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CN-121987675-A - Preparation method and application of spore-free fruiting body waste-derived ganoderma lucidum outer vesicles

CN121987675ACN 121987675 ACN121987675 ACN 121987675ACN-121987675-A

Abstract

The invention discloses a preparation method and application of ganoderma lucidum outer vesicles from spore-free fruiting body waste. The ganoderma lucidum external vesicle is used for preparing a medicament for treating ischemic cerebral apoplexy sequela, and can improve core symptoms such as post-apoplexy movement dysfunction (including limb paralysis, imbalance and gait abnormality), cognitive decline and the like. The spore-free fruiting body waste source ganoderma lucidum outer vesicle is prepared by pretreating raw materials, centrifuging at a differential speed, concentrating by tangential flow, and filtering by a filter membrane, wherein the spore-free fruiting body waste is completely released by spores after a ganoderma lucidum powder spraying period. The preparation method takes the wastes as raw materials to prepare the high-value medicinal outer vesicles, so that the green circulation of agricultural wastes-high-added-value medicinal raw materials is realized, the ganoderma lucidum outer vesicles can effectively penetrate through the blood brain barrier, the brain targeting delivery is realized, the problems that the blood brain barrier penetration rate of the traditional ganoderma lucidum active ingredient is low, and the effective treatment concentration is difficult to reach in brain tissues are solved, and the bottleneck of traditional drug delivery is broken through.

Inventors

  • ZHANG CHAO
  • KONG LINGYI
  • Ye Rigan
  • ZHANG ZHENZHEN

Assignees

  • 中国药科大学

Dates

Publication Date
20260508
Application Date
20260119

Claims (9)

