriol and 15-O-acetylcyathatriol compound at home and abroad.
Summary of the invention
The purpose of this invention is to provide a kind of microbial fermentation preparation formula II(11-O-acetylcyathatriol that utilizes) and/or formula III (15-O-acetylcyathatriol) shown in method and the special strain therefore thereof of compound.
(formula II) (formula III)
Special strain therefore provided by the present invention is C.africanus (Cyathus africanus) L38, and this strains separation is from Wutai, Shanxi Province, and the basidioma of L38 bacterial strain is back taper, high 6-8mm, the wide 9-11*6.5-8 μ of oral area m; Coated without obvious vertical stripe; Parcel tool individual layer cortex and film, oblate, 1.9-2.5*1.8-2.0mm; Sporidium is avette to have a little point, 9-12.0*6.5-8.0.
C.africanus (Cyathus africanus) L38 is on 07 04th, 2012, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (be called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), deposit number is CGMCC NO.6259.
The application of fermentation C.africanus (Cyathus africanus) L38 CGMCC NO.6259 in compound (15-O-acetylcyathatriol) shown in compound shown in the preparation formula II (11-O-acetylcyathatriol) and/or the formula III also belongs to protection scope of the present invention.
The present invention also provides the method for utilizing compound (15-O-acetylcyathatriol) shown in compound (11-O-acetylcyathatriol) shown in fermentation C.africanus (Cyathus africanus) the L38 CGMCC NO.6259 preparation formula II and the formula III.
The method of compound (15-O-acetylcyathatriol) shown in compound shown in the described preparation formula II (11-O-acetylcyathatriol) and the formula III may further comprise the steps:
1) C.africanus (Cyathus africanus) the L38 CGMCC NO.6259 that will ferment is inoculated into and carries out solid fermentation on the solid medium and cultivate;
2) behind the solid fermentation of step 1), add organic solvent extraction to substratum, obtain extracting solution, with described extracting solution underpressure distillation, obtain crude extract;
Wherein, in the step 1), solid medium is mixed to get by base material and water; Optional one or more arbitrary combination in wheat, polished rice, corn, glutinous rice, long-grained nonglutinous rice and the seed of Job's tears of described base material; The water content of described solid medium is the 100-150% of described base material gross weight.
In the described step 1), the condition that described solid fermentation is cultivated is 18-28 ℃, the static cultivation of lucifuge 20-60 days.The condition optimization that described solid fermentation is cultivated is 25 ℃, static cultivation 30 days.
Described step 2) in, described organic solvent is any one in ethyl acetate, acetone, methyl alcohol, ethanol, chloroform, Virahol and the propyl carbinol.
In the described method, also comprise and described crude extract is carried out the normal pressure silica gel column chromatography or gel chromatography separation obtains compound (15-O-acetylcyathatriol) shown in compound shown in the formula II (11-O-acetylcyathatriol) and/or the formula III.
Wherein, the method of normal pressure silica gel column chromatography, take the mixed solution of chloroform and acetone as solvent, that the gradient of 100:0,100:1,50:1,30:1,20:1,10:1,5:1,3:1,2:1 is carried out gradient elution successively according to the ratio of chloroform in the solvent and acetone, 1500 milliliters of each gradient elution solvents, per 350 milliliters are collected as a stream part; Be to be dissolved in the 10 ml methanol solution after second the stream part concentrating under reduced pressure drying of eluting solvent wash-out of 10:1 the ratio of chloroform and acetone, room temperature leaves standstill, and separates out colourless crystallization, obtains the compound shown in the formula II; Be to be dissolved in the 10 ml methanol solution after the 3rd the stream part concentrating under reduced pressure drying of eluting solvent wash-out of 10:1 the ratio of chloroform and acetone, the room temperature hold over night is separated out colourless crystallization, obtains the compound shown in the formula III.The used filler of described normal pressure silica gel column chromatography (silica gel column chromatography separation) is 200-300 purpose silica gel particle, and filler is 150g, and the silicagel column specification is Φ 4.5 * 80cm.
In the described step 1), also be included in and C.africanus (Cyathus africanus) L38CGMCC NO.6259 carried out preculture at the PDA solid medium before the solid fermentation, get mycelium after the preculture and be transferred in the liquid nutrient medium and cultivate; Pre-incubated condition is: 25-28 ℃, lucifuge was cultivated 5-7 days; The condition of liquid culture is: 25-28 ℃, lucifuge was cultivated 7-10 days.
