CN102703327B

CN102703327B - Cladosporium Sp. XJ-AC03 and application thereof

Info

Publication number: CN102703327B
Application number: CN 201210105591
Authority: CN
Inventors: 李勤凡; 杨凯; 梁洁; 孔祥雅; 夏爽
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2012-02-04
Filing date: 2012-04-12
Publication date: 2013-11-06
Anticipated expiration: 2032-04-12
Also published as: CN102703327A

Abstract

The invention discloses Cladosporium Sp. XJ-AC03 which is preserved in China center for type culture collection under the number of CCTCC NO.M2011483 on December 26 2011. A bacterial colony of the Cladosporium Sp. XJ-AC03 is circular with white regular edge. The bacterial colony is in milk white early in culture, and gradually turns gray to deep grayish green over time, and the center of the bacterial colony project slightly. Mycelia of the Cladosporium Sp. XJ-AC03 have transverse septa without longitudinal septa and are dense. Certain mycelia have conidiophore which is branched. The conidiophore is olivary with brown to dark brown color and is concentrated with 0-3 septa. The Cladosporium Sp. XJ-AC03 is obtained from root of Xinjiang Aconitum leucostomum by artificial separating and screening. The Cladosporium Sp. XJ-AC03 of aconitine can be synthesized effectively, and can be used as material for preparing antineoplastic drugs, anti-inflammatory drugs, and anti-rheumatic drugs.

Description

Produce cladosporium XJ-AC03 and the application thereof of napelline

Technical field

The invention belongs to microbial technology field, relate to a kind of new product napelline cladosporium ( Cladosporiumsp.) and use.

Background technology

The rhizome of Chinese monkshood is China's record poisonous plants the earliest, and rhizome of Chinese monkshood plant whole body is poisonous, belongs to root the most malicious, and its toxic ingredient is mainly Diterpenoid Alkaloids, and the rhizome of Chinese monkshood causes that poisoning is mainly to eat by mistake with medicinal improper.In general, local animal has recognition capability to the rhizome of Chinese monkshood, and the rhizome of Chinese monkshood has hormesis to oral mucosa, can initiatively not search for food, and the animal that introduce in the other places is owing to lacking judgement to poisonous weeds, initiatively searches for food and poisoning.

Belong to napelline toxicity in the main component of the diphtheria rhizome of Chinese monkshood maximum, napelline is mainly to the cardiovascular of animal and neuron excitotoxicity effect.Cardiovascular toxic action is mainly excited cardiac vagal, reduces the autorhymicity of sinus node and the conductivity of atrioventricular node, causes sinus bradycardia and atrioventricular block; Neurotoxicity is to make the central nervous system paralysis, makes blood pressure drops, akinesia.

Although napelline is toxic ingredient, be also the effective constituent of rhizome of Chinese monkshood plant simultaneously.Napelline is C ₁₉The diterpenes diester-type alkaloids, be present in aconitum plant, the main effective constituent in the Chinese medicine monkshood commonly used, radix aconiti agrestis, monkshood, Aconitum Chinese medicine is mainly derived from female root of the cohosh rhizome of Chinese monkshood and the northern rhizome of Chinese monkshood and the processed goods of lateral root thereof, has good antitumor, anti-inflammatory, rheumatism, analgesic activity.Yet the source of napelline is really very deficient, expensive, can not satisfy people's research and clinical treatment needs far away.

Summary of the invention

For problems of the prior art and defective, the object of the present invention is to provide a kind of cladosporium Cladosporium sp.XJ-AC03 that can effectively synthesize the product napelline of napelline,, anti-inflammatory antitumor for preparing, antirheumatic provide material.

For achieving the above object, technical scheme of the present invention is as follows:

A kind of cladosporium Cladosporium sp. XJ-AC03 that can synthesize the product napelline of napelline, be preserved in Chinese Typical Representative culture center on December 26th, 2011, preserving number is: CCTCC NO:M2011483, preservation address: Wuhan, China Wuhan University.

A further object of the invention is to provide the application of cladosporium XJ-AC03 in synthetic napelline of producing napelline.

A further object of the invention is to provide the application of cladosporium XJ-AC03 in the preparation antitumor drug of producing napelline.

A further object of the invention is to provide the application of cladosporium XJ-AC03 in the preparation anti-inflammatory drug of producing napelline.

A further object of the invention is to provide the application of cladosporium XJ-AC03 in the preparation analgesic of producing napelline.

[0011]The cladosporium XJ-AC03 that produces napelline colonizes in diphtheria rhizome of Chinese monkshood root tissue, sets up symbiotic relationship, can not cause the disease of the diphtheria rhizome of Chinese monkshood, finds that through vitro culture XJ-AC03 can produce napelline, illustrates that it is participating in the synthetic of diphtheria rhizome of Chinese monkshood mesaconitine.