  1. 1. The application of the spore-free fruiting body waste-derived ganoderma lucidum outer vesicle in the preparation of medicaments for treating ischemic cerebral apoplexy sequela is characterized in that the spore-free fruiting body waste-derived ganoderma lucidum outer vesicle is prepared by pretreating raw materials, centrifuging at a differential speed, concentrating by tangential flow and filtering by a filter membrane, wherein spores of the spore-free fruiting body waste are completely released after a ganoderma lucidum powder spraying period.
  2. 2. The use according to claim 1, wherein said ischemic stroke sequelae comprises post-stroke motor dysfunction, core symptoms of cognitive decline.
  3. 3. The application of the ganoderma lucidum outer vesicles from the spore-free fruit body waste is characterized in that ganoderma lucidum outer vesicles from the spore-free fruit body waste are prepared by the steps of taking ganoderma lucidum outer vesicles from the spore-free fruit body waste, washing with sterile water, cutting, mechanically crushing, removing impurities, adding PBS according to a feed liquid ratio of 1-4L/kg, soaking for 8-12 h under the conditions of 4 ℃ and 60-120 rpm to enable the outer vesicles to be fully dissolved out, filtering the outer vesicles by three layers of gauze to remove coarse residues, centrifuging the filtrate by 2000-4000 g and 4 ℃ for 15-25 min, discarding precipitates, filtering the supernatant by a 60-mesh screen, centrifuging by 10000-12000 g and 4 ℃ for 30-60 min, taking supernatant, further removing macromolecular impurities and cell fragments, collecting the supernatant, sequentially carrying out tangential flow concentration filtration by a hollow fiber column with a pore diameter of 0.22-0.45 mu m and a hollow fiber column with a cut-off molecular weight of 30-100 kDa, collecting permeate, filtering the permeate by a hollow fiber column with a cut-off molecular weight of 0.22-0.45 mu m, concentrating the permeate liquid by a cut-off filter membrane, concentrating the permeate liquid by a hollow fiber column with a cut-off molecular weight of 0.0-120 mu m, concentrating tube concentrating the permeate liquid by a concentrated liquid of 3500 m, concentrating the permeate liquid by a concentrated liquid, concentrating tube concentrating the permeate liquid by a concentrated liquid by a tube of 0.3500 m, concentrating tube, concentrating the permeate liquid by a concentrated liquid by a tube, concentrating liquid after concentrating fluid by a tube, concentrating fluid by concentrating fluid.
  4. 4. A preparation method of ganoderma lucidum outer vesicles from spore-free fruiting body waste comprises the steps of taking ganoderma lucidum spore-free fruiting body waste, cleaning with sterile water, cutting, mechanically crushing, removing impurities, adding PBS according to a feed liquid ratio of 1-4L/kg, soaking for 8-12 h at the conditions of 4 ℃ and 60-120 rpm, enabling the outer vesicles to be fully dissolved out, filtering with three layers of gauze to remove coarse residues, centrifuging the filtrate at 2000-4000 g and 4 ℃ for 15-25 min, discarding precipitates, filtering the supernatant with a 60-mesh screen, centrifuging the supernatant at 10000-12000 g and 4 ℃ for 30-60 min, further removing macromolecular impurities and cell fragments, collecting the supernatant, sequentially carrying out tangential flow concentration and filtration on hollow fiber columns with the aperture of 0.22-0.45 mu m and hollow fiber columns with the molecular weight of 30-100 kDa, finally concentrating the concentrate obtained after the tangential flow concentration and filtration by using a ultrafiltration tube of 0.45 mu m and 30kDa at 3500g, further concentrating the concentrate, collecting the concentrate, and filtering the concentrate by a filter membrane of 0.22 mu m, thus obtaining the ganoderma lucidum outer vesicles.
  5. 5. The method according to claim 4, wherein the tangential flow concentration filtration is performed by passing the supernatant through a hollow fiber column with a filtration diameter of 0.22-0.45 μm to collect the permeate, and then concentrating the permeate through a hollow fiber column with a molecular weight cut-off of 30-100 kDa to collect the concentrate.
  6. 6. The preparation method of the ganoderma lucidum spore powder is characterized by comprising the steps of taking spore-free ganoderma lucidum fruit body waste which is completely released by spores after a powder spraying period, washing, cutting, mechanically crushing, adding PBS (phosphate buffer solution) according to the proportion of 1:4 (w/v), soaking for 12 hours at the speed of 4 ℃, fully dissolving out outer vesicles, filtering by three layers of gauze to remove coarse residues, centrifuging the filtrate by 4000g of 20min, discarding precipitates, filtering the supernatant by a 60-mesh screen, centrifuging for 40 minutes by 10000g and 4 ℃, further removing macromolecular impurities and cell fragments, collecting supernatant, sequentially carrying out tangential flow concentration filtration on the collected supernatant by a hollow fiber column with the aperture of 0.45 μm and a hollow fiber column with the interception molecular weight of 30kDa, collecting permeate liquid, concentrating the permeate liquid by the hollow fiber column with the interception molecular weight of 30kDa, collecting concentrate, concentrating the concentrate obtained by tangential flow concentration filtration by using a 0.45 mu g of centrifugation tube, centrifuging for obtaining concentrate by using a centrifugation tube of 3500 ℃, centrifuging for 0.45 mu m, concentrating the concentrate by a centrifugation tube of 3500 ℃, filtering for 0.45 mu.m, concentrating the concentrate by the supernatant, and concentrating the concentrate by the method of 0.110 mu.m, and filtering under the condition of 3500 ℃, filtering the concentrate by the filter membrane.
  7. 7. The method according to any one of claims 4 to 6, wherein the spore-free fruiting body waste-derived ganoderma lucidum outer vesicles are obtained.
  8. 8. The spore-free fruiting body waste-derived ganoderma lucidum outer vesicle according to claim 7, wherein the particle size distribution is 30-150 nm, the concentration is not lower than 1 x 10 10 parts per mL, the morphology is a typical cup-shaped vesicle structure, and the membrane stability is good.
  9. 9. A pharmaceutical composition for treating ischemic stroke sequela, comprising the spore-free fruiting body waste-derived ganoderma lucidum outer vesicle of claim 7 or 8.