Consisting of of described PDA substratum: potato 200g, glucose 20g, agar powder 16g, water is settled to 1L; Consisting of of described liquid nutrient medium: glucose 4.0g/L, malt extract 10.0g/L, yeast 4.0g/L, water is settled to 1L.
The present invention utilizes superior strain C.africanus (Cyathus africanus) L38 fermentation preparation compound 11-O-acetylcyathatriol and 15-O-acetylcyathatriol, and adopt multiple condition to cultivate to this bacterium, detect the best of this compound and obtained period, all can obtain higher pure compound productive rate (be pure compound and crude extract mass ratio, wherein compound 11-O-acetylcyathatriol is generally more than 12%).Can realize the optimal yield of compound, and then have a good application prospect.
Embodiment
The below is described in further detail the present invention with embodiment, but content of the present invention is not limited to this.
Experimental technique described in the following embodiment if no special instructions, is ordinary method; Described reagent and biomaterial if no special instructions, all can obtain from commercial channels.
Embodiment 1, the acquisition of producing bacterial strain C.africanus (Cyathus africanus) L38 of cyathane class diterpene compound 11-O-acetylcyathatriol and 15-O-acetylcyathatriol
One, the separation of C.africanus (Cyathus africanus) L38
The inventor is on the high flux screening basis of Secondary Metabolites of Microorganisms active compound, and the extract that separates the solid fermentation of a strain C.africanus (Cyathus africanus) that obtains from Wutai, Shanxi Province Cyathus africanus sporophore has preferably anti-tumor activity; By the further separation and purification under the active tracking instruction, it is higher and easily be purified to the bacterial strain of compound to obtain output, and with this bacterial strain called after L38.
Specifically separation method is, at first, with ethanol for disinfection the sporophore that gathers is carried out surface sterilization, uses sterile water wash again, then, parcel in the sporophore is cut into small pieces, and is last, is seeded on the PDA substratum, and separation and purification obtains bacterial strain.
Two, the evaluation of C.africanus (Cyathus africanus) L38.
1, physiology and morphology biochemical identification
The basidioma of L38 bacterial strain is back taper, high 6-8mm, and the wide 5-8mm of oral area, the mycelia pad of base portion is obvious, diameter 5.5mm; Coated outside light color, inboard light brown, nothing is vertical stripe obviously, and peristoma is smooth without tasselled; Parcel tool individual layer cortex and film, oblate, 1.9-2.5*1.8-2.0mm; Sporidium, avette, have a little point, 9-12.0*6.5-8.0 μ m.
This L38 bacterial strain meets the feature that following C.africanus (Cyathus africanus) pattern DAOM200370 describes:
C.africanus (Cyathus africanus) belongs to Genus Cyathus (Cyathus), and its Main Morphology feature is that basidioma is cup-shaped, turbination or doline, high 5-8mm, and the wide 5-8mm of oral area, diameter can reach 6mm, base portion tool mycelia pad; Be coated with three layers (middle for paraplectenchyma), outside sandy soil look, dirty brown color, olive colour, light brown to brown, powder is yellow, yellowish by having, the thin wool of sandy soil look, brown color, often is close to coated wall, sometimes also forms tuftlet, smoothly without striped; The inboard is generally slightly more shallow than the outside, and most level and smooth, peristoma is smooth.A white thin epiphragma is stamped at the top, generally disappears when ripe.Parcel is flat, wide ellipse, and 2-2.5*1.8-2.3mm, grey or black, tool individual layer cortex, one deck is pale yellow to beige film.Sporidium is avette, the little point of most end tools, and 8.5-11.0 (14.0) * (5.5-) 6.5-8.0 (9.0) μ m wall is thinner, colourless, non-starchiness, general wall thickness; Hyphae colorless has obvious clamp connexion; Give birth on ground, rotten wood is given birth to, excrement is given birth to or be born on the litter layer.
Molecular Identification to the L38 bacterial strain shows, the sequence of its 18Sr DNA is shown in sequence in the sequence table 1, with American National biotechnology information center (NCBI) in accession number be the sequence similarity degree of DQ463342.1 C.africanus, reach 100%, according to above morphology, physiological and biochemical property and 18Sr DNA, bacterial strain L38 is accredited as C.africanus (Cyathus africanus).
