Preparation method and application of balanophora polyandra Griff. endophytic fungus antibacterium and/or oxidation resisting secondary metabolism products

A technology for secondary metabolites, endophytic fungi, used in microorganism-based methods, biochemical equipment and methods, antibacterial drugs, etc.

Pending Publication Date: 2019-04-02
SOUTH CHINA AGRI UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide the preparation method and application of the antibacterial and / or anti-oxidation secondary metabolites of the endophytic fungus of Snakeweed. Extraction of secondary metabolites of endophytic fungi from Snakeweed to obtain secondary metabolites with antibacterial and / or antioxidant activity

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  • Preparation method and application of balanophora polyandra Griff. endophytic fungus antibacterium and/or oxidation resisting secondary metabolism products
  • Preparation method and application of balanophora polyandra Griff. endophytic fungus antibacterium and/or oxidation resisting secondary metabolism products
  • Preparation method and application of balanophora polyandra Griff. endophytic fungus antibacterium and/or oxidation resisting secondary metabolism products

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0024] Experimental Example 1 Isolation, Purification and Preservation of Endophytic Fungi in Snakeweed

[0025] (1) Rinse Snakeweed with flowing tap water to remove silt dirt;

[0026] (2) Rinse 3 times with sterile water on the ultra-clean workbench, put it on a sterilized filter paper sheet to dry, and after the water is dry, use a sterile scalpel to cut Snakeweed into 0.5cm×0.5cm Small pieces of ×0.5cm;

[0027] (3) Rinse with 75% ethanol for 20s, and rinse with sterile water for 3 times;

[0028] (4) Then use 0.2% mercuric chloride surface disinfection for 20 minutes, and finally wash 3 times with sterile water;

[0029] (5) Each small piece was placed on the culture medium containing 500 μg / mL streptomycin sulfate, 3 pieces / dish, and placed in a fungal incubator at 28°C for upside-down cultivation. Inoculate on a new PDA plate, purify continuously for several times until the colony form is consistent, store at 4°C, and set aside;

experiment example 2

[0030] Experimental Example 2 Identification of Endophytic Fungi in Snakeweed

[0031] (1) According to the morphological characteristics of plant endophytic fungal colonies, including colony size, color, growth rate, surface characteristics and growth mode, etc., visually observe and classify the strains with obvious morphological differences. A total of 22 strains of endophytic fungi with different shapes were isolated from S. multistamen, and they were numbered Bpf-1~Bpf-22.

[0032] (2) Using molecular biology methods to further identify the 22 strains of endophytic fungi isolated

[0033] The endophytic fungus of S. multistamens is inserted into the liquid culture medium, cultured by shaking for 5-7 days, and the hyphae are collected after filtering.

[0034] The mycelium was fully ground to powder with liquid nitrogen, and its DNA was extracted using a kit method (Shanghai Sangon DNA Extraction Kit).

[0035] The primers for amplifying the ITS sequence are:

[0036] I...

experiment example 3

[0042] Experimental Example 3 Preparation of Secondary Metabolites of Endophytic Fungi in Snakeweed

[0043] (1) Activate the bacterial strains in the cryopreservation tube on the PDA plate for 4 to 7 days, then insert the activated bacterial strains into the Erlenmeyer flasks with 20mL potato liquid medium in each bottle, and cultivate 2 bottles for each bacterium;

[0044] (2) shaking culture at 28°C and 150rmp for 5 to 7 days, then insert the liquid seeds into the pre-sterilized rice culture medium, the culture temperature is 28°C, and the culture time is 60 days;

[0045] (3) After the fermentation is completed, the fermentation product is extracted by cold dipping in ethyl acetate three times, each time for 7 days, and then the extract is vacuum filtered and concentrated under reduced pressure to obtain a crude ethyl acetate extract.

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Abstract

The invention discloses a preparation method and application of balanophora polyandra Griff. endophytic fungus antibacterium and / or oxidation resisting secondary metabolism products. The method comprises the following steps of (1) separating endophytic fungi from balanophora polyandra Griff. by a tissue block separation method; (2) performing shaking culture on the endophytic fungi obtained through separation in a potato glucose liquid nutrient medium; (3) inoculating the sterilized rice culture medium with the liquid seeds obtained in the step (2) for culturing, performing cold soaking and extracting on rice culture medium fermentation products in ethyl acetate, and performing decompressed concentration to obtain balanophora polyandra Griff. endophytic fungus secondary metabolism products; and (4) adopting an TLC-MTT-bioautography and a DPPH method for screening the endophytic fungi having antibacterium and / or antioxidant activity. According to the method, the balanophora polyandra Griff. endophytic fungus secondary metabolism products can be extracted, and the secondary metabolism products having antibacterium and / or antioxidant activity can be obtained.

Description

technical field [0001] The invention relates to a preparation method of a secondary metabolite of an endophytic fungus, in particular to a preparation method and application of an anti-bacterial and / or anti-oxidation secondary metabolite of an endophytic fungus of Snakeweed. Background technique [0002] Balanophora polyandra Griff. (Balanophora polyandra Griff.) is a perennial fleshy parasitic herbaceous plant of the genus Balanophora in the genus Balanophora. On the root, it is mainly distributed in Yunnan, Sichuan, Guangdong and Hainan in my country, and also in Nepal, India, Myanmar and other places (Flora of China, 1988). Snakeweed has the effects of clearing away heat, detoxifying, stopping bleeding, and nourishing blood. It has certain efficacy and no obvious side effects. [0003] In recent years, domestic and foreign researches on Snakeweed mainly focus on the study of phytochemical components and biological activities. The existing research results show that the a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P1/02C12N1/14C12N1/02A61P31/04A61P39/06A01P1/00A01P3/00C12R1/77C12R1/645
CPCA61P31/04A61P39/06C12N1/02C12N1/14C12P1/02
Inventor 单体江韩小腾毛子翎吴春银王军黄永芳伍慧雄谢银燕
Owner SOUTH CHINA AGRI UNIV
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