The Material Basis Identification And Antifungal Investigation Of Chinese Herbal Puffball Spore Powder Fermentation Against Fungus

Bovistella radicata（Mont.）Pat is a kind of puffball,which is an edible medicinal fungus and has a variety of biological activities such as hemostasis,anti-tumor,antibacterial,anti-inflammatory.B.radicata is also used as folk medicine in the treatment of tinea pedis.Tinea pedis is an epidermal dermatoses caused by pathogenic fungi and Trichophyton rubrum is the main pathogenic fungus.Chemotherapeutic drugs for the treatment of tinea pedis are emerging in an endless stream in recent years.Human body can build up tolerance to chemotherapeutic drugs due to long term treatment.The antifungal secondary metabolites obtained from plants or microorganisms have low-side-effect,less-drug-resistance and rich natural resources.In this paper,the spore powder of B.radicata was used as raw materials,the pharmacodynamic substance basis of B.radicata spore powder against tinea pedis were inverstigated.Experimental study of treatment of traditional chinese medicine on tinea pedis has become a top priority to avoid and overcome antibiotic resestance and side effects.The main research methods and conclusions are as follows:1.A comparative analysis of the antimicrobial activity of B.radicata picked from Jiangxi and different puffball on the market confirmed that B.radicata spore powder has antifungal activity.The study found that the fermentation broth of B.radicata spore powder expressed a higher antifungal activity.The fermentation conditions of B.radicata spore powder were optimized and antimicrobial effect was evaluated.The best nitrogen source,carbon source and ion for the fermentation of B.radicata spore powder were peptone peptone（2 g/L）,sucrose（4 g/L）,and Mg SO4（0.4 g/L）,respectively.The single factor experiment and response surface method are mainly used to obtained the optimium fermentation condition were the initial p H 5.9,fermentation temperature23.2°C,the shaker rotation speed 124.6 rpm,and the fermentation time 69.6 hours.At this time,the size of inhibition zone was 3.05 cm against T.rubrum.2.The antifungal active component in SPAF（inclusding the alcohol-soluble extracts SPAF-1 and the water-soluble extracts SPAF-2）was determined through analyzing the components of the fermentation broth.The SPAF-1 was obtained by 90%ethanol desorption.The active component in SPAF-1 which desorpted from macroporous adsorption resin D-101 with 90%ethanol were identified,and decanol and decanal were determinated to be the main antifugal active components in SPAF-1.The minimum inhibitory concentration（MIC）,minimum fungicide concentration（MFC）and fungicide effect（MFC/MIC）of SPAF-1 against T.rubrum was 62.4 mg/L,125 mg/L and 2.0,respectively,while the three corresponding indexes of positive control was 15.6mg/L,93.6 mg/L and 6.0,respectively.The fungicide effect（MFC/MIC）of SPAF-1 was better than that of positive control.3.Through separation and purification,the structure and molecular formula(C₃₇H₄₃NO₁₀)of the main component against T.rubrum in SPAF-2 were identified and analyzed by four major spectra.The MIC,MFC and MFC/MIC values of SPAF-2against T.rubrum was 31.2 mg/L,31.2 mg/L and 1,while the three corresponding indexes of positive control was 15.6 mg/L,93.6 mg/L and 6.0,respectively.The fungicide effect（MFC/MIC）of SPAF-2 was also better than that of positive control.4.The mechanism of SPAF on T.rubrum was studied from the aspects of cell wall membrane,cytoplasmic membrane,wall membrane composition and microscopic morphology.Laser confocal fluorescence microscopy,and electrical conductiveity,and extracellular nucleic acid detection results showed that the cell wall membrane components and cytoplasmic inner membrane of T.rubrum were obviously damaged when treatment with SPAF.Nucleic acid dye was bound to the DNA of dead pathogenic fungi,and the increase of extracellular OD_260nmvalue indicated that the leakage of nucleic acid.The decrease of ergosterol content indicated that the synthesis of ergosterol was blocked,which further result in an increase of cell permeability.FT-IR analysis of T.rubrum wall membrane components showed that the phospholipid and protein components in T.rubrum cell wall membranes were different with or without SPAF treatment.The results of the mechanism study indicated that wall membrane and intracellular membrane system were the main targets of SPAF against T.rubrum.In addition,toxicity experiments on human foreskin fibroblast（HFF）show that SPAF is safe and reliable for human cells within the effective inhibitory concentration range.To sum up,in this paper,the alcohol-soluble active ingredients（including decanol and decanal）and water-soluble active ingredient（griseococcin（1））were obtained from the fermentation broth of B.radicata spore powder.In vitro antifungal experiments indicated that the antifungal effects of SPAF were superior.Fungicidal mechanism study indicated that SPAF could increase cell permeability,destroyed the integrity of cell wall and plasma membrane,and changed the components of pathogenic fungi cell membrane.The cytotoxicity test incated that SPAF was non-toxic action to the HFF cells at the concentration range from 0 mg/L to 250 mg/L.The experiments provide important theoretical and technical support for new drug development on tinea pedis and improving the quality of drug.