C.africanus (Cyathus africanus) L38 is on 07 04th, 2012, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (be called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), deposit number is CGMCC NO.6259.
Embodiment 2, utilize C.africanus (Cyathus africanus) L38 to produce 11-O-acetylcyathatriol and 15-O-acetylcyathatriol
One, bacterial strain activation, fermentation culture
1, the acquisition of seed liquor:
C.africanus (Cyathus africanus) L38 is inoculated on the PDA test tube nutrient agar inclined-plane, carries out preculture; Pre-incubated condition is: 25 ℃, lucifuge is cultivated 7 days (the PDA substratum forms: potato 200g, glucose 20g, agar powder 16g, pure water are settled to 1L (pH nature)).After mycelia is covered with whole inclined-plane, under aseptic condition, mycelium is transferred in the liquid nutrient medium and cultivates, obtain seed culture fluid; The condition of liquid culture is: 25 ℃, lucifuge was cultivated 7 days.Liquid nutrient medium: glucose 4.0g/L, malt extract (Malt Extract) 10.0g/L, yeast (Yeast Extract) 4.0g/L, water is settled to 1L; Yeast extract (Yeast Extract) is available from Oxoid Ltd., and lot number is 1074139.
2, solid fermentation is cultivated:
Get 10 milliliters of seed culture fluids and be inoculated into respectively through being equipped with in solid medium 500 milliliters of triangular flasks of (solid medium is comprised of 80g long-grained nonglutinous rice and 120ml water) of sterilizing, inoculate 10 bottles, 25 ℃, lucifuge fermentation culture 30 days.(mycelium dry weight after each triangular flask fermentation is 75.5g).
Two, the extraction of compound separates
1, organic solvent extraction
Solid fermentation is added to 300 milliliters of ethyl acetate respectively in each triangular flask after finishing, and soak at room temperature extracts a week, repeat to extract 3 times, combined ethyl acetate extracting solution vacuum distillation drying gets medicinal extract, i.e. 12.5g ethyl acetate extract (extraction yield is 12.5/755=1.66%).
2, the separation and purification of compound
Ethyl acetate extract is opened column chromatography (200-300 of Qingdao Marine Chemical Co., Ltd. order, column chromatography silica gel 150g through silica gel; Φ 4.5 * 80cm) separates, and chloroform-acetone mixed solvent gradient is washed (100:0,100:1,50:1,30:1,20:1,10:1,5:1,3:1,2:1; Volume ratio), 1500 milliliters of each gradient elution solvents, per 350 milliliters are collected as a stream part.Be dissolved in the 10 ml methanol solution after second the stream part concentrating under reduced pressure drying with chloroform-acetone 10:1 wash-out, the room temperature hold over night, separate out colourless crystallization, obtain compound shown in the formula II (11-O-acetylcyathatriol, 2.1g(pure compound productive rate is 2.1/12.5=16.8%)).Be dissolved in the 10 ml methanol solution after the 3rd the stream part concentrating under reduced pressure drying with chloroform-acetone 10:1 wash-out, the room temperature hold over night is separated out colourless crystallization, obtains the compound (15-O-acetylcyathatriol shown in the formula III, 0.32g the pure compound productive rate is 0.32/12.5=2.56%).
3, compound structure conclusive evidence
Formula II compound: clear crystal; The specific rotatory power value
HRESIMS m/z385.2357[M+Na]
+The hydrogen spectrum (
1H-NMR) and carbon spectrum (
13C-NMR) see Table 1.According to the physico-chemical property of compound, hydrogen spectrum, carbon spectrum data, and with the reference numeric ratio to (Can.J.Chem., 1979,57 (24), 3332-3337; Biosci.Biotechnol.Biochem., 2004,68 (8), 1786-1789.), determine that the structure of this compound is identical with 11-O-acetylcyathatriol.
Formula III compound: colorless oil; The specific rotatory power value
HRESIMS m/z385.2340[M+Na]
+The hydrogen spectrum (
1H-NMR) see Table 1.According to the physico-chemical property of compound, hydrogen spectrum data, and with the reference numeric ratio to (Can.J.Chem., 1979,57 (24), 3332-3337), determine that the structure of this compound is identical with 15-O-acetylcyathatriol.