Description

Preparation method and application of spore-free fruiting body waste-derived ganoderma lucidum outer vesicles Technical Field The invention belongs to the field of traditional Chinese medicines, and relates to a preparation method and application of ganoderma lucidum exovesicles from spore-free fruiting body waste. Background The ganoderma lucidum outer vesicles (GLEVs) are extracellular vesicles (Extracellular Vesicles, EVs) with the particle size of 30-150nm extracted and separated from fruiting bodies of ganoderma lucidum (Ganoderma lucidum), and GLEVs has the characteristics of good biocompatibility and strong targeted delivery capability of active substances of the outer vesicles while carrying ganoderma lucidum specific active ingredients such as ganoderma lucidum polysaccharide, triterpene and the like, and are important carriers for developing natural active preparations. The related technology of the ganoderma lucidum external vesicles mainly focuses on preparation method optimization and application in specific fields, namely patent CN120361065A discloses application of the ganoderma lucidum external vesicles in breast cancer resisting products, patent CN202510420349.9 discloses a preparation method of the ganoderma lucidum external vesicles containing medicinal and edible active ingredients, the yield of the external vesicles is improved through extraction process improvement, and application aspects are expanded to the fields of cornea injury repair (CN 202511353631.6), skin protection (CN 202511223696.9), liver protection (CN 202510798017.4) and the like, but the application does not relate to treatment of stroke sequelae such as behavior disorder and the like. The existing preparation of the ganoderma lucidum outer vesicle has two core problems that firstly, the raw materials do not utilize industrial waste, the prior art takes fresh ganoderma lucidum mycelia or sporophores which do not release spores as raw materials, the spore-free sporophore waste generated after the powder spraying of ganoderma lucidum is usually discarded and is not utilized in a high-value mode, secondly, the existing extraction process of the ganoderma lucidum outer vesicle mostly adopts a differential centrifugation method, the extraction efficiency is low, the period is long, the product purity is poor, the large-scale preparation cannot be realized, thirdly, the application scene is limited, the potential of the ganoderma lucidum outer vesicle in the fields of neuroprotection and apoplexy sequelae (such as unilateral limb disorder) is not excavated in the prior art, and the existing medicines for treating the apoplexy sequelae (such as ginkgo leaf extract injection) have the defects of weak blood brain barrier penetrability and obvious side effects and lack of natural-source efficient intervention preparations. Disclosure of Invention The invention aims at overcoming the defects in the prior art and provides an application of ganoderma lucidum outer vesicles from spore-free fruiting body waste. The invention also aims to provide a preparation method of the ganoderma lucidum exovesicle from the spore-free fruiting body waste. The aim of the invention can be achieved by the following technical scheme: the application of the spore-free fruiting body waste-derived ganoderma lucidum outer vesicle in the medicament for treating ischemic cerebral apoplexy sequela is characterized in that the spore-free fruiting body waste-derived ganoderma lucidum outer vesicle is prepared by pretreating raw materials, centrifuging at a differential speed, concentrating by tangential flow and filtering by a filter membrane, wherein spores of the spore-free fruiting body waste are completely released after a ganoderma lucidum powder spraying period. Preferably, the ischemic stroke sequela comprise core symptoms of post-stroke motor dysfunction and cognitive decline. The ganoderma lucidum external vesicle is used for preparing a medicament for treating ischemic cerebral apoplexy sequela, can improve core symptoms such as post-apoplexy movement dysfunction (including limb paralysis, imbalance and gait abnormality), cognitive decline and the like, and can be expanded to prepare auxiliary medicaments for treating neurodegenerative diseases such as Alzheimer disease, parkinsonism and the like and relieve the accompanying behavior disorder and nerve function defect. The ganoderma lucidum external vesicles treat cerebral apoplexy sequelae through the multi-way synergistic effect of resisting oxidative stress, resisting inflammation and repairing damaged nerve cells. The ganoderma lucidum external vesicle is administrated by adopting a tail vein injection way, the administration dosage is 1X 10 7~1×1010 parts per time, 1 time a day, and the administration is carried out continuously for 7-14 days. The preparation method comprises the steps of taking ganoderma lucidum spore-free fruit body waste, cleaning with sterile water, cutting, mechanically cru