The cladosporium XJ-AC03 bacterial strain that produces napelline can the amount reproduction amplification through vitro culture, and can prepare antitumor, anti-inflammatory under the condition of artificial culture, the analgesic napelline is supplied raw materials.

Description of drawings

The colonial morphology photo of Fig. 1 XJ-AC03 bacterial strain;

The mycelia microscope figure of Fig. 2 XJ-AC03 bacterial strain;

The conidium microscope figure of Fig. 3 XJ-AC03 bacterial strain;

Fig. 4 utilizes MEGA software component phyletic evolution tree structure diagram;

The high-efficient liquid phase chromatogram of Fig. 5 napelline standard substance;

The high-efficient liquid phase chromatogram of Fig. 6 XJ-AC03 mycelia extract.

Embodiment

The accompanying drawing that provides below in conjunction with the contriver illustrates that with concrete test example and specific embodiment the present invention produces beneficial effect and the preparation method of the cladosporium XJ-AC03 of napelline.

[0015The separation that embodiment 1 produces the cladosporium XJ-AC03 of napelline

That diphtheria rhizome of Chinese monkshood root that lifts the prairiedir that 1) will pick up from Xinjiang Uygur autonomous region Xinyuan County spends ionized water the silt on surface is rinsed well, carries out surface sterilization;

2) suck moisture on the root tissue of surface sterilization with sterilization filter paper, then it is cut into the tissue block of 5 mm * 5 mm * 5 mm sizes, then it is inoculated in PDA solid culture primary surface, put dark culturing in 20 ℃ of incubators;

3) when the tissue block edge has mycelia to grow, the mycelium inoculation at picking colony edge is in PDA solid culture primary surface, and dark culturing in 20 ℃ of incubators is until purifying is single bacterium colony; Collect the hypha,hyphae of cultivating, with its alkaloid of organic solvent extraction; Application of thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) detect the alkaloid that extracts in mycelia, detect whether to contain napelline, and screening obtains synthesizing the cladosporium XJ-AC03 of the product napelline of napelline.

The evaluation of embodiment 2 cladosporium XJ-AC03

1) morphological feature

As Fig. 1, Fig. 2, shown in Figure 3, the bacterium colony of this bacterial strain is rounded, and the edge is more regular to be white in color; Initial stage of culture is oyster white, and along with the passing of incubation time, grizzle is to spruce gradually for bacterium colony, and central authorities are projection slightly; The mycelium tool tabula of this bacterial strain, without mediastinum, than comparatively dense, some mycelia tool conidiophores, and conidiophore has branch; The conidium olive shape is brown to Vandyke brown, concentrates to distribute, and 0 ~ 3 barrier film does not wait;

2) cultivate feature

This bacterial strain is grown moderate on the PDA solid medium, and optimum growth temperature is 20 ℃, and time growth is suppressed higher than 28 ℃ when temperature;

The component of described PDA solid medium and proportioning are: potato 200 g, glucose 20 g, agar 17 g, 121 ℃ of autoclaving 30 min;

3) molecular biology identification

XJ-AC03 bacterial strain ITS-5.8S rDNA sequence is carried out homology relatively with the Blast in NCBI and the ITS-5.8S rDNA sequence in Genbank, with cladosporium ( Cladosporiumsp.) bacterial strain sequence construct systematic evolution tree.Use ClustalX 1.83 Version and MEGA 5.0 softwares employing adjacent method (neighbor-johning, NJ) phylogenetic tree construction (Fig. 4), XJ-AC03 bacterial strain and Cladosporium are nearest, and this bacterium cladosporium for the product napelline in the molecular biology classification is described.

According to morphological feature, cultural characters, Phylogenetic Analysis, and in conjunction with bibliographical information, determine that this bacterium is for producing the cladosporium XJ-AC03 of napelline.

Test example 1 is produced the test of the synthetic napelline of cladosporium XJ-AC03 of napelline

The XJ-AC03 inoculation on the PDA solid medium, is put 20 ℃ and cultivated 20 d, then mycelia is scraped 50 ℃ of oven dry, then weighing mycelia.After the mycelia of drying is ground to form powdery, with filter paper, it is wrapped with ether extraction 24 h, ether extracted liquid is volatilized obtain residue, with methylene dichloride, residue is dissolved.With methylene dichloride, the alkaloid that extracts in napelline standard substance, XJ-AC03 is dissolved, and point sample on silica gel thin-layer plate, detects with thin-layer chromatography respectively.If the mycelia alkaloid have after Thin-layer separation with the color of napelline standard substance and displaced phase with spot, can determine that its corresponding fungi can synthesize napelline.Use the Content of Aconitine in high performance liquid chromatography detection mycelia, its high-efficient liquid phase chromatogram is as shown in 5,6.

