CN106047715A - Nothapodytes pittosporoides endophyte trichoderma sp. strain and method thereof for extracting camptothecin - Google Patents

Nothapodytes pittosporoides endophyte trichoderma sp. strain and method thereof for extracting camptothecin Download PDF

Info

Publication number
CN106047715A
CN106047715A CN201610316234.6A CN201610316234A CN106047715A CN 106047715 A CN106047715 A CN 106047715A CN 201610316234 A CN201610316234 A CN 201610316234A CN 106047715 A CN106047715 A CN 106047715A
Authority
CN
China
Prior art keywords
trichoderma
gzu
bcec
strain
extract
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.)
Pending
Application number
CN201610316234.6A
Other languages
Chinese (zh)
Inventor
雷帮星
康冀川
吴璇
钱鑫
钱一鑫
文庭池
周思璇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guizhou University
Original Assignee
Guizhou University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Guizhou University filed Critical Guizhou University
Priority to CN201610316234.6A priority Critical patent/CN106047715A/en
Publication of CN106047715A publication Critical patent/CN106047715A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/885—Trichoderma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Botany (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to the technical field of microorganisms, in particular to a nothapodytes pittosporoides endophyte trichoderma sp. strain and a method thereof for extracting camptothecin. The nothapodytes pittosporoides endophyte is separated from living nothapodytes pittosporoides plants which are collected from suburbs of Guiyang, Guizhou Province by virtue of an endophyte separation technology; the strain is identified by microbial systematics and is named as trichoderma sp. GZU-BCEC-GX8, and the strain is preserved in China Center for Type Culture Collection on September 14, 2015 with preservation number of CCTCC NO: M2015529; the trichoderma sp. GZU-BCEC-GX8 strain disclosed by the invention has the most most remarkable characteristic that the strain has an effect of generating the camptothecin; and in combination with the method for extracting the camptothecin from the strain provided by the invention, another source is provided for the camptothecin and derivatives thereof; therefore, a microbial strain is provided for developing a new antineoplastic medicine source in medicine.