The NMR data of table 1. compound
a?
1H?NMR?at?500Hz,
13C?NMR?at?125Hz?in?CD
3OD.
b?
1H?NMR?at?500Hz?in?CDCl
3.
Embodiment 3, utilize C.africanus (Cyathus africanus) L38 to produce 11-O-acetylcyathatriol and 15-O-acetylcyathatriol
Embodiment 3 methods are with embodiment 2, difference: one, and the solid culture based formulas, its prescription is: the 80g seed of Job's tears, 120ml distilled water; Two, incubation time is also different, is 40 days; Three, the mycelium dry weight after each triangular flask fermentation is 74.3g; Four, solvent for use is acetone in the organic solvent extraction, obtains 13.2g extract (extraction yield is 13.2/743=1.77%).Separate according to the method for the step 2 of embodiment 2 and to obtain compound (11-O-acetylcyathatriol shown in the formula II, 1.7g(the pure compound productive rate is 12.9%)), compound shown in the formula III (15-O-acetylcyathatriol, 0.31g, pure compound productive rate are 2.35%)
Embodiment 4, utilize C.africanus (Cyathus africanus) L38 to produce 11-O-acetylcyathatriol and 15-O-acetylcyathatriol
Embodiment 4 methods are with embodiment 2, difference: one, and the solid culture based formulas, its prescription is: corn 40g, wheat 40g, 120ml distilled water; Two, the mycelium dry weight after each triangular flask fermentation is 75.7g; Three, solvent for use is methyl alcohol in the organic solvent extraction, obtains 15.0g extract (extraction yield is 15.0/757=1.98%).Compound shown in the formula II (11-O-acetylcyathatriol, 1.83g(pure compound productive rate is 12.2%)), the compound shown in the formula III (15-O-acetylcyathatriol, 0.28g, pure compound productive rate are 1.87%)
Embodiment 5, utilize C.africanus (Cyathus africanus) L38 to produce 11-O-acetylcyathatriol and 15-O-acetylcyathatriol
Embodiment 5 methods are with embodiment 2, difference: one, and the solid culture based formulas, its prescription is: corn 80g, 100ml distilled water; Two, incubation time is also different, is 40 days; Three, the mycelium dry weight after each triangular flask fermentation is 76.4g; Four, solvent for use is ethanol in the organic solvent extraction, obtains 17.7g extract (extraction yield is 17.7/764=2.32%).Compound shown in the formula II (11-O-acetylcyathatriol, 2.2g(pure compound productive rate is 12.4%)), the compound shown in the formula III (15-O-acetylcyathatriol, 0.44g, pure compound productive rate are 2.45%)
Claims (8)
1. C.africanus (Cyathus africanus) L38, its deposit number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC NO.6259.
2. C.africanus (Cyathus africanus) L38 CGMCC NO.6259 is in the application in the compound shown in compound and/or the formula III shown in the preparation formula II;
(formula II) (formula III)
3. the method for compound shown in compound shown in the preparation formula II and the formula III may further comprise the steps:
1) C.africanus (Cyathus africanus) the L38 CGMCC NO.6259 that will ferment is inoculated into and carries out solid fermentation on the solid medium and cultivate;
2) add organic solvent in the system after the step 1) fermentation and soak the extracting solution that extraction obtains containing compound shown in compound shown in the formula II and the formula III, with described extracting solution underpressure distillation, obtain containing the crude extract of compound shown in compound shown in the formula II and the formula III;
Wherein, in the step 1), described solid medium is mixed to get by base material and water, and described base material is selected from one or more arbitrary combination in wheat, polished rice, corn, glutinous rice, Gorgon fruit, long-grained nonglutinous rice and the seed of Job's tears; The water content of described solid medium is the 100-150% of described base material gross weight.
4. method according to claim 3 is characterized in that: in the described step 1), the culture condition that described solid fermentation is cultivated is 18-28 ℃, the static cultivation of lucifuge 20-60 days.
5. method according to claim 4 is characterized in that: in the described step 1), the culture condition that described solid fermentation is cultivated is 25 ℃, the static cultivation of lucifuge 30 days.
6. according to claim 4 or 5 described methods, it is characterized in that: described step 2), described organic solvent is any one in ethyl acetate, acetone, methyl alcohol, ethanol, chloroform, Virahol and the propyl carbinol.