Test example 2 is produced the antitumor test of the synthetic napelline of cladosporium XJ-AC03 of napelline

Adopt four nitrogen bromine salt (MTT) reduction methods measure from diphtheria aconite tuber part from napelline that endogenetic fungus produces to the restraining effect of tumour cell.Cell strain is human prostata cancer (PC-3), action times 72 h.With tumor cell inoculation in Tissue Culture Plate, 37 ℃, 5% CO ₂Cultivate in incubator, after waiting for cell attachment, add different concns 10 in every hole ^-4, 10 ^-5, 10 ^-6, 10 ^-7, 10 ^-8The napelline of mol/L.Negative control group adds equal-volume RPMI 1640 complete culture solutions, and the blank hole (only add complete culture solution, do not add cell) of returning to zero is set, and each group is equipped with 4 parallel holes, at 37 ℃, and 5% C0 ₂Condition under cultivate respectively 72 h in incubator, experiment stops the MTT storage liquid 10 μ L that front 4 h add 5 mg/mL again, continue again to cultivate 4 h after mixing, remove supernatant liquor, every hole adds the DMSO of 150 μ L again, 10 min that vibrate, when measuring wavelength and be 492 nm with microplate reader, the OD value in each hole, obtain inhibitory rate of cell growth.Inhibitory rate of cell growth (%): (the average OD value of the average OD value-experimental group of the control group)/average OD value of control group * 100%, result evaluation: (%) 〉=50% is effective for inhibitory rate of cell growth.Result shows that concentration is 10 ^-4The mol/L napelline illustrates that to PC-3 inhibitory rate of cell growth (%)=72.9% napelline has certain antitumous effect.

Test example 3 is produced the anti-inflammatory test of the synthetic napelline of cladosporium XJ-AC03 of napelline

With the Kunming mouse of 20 body weight 18～20 g, be divided at random 2 groups by body weight: the blank group of Vaseline and napelline medicine group.Use etherization, at the scorching liquid of the front and back of mouse right ear two sided coatings 100% caused by dimethylbenzene xylene, 0.02 mL/ only after 30 min, is coated with blank group and napelline medicine group equivalent at the mouse right ear inflammation part respectively, and dosage 0.06 mg/, left ear is left intact.After 4 h, mouse is put to death in the cervical vertebra dislocation, cut immediately ears, with 8 mm punchings, deduct left auricle weight as swelling take auris dextra sheet weight, (auris dextra weight-left ear is heavy)/left ear is heavily swelling rate, take (the average swelling of control group-average swelling of administration group)/average swelling of model group as inhibitory rate of intumesce.As following table (n=10, x± s, * P＜0.01):

Group	Left ear weight/mg	Auris dextra weight/mg	Mouse ear swelling degree/mg	Mouse ear swelling rate/%	Inhibitory rate of intumesce/%
						Control group	9.97±0.99	19.83±0.45	10.07±0.66	104.69±17.18	?
Experimental group	9.91±1.22	15.03±0.95	5.11±0.41	52.56±9.74＊	49.79

As can be seen from the table, compare with the blank group, the napelline p-Xylol causes scorching inhibiting rate greater than 30%, illustrates that napelline p-Xylol induced mice auricle edema has obvious restraining effect.

Test example 4 is produced the analgesic test of the synthetic napelline of cladosporium XJ-AC03 of napelline

With the Kunming mouse of 20 body weight 18～20 g, the experiment preabdomen loses hair or feathers with 8% sodium sulphite.Be divided into immediately blank group and napelline medicine group after 24 h.Measure in accordance with regulations medicine is coated the district of losing hair or feathers, after 1 h, equal abdominal injection 0.6% acetum 0.01 mL/g, writhing time of occurrence and writhing number of times in observed and recorded 20 min.Mouse writhing experimental data such as following table (n=10, x± s, * P＜0.01):

Group	Dosage/(mg /Only)	Pain latent period/min	Writhing response/time
				Control group	0.06	3.14±0.90	25.86±4.67
Experimental group	0.06	?7.14±0.69＊	?13.14±2.12＊

In table, the demonstration experimental group is compared with control group, on latent period, writhing number of times, significant difference is arranged all, can obviously reduce the number of times of mouse writhing, the latent period of prolongation mouse writhing.Experiment shows, the pain that napelline causes Glacial acetic acid has obvious analgesic activity.

Claims

1. produce napelline cladosporium ( Cladosporium sp.) XJ-AC03, preserving number is CCTCC NO:M2011483.

The cladosporium of product napelline claimed in claim 1 ( Cladosporium sp.) application of XJ-AC03 in synthetic napelline.

The cladosporium of product napelline claimed in claim 1 ( Cladosporium sp.) application of XJ-AC03 in the preparation antitumor drug.

The cladosporium of product napelline claimed in claim 1 ( Cladosporium sp.) application of XJ-AC03 in the preparation anti-inflammatory drug.

The cladosporium of product napelline claimed in claim 1 ( Cladosporium sp.) application of XJ-AC03 in the preparation analgesic.