Description

A kind of Cortex Nothapodytis Radicis endophyte Trichoderma strain and the method extracting camptothecine thereof
Technical field
The present invention relates to microbial technology field, be specifically related to the raw Trichoderma strain of bacterium and extraction happiness thereof in a kind of Cortex Nothapodytis Radicis The method of tree alkali.
Background technology
The most over one hundred kind of the cancer therapy drug of world community approved listing at present.In numerous anticarcinogens, botanical anticancer Medicine is big class leading products in International Contre pharmaceutical market.Wherein camptothecine (Camptothecin, CPT) is famous anticancer One of lead compound of medicine is it is considered to be there is one of phytochemicals production anticarcinogen of development and application prospect.
Camptothecine belongs to neutrophilous organism alkali, and camptothecine has inhibitory action to the growth of many tumors, is applied to digestive tract Tumor, leukemia, chorionic epithelioma, bladder cancer treatment on have remarkable result.At present, camptothecine is from the fruit of Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae) Extracting, its source is limited, and content is low.Along with the continuous extension of research, find that the camptothecine content of Cortex Nothapodytis Radicis is higher, In the Cortex Nothapodytis Radicis of some areas plantation, camptothecine content is up to dry weight 0.3%.
Traditional camptothecine extracting method, mainly uses ion-exchange-resin process directly to extract from Cortex Nothapodytis Radicis, wherein, Employing ion exchange resin column chromatographs, and cation exchange resin is too strong to camptothecine absorption, need to wash with alkaline water liquid De-, under strong base solution, partial amino-acid and protein solubility increase, and absorption these macromole impurity in post are also eluted Getting off, cause product purity low, and eluent is high due to protein content, in concentration process, product is easy to foaming, adds concentration Difficulty.
In the prior art, occurred and utilized fermentable extractive technique.Such as Patent No. 201110368489.4 Disclose and a kind of utilize solid microbe fermentation technology to extract the method for camptothecine in Cortex Nothapodytis Radicis, main use inoculating microbe to enter Row fermented extracted.
And about Cortex Nothapodytis Radicis endogenetic fungus produce camptothecine research less, further relate to Cortex Nothapodytis Radicis endogenetic fungus point From and the research report of Cortex Nothapodytis Radicis endogenous fungus metabolite more rarely have.
Summary of the invention
The present invention solves above-mentioned technical problem, it is provided that a kind of Cortex Nothapodytis Radicis endophyte Trichoderma strain and extraction camptothecine thereof Method.
It is achieved especially by below scheme:
A kind of Cortex Nothapodytis Radicis endophyte Trichoderma strain, isolated from Cortex Nothapodytis Radicis, named trichoderma (Trichoderma sp.) GZU-BCEC-GX8 bacterial strain, is preserved in China typical culture collection center, and preservation date is 2015 On JIUYUE 14, deposit number is CCTCC NO:M2015529；Depositary institution address: Wuchang District, Wuhan City, Hubei Province Bayi Road No. 299 Wuhan Universitys in the school, Wuhan University's preservation center.
Described trichoderma (Trichoderma sp.) GZU-BCECX-GX8 bacterial strain, wherein, the separation and Culture of this bacterial strain Method comprises the following steps:
(1) preparation of tissue: take the fresh young stem of Cortex Nothapodytis Radicis, leaf, petiole, bud, root bark, root core one or more, use Tap water rinses, and removes the raw microorganism of table of sample surfaces；
(2) surface sterilization and Sterility testing: be placed on superclean bench by tissue, the tween 80 sterilized water with 1 ‰ is by material Material is washed 2～3 times, then is washed till non-foam with sterilized water, after sucking tissue surface moisture with aseptic filter paper, is soaked in 75% ethanol molten Liquid 3min, then with aseptic water washing 2-3 time, then use 0.1%HgCl2Solution soaking 1～3min, then rushes extremely with sterilized water Flushing water is aseptic, after blotting tissue segments remained on surface water droplet with aseptic filter paper, is cut into a length of 0.5cm, and the tissue of a width of 0.5cm is cut Section；