7. the described method of any one according to claim 3-6, it is characterized in that: in the described method, comprise that also the crude extract that step 2 is obtained separates with silica gel column chromatography, take the mixed solution of chloroform and acetone as solvent, that the gradient of 100:0,100:1,50:1,30:1,20:1,10:1,5:1,3:1,2:1 is carried out gradient elution successively according to the ratio of chloroform in the solvent and acetone, 1500 milliliters of each gradient elution solvents, per 350 milliliters are collected as a stream part; Be to be dissolved in the 10 ml methanol solution after second the stream part concentrating under reduced pressure drying of eluting solvent wash-out of 10:1 the ratio of chloroform and acetone, room temperature leaves standstill, and separates out colourless crystallization, obtains the compound shown in the formula II; Be to be dissolved in the 10 ml methanol solution after the 3rd the stream part concentrating under reduced pressure drying of eluting solvent wash-out of 10:1 the ratio of chloroform and acetone, the room temperature hold over night is separated out colourless crystallization, obtains the compound shown in the formula III.
8. method according to claim 7, it is characterized in that: in the described step 1), also be included in and C.africanus (Cyathus africanus) L38 CGMCC NO.6259 carried out preculture at the PDA solid medium before the solid fermentation, get mycelium after the preculture and be transferred in the liquid nutrient medium and cultivate; Pre-incubated condition is: 25-28 ℃, lucifuge was cultivated 5-7 days; The condition of liquid culture is: 25-28 ℃, lucifuge was cultivated 7-10 days;
Consisting of of described PDA substratum: potato 200g, glucose 20g, agar powder 16g, water is settled to 1L; Consisting of of described liquid nutrient medium: glucose 4.0g/L, malt extract 10.0g/L, yeast 4.0g/L, water is settled to 1L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210352773.7A CN102851218B (en) | 2012-09-20 | 2012-09-20 | Production method of 11-O-acetylcyathatriol and 15-O-acetylcyathatriol, and special bacterial strain thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210352773.7A CN102851218B (en) | 2012-09-20 | 2012-09-20 | Production method of 11-O-acetylcyathatriol and 15-O-acetylcyathatriol, and special bacterial strain thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102851218A true CN102851218A (en) | 2013-01-02 |
| CN102851218B CN102851218B (en) | 2014-01-01 |
Family
ID=47398198
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210352773.7A Expired - Fee Related CN102851218B (en) | 2012-09-20 | 2012-09-20 | Production method of 11-O-acetylcyathatriol and 15-O-acetylcyathatriol, and special bacterial strain thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102851218B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472326A (en) * | 2003-06-03 | 2004-02-04 | 云南大学 | Metabolist product of cyathus against mycobacterium tuberculosis and preparation thereof |
| WO2007123027A1 (en) * | 2006-04-10 | 2007-11-01 | Geol Chemical Co., Ltd. | Anti-cancer agent comprising cyathane derivative |
| KR100811614B1 (en) * | 2006-09-29 | 2008-03-31 | 한국생명공학연구원 | Antioxidant extract of fermented mushroom cyathus stercoreus, antioxidant compounds isolated therefrom and a method for preparation of the same |
| CN101306998A (en) * | 2007-10-26 | 2008-11-19 | 河南农业大学 | Nest alkane type diterpenoid and abstraction and separation method thereof |
| WO2011151831A1 (en) * | 2010-06-03 | 2011-12-08 | Carmel-Haifa University Economic Corporation Ltd | Extracts of cyathus striatus mushrooms, pharmaceutical compositions comprising them and a new cyathus striatus strain |
-
2012
- 2012-09-20 CN CN201210352773.7A patent/CN102851218B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472326A (en) * | 2003-06-03 | 2004-02-04 | 云南大学 | Metabolist product of cyathus against mycobacterium tuberculosis and preparation thereof |
| WO2007123027A1 (en) * | 2006-04-10 | 2007-11-01 | Geol Chemical Co., Ltd. | Anti-cancer agent comprising cyathane derivative |
| KR100811614B1 (en) * | 2006-09-29 | 2008-03-31 | 한국생명공학연구원 | Antioxidant extract of fermented mushroom cyathus stercoreus, antioxidant compounds isolated therefrom and a method for preparation of the same |
