AchE (acetylcholinesterase) inhibiting secondary metabolite of endophytic cladosporium sp. Pt-2 of portunus trituberculatus and preparation method thereof
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A technology of secondary metabolites and branch mold, applied in the field of biology
Inactive Publication Date: 2013-04-24
QINGDAO AGRI UNIV
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There are relatively few reports on endophytic fungi from marine sources against Alzhei
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Embodiment 1
[0045] Example 1: The screening of shoot mold Pt-2 (Cladosporium sp.Pt-2) obtains:
[0046] 1. Isolation of strains
[0047] 1.1. Medium
[0048] Medium for separation: Potato agar (PDA) medium, adding penicillin potassium and 150U / ml streptomycin sulfate at a final concentration of 200U / mL.
[0049] Liquid fermentation medium (modified Chase medium): 30g sucrose, 3g NaNO 3 , 0.5g KCL, 0.5g MgSO 4 ·7H 2 O, 0.01g FeSO 4 , 1g K 2 HPO 4 Add 1000mL seawater in turn, stir well to dissolve, then add 1g of yeast extract to slightly heat to dissolve, and sterilize at 121°C for 20min for later use.
[0050] 1.2. Strain isolation method
[0051] Shell the crab, take its gills and gastrointestinal parts, first soak in 75% alcohol for 5 minutes for surface disinfection, then cut into pieces and add 3mL sterile water to grind, spread on the separation medium, place the plate in a constant temperature incubator, Inverted culture at 28°C. When mycelium grows on the plate, the end...
Embodiment 2
[0092] Embodiment 2, Preparation of secondary metabolites of AchE inhibited by Endophytic Cladomycetes Pt-2 in Portunus trituberculatus:
[0093] The preparation process of the secondary metabolites of AchE inhibited by endogenous Cladomycetes trituberculatus Pt-2 is as follows:
[0094] (1), with endophytic branch mold Pt-2 of portunus trituberculatus as bacterial classification, adopt the method for liquid fermentation, under the conditions of 10% inoculum size, 200mL / 500mL Erlenmeyer flask, 28 ℃ of conditions in the fermentation medium Fermented in medium for 9 days to obtain the fermented product; the formula of the fermentation medium is: 30g glucose, 3g NaNO 3 , 0.5g KCl, 0.5g MgSO 4 ·7H 2 O, 0.01g FeSO 4 , 1g K 2 HPO 4 , 1g yeast extract, 1000mL sea water;
[0095] (2), concentrating the fermentation broth in vacuum at 40°C to 1 / 3 of the original volume, then extracting with ethyl acetate, and concentrating the extract to dryness;
[0096] (3) The organic phase...
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Abstract
The invention provides an AchE (acetylcholinesterase) inhibiting secondary metabolite of endophytic cladosporium sp. Pt-2 of portunus trituberculatus and a preparation method thereof, belonging to the technical field of biology. The preparation method comprises the following steps of: (1) carrying out liquid fermentation on endophytic cladosporium sp. Pt-2 of portunus trituberculatus taken as a strain to obtain a fermented product; (2) carrying out vacuum concentration on fermentation liquor at 40 DEG C until the volume of the fermentation liquor is 1/3 of the original volume, then extracting the fermentation liquor with ethyl acetate and concentrating extract liquor until the extract liquor is dry; and (3) carrying out silica column chromatography on the organic phase extract, which has been concentrated until the extract is dry, of the cladosporium sp. Pt-2 fermentation product, carrying out gradient elution with a chloroform/methanol system to obtain six polarity segments and separating the segment with the highest activity by adopting a C18 reversed-phase column and sephadex chromatography, thus obtaining the secondary metabolite with AchE inhibition ratio up to 70-90%. The secondary metabolite and the preparation method have the advantages that the defects that current drugs are expensive and have side effects and high chemical synthesis difficulty are overcome; and the new drug and preparation method are provided to provide theoretical bases for development of senile dementia preventing biological drugs.
Description
technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a secondary metabolite of AchE inhibited by Endophytic Cladomycetes Pt-2 in Portunus trituberculatus and a preparation method thereof. Background technique [0002] Alzheimer's disease (AD) is a common senile brain neurodegenerative disease with a relatively high incidence, and has become one of the diseases that seriously threaten the lives of the elderly in modern society. Its death rate ranks fourth after cardiovascular disease, cancer and stroke. The pathogenesis of AD is complex, and it is a multi-etiological disease. Typical pathological changes include β-amyloid protein (β-amyloid protein, Aβ) deposition to form senile plaques (senile plaques, SP), neurofibrillary tangles (neurofibrillary tangles, NFTs) ), cholinergic dysfunction in the basal forebrain and extensive neuronal loss and changes in synaptic morphology in the cortex and hippocampus. In recent ...
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