(3) endogenetic fungus is isolated and purified: the tissue cutting after step (2) being sterilized is inoculated in dual anti-PDA++Culture medium is put down On plate, constant temperature culture 4～7 days under the conditions of being placed in 25-30 DEG C, grow from piece of tissue to mycelium, use edge mycelia picking Method, is transferred to mycelia in PDA medium slant, carries out constant temperature culture, treat that bacterium colony grows under the conditions of 25-30 DEG C, then transfers 2 ～3 times, to ensure that gained bacterium colony, as pure culture, obtains Cortex Nothapodytis Radicis endogenetic fungus.
Described PDA culture medium is peeled potatoes 200g, glucose 20g, agar 20g.Rhizoma Solani tuber osi is cut into small pieces, adds water Boil 30min, four layers of filtered through gauze, filtrate is added distilled water and complements to 1L, 121 DEG C, sterilizing 30min.
Described dual anti-PDA++Culture medium contains streptomycin that concentration is 40U/mL and concentration is the penicillin of 30U/mL.
The extracting method of camptothecine in described trichoderma (Trichoderma sp.) GZU-BCECX-GX8 metabolite, Comprise the following steps:
A: liquid fermentation and culture: by the trichoderma GZU-BCECX-GX8 inoculation of separation preservation in PDA culture medium, Under the conditions of 28 DEG C, cultivate 5d, be then inoculated in the triangular flask equipped with PD culture medium or fermentation medium, then by three Angle bottle is placed on shaking table, at 120 ± 3rpm, cultivates under the conditions of 28 ± 2 DEG C, obtains strain fermentation product；
B: the method extracting camptothecine in metabolite:
1. the strain fermentation product of step (1) gained is used buchner funnel vacuum filtration, separate to obtain mycelium and fermentation Liquid；Use distilled water flushing mycelium, mycelium is placed under conditions of temperature is 45 ± 3 DEG C and dries, ground, 60 mesh sieves excessively, Obtain mycelium powder；Fermentation liquid is concentrated in vacuo under conditions of temperature is 45 ± 3 DEG C the 10%-20% of original volume, obtains concentration Fermentation liquid；
2. in concentrated broth, add 95% ethanol of 3 times of volumes, precipitate with ethanol, stand overnight under low temperature, buchner funnel sucking filtration Removal macromole precipitates, and takes supernatant and is concentrated in vacuo to former concentration volume in 45 ± 3 DEG C, obtains concentrating and impurity removing fermentation liquid；
3. take 1mL concentrating and impurity removing fermentation liquid, add extractant 2-4mL, after ultrasonic extraction 30min, lucifuge vibration 12h, then Ultrasonic extraction 30min, stands, after extract and separation of fermentative broth, it is thus achieved that extract A；Extractant 2-is added again in fermentation liquid 4mL, ultrasonic extraction 30min, stand, after extract and separation of fermentative broth, it is thus achieved that extract B；
4. taking 1g mycelium powder, add extractant 2-5mL, after being placed in ultrasonic extraction 30min, lucifuge stands 12h, more ultrasonic Extraction 30min；Filter, it is thus achieved that extract C and filtering residue；Take filtering residue, add extractant 2-5mL, ultrasonic extraction 30min, filter, obtain Obtain extract D；
5. trichoderma (Trichoderma sp.) GZU-BCECX-GX8 bacterial strain is i.e. obtained after extract A, B, C and D being mixed The extract of camptothecine in metabolite.
PD culture medium in described step A is removal agar composition in PDA culture medium；Fermentation medium is carbon source 10- 25g, nitrogen source 0.5-2.5g, K2HPO41g, MgSO41g, 1L supplied by distilled water；Described PD culture medium or fermentation medium are three Liquid amount in the bottle of angle is the 30% of triangular flask volume.
Any one during the carbon source of fermentation medium is soluble starch, glucose and mannitol in described step A, nitrogen Source is (NH4)2SO4, peptone and NH4Any one in Cl.
In described step A, incubation time is 7～14 days.
Described extractant is that the ratio of (1-4) 1 is made by volume by chloroform, methanol.
Described ultrasonic extraction, its power is 90W, frequency is 59KHz.
Beneficial effects of the present invention
One be the present invention separate from Cortex Nothapodytis Radicis metabolite have produce camptothecine effect trichoderma (Trichoderma sp.) GZU-BCECX-GX8 bacterial strain, this can be as the way, an other source of camptothecin analogues Footpath, this developing in pharmaceutically anti-cancer agent source provides a kind of microbial strains.