| CN101306998A (en) * | 2007-10-26 | 2008-11-19 | 河南农业大学 | Nest alkane type diterpenoid and abstraction and separation method thereof |
| WO2011151831A1 (en) * | 2010-06-03 | 2011-12-08 | Carmel-Haifa University Economic Corporation Ltd | Extracts of cyathus striatus mushrooms, pharmaceutical compositions comprising them and a new cyathus striatus strain |
Non-Patent Citations (4)
| Title |
|---|
| JUNJIE HAN ET AL: "Anti-inflammatory and cytotoxic cyathane diterpenoids from the medicinal fungus Cyathus africanus", 《FITOTERAPIA》, vol. 84, 14 October 2012 (2012-10-14), pages 22 - 31 * |
| WILLIAM A.AYER ET AL: "Metabolites of bird"s nest fungi. Part 9. Diterpenoid metabolites of Cyathusafricanus Brodie", 《CAN.J.CHEM》, 31 December 1979 (1979-12-31), pages 2113 - 2120 * |
| WILLIM A.AYER: "Metabolites of bird"s nest fungi. Part 11. Diterpenoid metabolites of Cyathusearlei Lloyd", 《CAN.J.CHEM.》, 31 December 1979 (1979-12-31), pages 3332 - 3337 * |
| 韩俊杰 等: "一株非洲黑蛋巢菌中鸟巢烷型二萜的结构及活性研究", 《2012年中国菌物学会学术年会会议摘要》, 10 August 2012 (2012-08-10), pages 154 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102851218B (en) | 2014-01-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102154116B (en) | Endophytic fungus Phomopsis sp. and use thereof | |
| CN101445784B (en) | Eupenicillium brefeldianum variety ZJB082702 and application thereof in preparation of Brefeldin A by fermentation | |
| CN103255061B (en) | Penicillium griseofulvum, antibacterial active compound generated thereby and application | |
| CN107353274A (en) | Come from the secalonic acid I of penicillium oxalicum and prepare the application of anti-human oesophagus cancer drug | |
| CN103937678A (en) | Marine fungi penicillium crustosum bacterial strain, quinolinone compounds derived from marine fungi penicillium crustosum, and preparation and applications of quinolinone compounds | |
| CN107298672A (en) | The secalonic acid I for coming from penicillium oxalicum is preparing the application of anti-human colon cancer drug | |
| CN101857841B (en) | Marine fungi aspergillus unguis strain, active extract thereof and preparation method and use of active extract thereof and active components thereof | |
| CN101720772B (en) | Macrolide composition for preventing and controlling fungal disease of crop and preparation process thereof | |
| CN102226152B (en) | Cylindrocarpon didymium and method for preparing ginsenoside Rh2 by using same | |
| CN102846588B (en) | Application of cyathane diterpene compound in antitumor drug | |
| CN107298669A (en) | Come from the secalonic acid I of penicillium oxalicum and anti-human oral cavity epidermoid carcinoma medicinal application | |
| CN101720781A (en) | New phosphorus and nitrogen mycin A for preventing and controlling fungal disease of crop and preparation process thereof | |
| CN103283482B (en) | Cordyceps militaris, cultivation method and separation method | |
| CN102851218B (en) | Production method of 11-O-acetylcyathatriol and 15-O-acetylcyathatriol, and special bacterial strain thereof | |
| CN103146594B (en) | Sorangiumcellulosum strain and application thereof to synthesis of epothilone | |
| CN111689895B (en) | Two-branch chain isomerization piericins compound and application thereof in preparation of anti-renal cancer drugs | |
| CN105316238B (en) | A method of the culture screening taxol-producing fungi from Chinese yew | |
| CN100387609C (en) | Novel antibiotic preparing process | |
| CN107312014B (en) | A kind of mould chlorins compound of lattice Féraud and its preparation method and application | |
| CN108660169A (en) | A method of fermentation prepares spine spore bacteriums antibiotic | |
| CN113402453B (en) | Pyridone piericin, preparation method thereof and application thereof in preparation of anti-cancer drugs | |
| CN107058137A (en) | A kind of wet graceful mould and its method for producing wortmannin | |
| Badal et al. | Fungal ms: a focus on endophytes | |
| CN101709058B (en) | Polyene macrolides compound, preparation and application thereof | |
| CN107129936B (en) | A kind of paclitaxel produced mould BP6T3 and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140101 |