Two is to the invention provides to utilize trichoderma (Trichoderma sp.) GZU-BCECX-GX8 metabolite to extract The method of camptothecine, and camptothecine can be applicable to the preparation of antitumor drug, the exploitation newly originated for medical adds newly Approach.
Accompanying drawing explanation
Fig. 1: the colonial morphology of bacterial strain GZU-BCEC-GX8；
Fig. 2: the conidial fructification of bacterial strain GZU-BCEC-GX8.
Detailed description of the invention
Below in conjunction with specific embodiment, technical scheme is further limited, but claimed Scope is not only limited to description.
Embodiment 1 (separation of trichoderma (Trichoderma sp.) GZU-BCECX-GX8)
The present invention gathers medicinal plants Cortex Nothapodytis Radicis root core in March, 2014 in suburb, Guiyang City, Guizhou Province, through surface The steps such as sterilization, endogenetic fungus separate, cultivate, ferment, morphologic observation and screening, therefrom obtain trichoderma (Trichoderma Sp.) GZU-BCECX-GX8, preserves.
The preparation of culture medium:
PDA culture medium: Rhizoma Solani tuber osi 200g, glucose 20g, agar 20g；Rhizoma Solani tuber osi after peeling is cut into small pieces, adds water Boil 30min, four layers of filtered through gauze, filtrate is added distilled water and complements to 1L；Described sterilising conditions: 121 DEG C, 30min；
Dual anti-PDA++ culture medium: be cooled to 45 DEG C before subpackage after PDA medium sterilization, by streptomycin before not solidifying 40U/mL, penicillin 30U/mL consumption adds PDA culture medium, and mixing is down flat plate；Described sterilising conditions: 121 DEG C, 30min；
Cha Shi solid medium: NaNO32g, K2HPO41g, KCl 0.5g, MgSO40.5g, FeSO40.01g, sucrose 30g, agar 20g, distilled water 1L, pH value is natural；Described sterilising conditions: 121 DEG C, 30min；
Concrete isolated culture method is:
(1) preparation of tissue: take the fresh young stem of Cortex Nothapodytis Radicis, leaf, petiole, bud, root bark, root core one or more, use Tap water rinses, and removes the raw microorganism of table of sample surfaces；
(2) surface sterilization and Sterility testing: be placed on superclean bench by tissue, the tween 80 sterilized water with 1 ‰ is by material Material is washed 2 times, then is washed till non-foam with sterilized water, after sucking tissue surface moisture with aseptic filter paper, is soaked in 75% ethanol solution 3min, then with aseptic water washing 3 times, then use 0.1%HgCl2Solution soaking 2min, then with aseptic water washing to rinsing Water is aseptic, after blotting tissue segments remained on surface water droplet with aseptic filter paper, is cut into the tissue cutting of a length of 0.5cm, a width of 0.5cm；
(3) endogenetic fungus is isolated and purified: the tissue cutting after step (2) being sterilized is inoculated in dual anti-PDA++Culture medium is put down On plate, described dual anti-PDA++Culture medium is to be cooled to 45 DEG C, by strepto-before not solidifying before subpackage after PDA medium sterilization Element 40U/mL, penicillin 30U/mL consumption adds PDA culture medium, and mixing is down flat plate；It is placed in 28 DEG C of constant temperature culture 7 days, to mycelia Body grows from piece of tissue, uses edge mycelia picking method, is transferred to by mycelia in PDA medium slant, under the conditions of 28 DEG C Carry out constant temperature culture, treat that bacterium colony grows, then transfer 2～3 times, to ensure that gained bacterium colony, as pure culture, obtains Cortex Nothapodytis Radicis Nei Shengzhen Bacterium；
The morphologic observation of bacterial strain GZU-BCEC-GX8:
By inoculation on Cha Shi solid medium, inoculate with spot planting, i.e. plant with a small amount of mycelia point of Inoculating needle picking In the center of flat board, then cultivate 7d or 14d in 28 DEG C and observe；
Observed result: bacterial strain GZU-BCEC-GX8 is initially white substrate mycelia in Cha Shi culture medium, growth is rapid, and After green statin shape closely knit gonimoblast bundle district occurs, bacterium colony reverse side is colourless, and mycelia is transparent, have every, branch is complicated, conidiophore without Color, conidiophore is the short lateral branch of mycelia, and conidium is spherical, the most single spore light green, therefore bacterial strain The morphological characteristic of GZU-BCEC-GX8 and trichoderma (Trichoderma) fungal morphology feature are more identical, bacterial strain GZU-BCEC- The colonial morphology of GX8 and conidial fructification are the most as depicted in figs. 1 and 2.
Embodiment 2 (extracts the side of camptothecine in trichoderma (Trichoderma sp.) GZU-BCECX-GX8 metabolite Method)
(1) liquid fermentation and culture: by the trichoderma GZU-BCECX-GX8 inoculation of separation preservation in PDA culture medium, Constant temperature culture 5d under conditions of 28 DEG C, is inoculated in equipped with in the 250mL triangular flask of 75mL fermentation medium, then by three Angle bottle is placed on shaking table, at 120 ± 3rpm, cultivates 12 days under the conditions of 28 ± 2 DEG C；
(2) method extracting camptothecine in metabolite:
1. the strain fermentation product of step (1) gained is used buchner funnel vacuum filtration, separate to obtain mycelium and fermentation Liquid；Use distilled water flushing mycelium, mycelium is placed under conditions of temperature is 45 ± 3 DEG C and dries, ground, 60 mesh sieves excessively, Obtain mycelium powder, fermentation liquid is concentrated in vacuo under conditions of temperature is 45 ± 3 DEG C the 20% of original volume, fermentation must be concentrated Liquid；
2. in concentrated broth, add 95% ethanol of 3 times of volumes, precipitate with ethanol, stand overnight under low temperature, buchner funnel sucking filtration Removal macromole precipitates, and takes supernatant and is concentrated in vacuo to former concentration volume in 45 ± 3 DEG C, obtains concentrating and impurity removing fermentation liquid；
3. take 1mL concentrating and impurity removing fermentation liquid, add extractant 2mL, after ultrasonic extraction 30min, lucifuge vibration 12h, then surpass Sound extraction 30min, stands, after extract and separation of fermentative broth, it is thus achieved that extract A；Extractant 2mL is added again to fermentation liquid, super Sound extraction 30min, stands, and after extract and separation of fermentative broth, collects and obtains extract B；
4. taking 1g mycelium powder, add extractant 2mL, after being placed in ultrasonic extraction 30min, lucifuge stands 12h, more ultrasonic Extraction 30min；Filter, it is thus achieved that extract C and filtering residue；Take filtering residue, add extractant 2mL, ultrasonic extraction 30min, filter, it is thus achieved that Extract D；
5. trichoderma (Trichoderma sp.) GZU-BCECX-GX8 bacterial strain is i.e. obtained after extract A, B, C and D being mixed The extract of camptothecine in metabolite；
Described fermentation medium is soluble starch 20g, ammonium sulfate 1.5g, K2HPO41g, MgSO41g, distilled water is mended Foot 1L；
Described extractant is that the ratio of 4:1 is made by volume by chloroform, methanol；
Described ultrasonic extraction, its power is 90W, frequency is 59KHz；
By the GZU-BCEC-GX8 bacterial strain extract of extract A, B, C and D mixing gained, send in the Chinese Academy of Sciences of Guizhou Province Natural product key lab and Guizhou University's fine chemistry industry center, by camptothecin standard product, GZU-BCEC-GX8 bacterial strain Extract and GZU-BCEC-GX8 bacterial strain extract carry out HPLC-MS analysis with camptothecin standard product aggregate sample, and Mass Spectrometry Conditions is such as Under:
Positive ion mode；Capillary pressure 3.5kV；Desolventizing temperature 350 DEG C；Nebulizer pressure 50PSI；Flow 10L/h；
Result: the appearance time of camptothecin standard product is TMarkThe guarantor of=4.345min, GZU-BCEC-GX8 bacterial strain extract Staying the time is TGX8=4.512min, GZU-BCEC-GX8 bacterial strain extraction liquid phase appearance time and camptothecin standard product appearance time Close, and by mixing sample detection, the sample peak of GZU-BCEC-GX8 bacterial strain extract and the standard substance peak weight of camptothecine Closing, retention time is TGX8+ mark=4.394min, then pass through API-ES collection of illustrative plates and the Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae) of GZU-BCEC-GX8 bacterial strain extract The API-ES collection of illustrative plates of alkali standard substance contrasts, the API-ES collection of illustrative plates of GZU-BCEC-GX8 bacterial strain extract have also discovered 349.4 [M+H]+Molecular weight material exist, illustrate: GZU-BCEC-GX8 bacterial strain substantially have metabolism generation camptothecine compounds Ability.

Claims (10)

1. a Cortex Nothapodytis Radicis endophyte Trichoderma strain, it is characterised in that named trichoderma (Trichoderma sp.) GZU- BCEC-GX8 bacterial strain, is preserved in China typical culture collection center, and preservation date is JIUYUE in 2015 14, and deposit number is CCTCC NO:M2015529。
2. trichoderma (Trichoderma sp.) GZU-BCEC-GX8 bacterial strain as claimed in claim 1, wherein, this bacterial strain Isolated culture method comprises the following steps:
(1) preparation of tissue: take the fresh young stem of Cortex Nothapodytis Radicis, leaf, petiole, bud, root bark, root core one or more, with from the beginning Water is rinsed well；
(2) surface sterilization and Sterility testing: be placed on superclean bench by tissue, washes material with the tween 80 sterilized water of 1 ‰ 2～3 times, then it is washed till non-foam with sterilized water, after sucking tissue surface moisture with aseptic filter paper, it is soaked in 75% ethanol solution 3min, then with aseptic water washing 2-3 time, then use 0.1%HgCl2Solution soaking 1～3min, then with aseptic water washing to rinsing Water is aseptic, after blotting tissue segments remained on surface water droplet with aseptic filter paper, is cut into the tissue cutting of a length of 0.5cm, a width of 0.5cm；
(3) endogenetic fungus is isolated and purified: the tissue cutting after step (2) being sterilized is inoculated in dual anti-PDA++Culture medium flat plate On, constant temperature culture 4～7 days under the conditions of being placed in 25-30 DEG C, grow from piece of tissue to mycelium, use edge mycelia picking method, Mycelia is transferred in PDA medium slant, under the conditions of 25-30 DEG C, carries out constant temperature culture, treat that bacterium colony grows, then transfer 2～3 Secondary, to ensure that gained bacterium colony, as pure culture, obtains Cortex Nothapodytis Radicis endogenetic fungus.
3. trichoderma (Trichoderma sp.) GZU-BCEC-GX8 bacterial strain as claimed in claim 2, it is characterised in that institute Stating PDA culture medium is peeled potatoes 200g, glucose 20g, agar 20g.Rhizoma Solani tuber osi is cut into small pieces, adds water boil 30min, Four layers of filtered through gauze, add distilled water by filtrate and complement to 1L, 121 DEG C, sterilizing 30min.
4. trichoderma (Trichoderma sp.) GZU-BCEC-GX8 bacterial strain as claimed in claim 2 or claim 3, it is characterised in that Described dual anti-PDA++Culture medium contains streptomycin that concentration is 40U/mL and concentration is the penicillin of 30U/mL.
5. Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae) in trichoderma as claimed in claim 1 (Trichoderma sp.) GZU-BCEC-GX8 metabolite The extracting method of alkali, comprises the following steps:
(1) liquid fermentation and culture: trichoderma GZU-BCEC-GX8 inoculation separation preserved is in PDA culture medium, 28 Cultivate 5d under the conditions of DEG C, be then inoculated in the triangular flask equipped with PD culture medium or fermentation medium, then by triangular flask It is placed on shaking table, at 120 ± 3rpm, cultivates under the conditions of 28 ± 2 DEG C, obtain strain fermentation product；
(2) method extracting camptothecine in metabolite:
1. the strain fermentation product of step (1) gained is used buchner funnel vacuum filtration, separate to obtain mycelium and fermentation liquid；With Distilled water flushing mycelium, is placed in mycelium under conditions of temperature is 45 ± 3 DEG C and dries, ground, 60 mesh sieves excessively, obtains mycelia Body powder；Fermentation liquid is concentrated in vacuo under conditions of temperature is 45 ± 3 DEG C the 10%-20% of original volume, obtains concentrated broth；
2. adding 95% ethanol of 3 times of volumes, precipitate with ethanol in concentrated broth, stand overnight under low temperature, buchner funnel sucking filtration is removed Macromole precipitates, and takes supernatant and is concentrated in vacuo to former concentration volume in 45 ± 3 DEG C, obtains concentrating and impurity removing fermentation liquid；
3. take 1mL concentrating and impurity removing fermentation liquid, add extractant 2-4mL, after ultrasonic extraction 30min, lucifuge vibration 12h, more ultrasonic Extraction 30min, stands, after extract and separation of fermentative broth, it is thus achieved that extract A；Extractant 2-4mL is added again in fermentation liquid, Ultrasonic extraction 30min, stands, and after extract and separation of fermentative broth, obtains extract B；
4. taking 1g mycelium powder, add extractant 2-5mL, after being placed in ultrasonic extraction 30min, lucifuge stands 12h, then ultrasonic extraction 30min；Filter, it is thus achieved that extract C and filtering residue；Take filtering residue, add extractant 2-5mL, ultrasonic extraction 30min, filter, it is thus achieved that extraction Take liquid D；
5. i.e. obtain trichoderma (Trichoderma sp.) GZU-BCEC-GX8 bacterial strain metabolism after extract A, B, C and D being mixed to produce The extract of camptothecine in thing.
6. camptothecine in trichoderma (Trichoderma sp.) GZU-BCEC-GX8 metabolite as claimed in claim 5 Extracting method, it is characterised in that the PD culture medium in described step (1) is removal agar composition in PDA culture medium；Fermentation culture Base is carbon source 10-25g, nitrogen source 0.5-2.5g, K2HPO41g, MgSO41g, 1L supplied by distilled water；Described PD culture medium or send out Ferment culture medium liquid amount in triangular flask is the 30% of triangular flask volume.
7. camptothecine in trichoderma (Trichoderma sp.) the GZU-BCEC-GX8 metabolite as described in claim 5 or 6 Extracting method, it is characterised in that in described step (1), the carbon source of fermentation medium is soluble starch, glucose and manna Any one in alcohol, nitrogen source is (NH4)2SO4, peptone and NH4Any one in Cl.
8. camptothecine in trichoderma (Trichoderma sp.) GZU-BCEC-GX8 metabolite as claimed in claim 5 Extracting method, it is characterised in that in described step (1), incubation time is 7～14 days.
9. camptothecine in trichoderma (Trichoderma sp.) GZU-BCEC-GX8 metabolite as claimed in claim 5 Extracting method, it is characterised in that described extractant is that the ratio of (1-4) 1 is made by volume by chloroform, methanol.
10. camptothecine in trichoderma (Trichoderma sp.) GZU-BCEC-GX8 metabolite as claimed in claim 5 Extracting method, it is characterised in that described ultrasonic extraction, its power is 90W, frequency is 59KHz.
CN201610316234.6A 2016-05-13 2016-05-13 Nothapodytes pittosporoides endophyte trichoderma sp. strain and method thereof for extracting camptothecin Pending CN106047715A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610316234.6A CN106047715A (en) 2016-05-13 2016-05-13 Nothapodytes pittosporoides endophyte trichoderma sp. strain and method thereof for extracting camptothecin

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610316234.6A CN106047715A (en) 2016-05-13 2016-05-13 Nothapodytes pittosporoides endophyte trichoderma sp. strain and method thereof for extracting camptothecin

Publications (1)

Publication Number Publication Date
CN106047715A true CN106047715A (en) 2016-10-26

Family

ID=57176246

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610316234.6A Pending CN106047715A (en) 2016-05-13 2016-05-13 Nothapodytes pittosporoides endophyte trichoderma sp. strain and method thereof for extracting camptothecin

Country Status (1)

Country Link
CN (1) CN106047715A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106613276A (en) * 2017-01-09 2017-05-10 重庆市中药研究院 Artemisia apiacea seedling cultivating method and special trichoderma atroviride fertilizer thereof
CN110511876A (en) * 2019-07-11 2019-11-29 长春中医药大学 A kind of korean epimedium herb endophyte, cultural method and its metabolite
CN113151538A (en) * 2021-03-09 2021-07-23 贵州大学 Candidate DNA bar code, primer and method for identifying cordyceps fungus

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102417883A (en) * 2010-09-27 2012-04-18 中国科学院过程工程研究所 Screening of new strain capable of producing camptothecin and method for preparing camptothecin
CN103074236A (en) * 2012-12-31 2013-05-01 中国科学院成都生物研究所 Camptotheca endophytic fungi for producing camptothecin and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102417883A (en) * 2010-09-27 2012-04-18 中国科学院过程工程研究所 Screening of new strain capable of producing camptothecin and method for preparing camptothecin
CN103074236A (en) * 2012-12-31 2013-05-01 中国科学院成都生物研究所 Camptotheca endophytic fungi for producing camptothecin and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
孙燕: "《微生物学实验指导》", 31 August 2015 *
王洋等: "《喜树幼株的喜树碱研究》", 31 May 2006 *
陈旭等: "马比木植物内生真菌Trichoderma sp.抗癌活性产物的液体发酵工艺优化", 《山地农业生物学报》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106613276A (en) * 2017-01-09 2017-05-10 重庆市中药研究院 Artemisia apiacea seedling cultivating method and special trichoderma atroviride fertilizer thereof
CN110511876A (en) * 2019-07-11 2019-11-29 长春中医药大学 A kind of korean epimedium herb endophyte, cultural method and its metabolite
CN113151538A (en) * 2021-03-09 2021-07-23 贵州大学 Candidate DNA bar code, primer and method for identifying cordyceps fungus

Similar Documents

Publication Publication Date Title
CN105820956B (en) One plant of Antrodia camphorata bacterial strain and Antrodia camphorata liquid state fermentation method
CN107298671A (en) Come from the secalonic acid H of penicillium oxalicum and prepare the application of anti-human colon cancer drug
CN101702984B (en) Phellinus igniarius mycelium liquid fermentation method by utilizing fungal elicitor for improving yield of flavone of phellinus igniarius
CN102676392B (en) Endophytic fungus in salvia miltiorrhiza bunge and application thereof
CN102559828B (en) Method for preparing astragaloside IV by converting total saponins of astragalus by microorganisms
CN101492706A (en) Method for improving cordyceps sinensis bacterium native volume of production with cordyceps militaris link liquid fermentation
CN106047715A (en) Nothapodytes pittosporoides endophyte trichoderma sp. strain and method thereof for extracting camptothecin
CN103172507B (en) Ophiobollin sesterterpine compound as well as preparation method and application thereof
CN106086147A (en) Fungal elicitor is utilized to induce the method extracting betulic acid from Inonqqus obliquus
CN112322687B (en) Microbial inoculum for preparing diosgenin and application thereof
CN104357332B (en) Aspergillus niger JH-2 and application to biotransformation and synthesis of asiatic acid
CN106010980A (en) Endophytic fungus paraconiothyrium brasiliense strain and application thereof
CN106047713A (en) Talaromyces pinophilum strain Li-93 and application thereof
CN101628931B (en) Antitumor antibiotics, pharmaceutically acceptable salts thereof, preparation method thereof and use thereof
CN107298670A (en) Come from penicillium oxalicum secalonic acid H and prepare anti-human oral cavity epidermoid carcinoma medicinal application
CN106701604B (en) Dongxiang wild rice endophytic fungus for efficiently converting glycyrrhizic acid to produce GAMG and application thereof
CN112195105B (en) Aspergillus tiannasensis and application thereof
CN102363796A (en) Method for producing glycyrrhetinic acid through microbial fermentation transformation
CN105296357A (en) Method for increasing yield of cordyceps sinensis liquid fermentation mycelia by supplementing materials
CN104726347A (en) Penicillium vulpinum fungi strain and method for preparing levo 7-hydroxyl butylphthalide employing fungi strain
CN103981104A (en) Endophytic fungi and method thereof for bio-transforming glycyrrhizinic acid into liquiritin
CN107298669A (en) Come from the secalonic acid I of penicillium oxalicum and anti-human oral cavity epidermoid carcinoma medicinal application
CN103724290A (en) Cyclopeptide compound clavatustide A as well as producing strain, preparation method and application thereof
CN101603011A (en) The method and the special strain therefore thereof that prepare the two glucosides of epipodophyllotoxin
CN102584615A (en) Alkaloid compound as well as preparation method and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20161026