Screening of Antifungal Substances from Bovistella Radicata (Mont.) Pat and Their Antifungal Effect

Screening of Antifungal Substances from Bovistella Radicata (Mont.) 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Pat and Their Antifungal Effect Yong Ye, Qinghua Zeng, Kun Liu, zeng qingmei This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-33942/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background To analyse the antifungal active compounds in B. radicata alcohol extract, the alcohol extract was purified by column chromatography (macroporous resinD-101) and the active compounds was named as SPAF-1(the spore powder active fraction). Results Alcohol extracts and SPAF-1 were submitted to GC-MS analysis, there were two characteristic peaks(peak1 and peak2) in Gas chromatogram. By comparing in NIST, the compound were 2-propyl-1-pentanol corresponding to peak1 and decanal, n-decanol and 2E-decanol corresponding to peak2. The main constituents were decanal(24.3%) n-decanol(27.9%), 2E-decenol (21.2%) and 2-Propyl-1-pentanol (13.6%). Their MIC values were 62.5 µg/ml, 31.2 µg/ml, 31.2 µg/ml and 250 µg/ml against T. rubrum respectively. Furthermore, transmission electron microscope (TEM) analysis showed altered surface morphology in the majority of T. rubrum cells after treatment with SPAF-1. Conclusions In this paper, we successfully separate SPAF-1 from alcohol extract of B. radicata . The antifungal effect of SPAF-1 is similar to positive control, the main component were decanal, n-decanol, 2E-decenol and 2-Propyl-1-pentanol, the anti-tinea pedis effect of them was obvious. General Microbiology Applied & Industrial Microbiology antifungal activity extracellular metabolites HPLC FT-IR GC–MS decanol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Tinea pedis is a chronic fungal infection of the feet [1] . Patients that have tinea pedis may be affected by several pathogens, including filamentous fungi named Trichophyton rubrum and Trichophyton mentagrophytes [2] , as well as a yeast named Candida albicans [3] . T. rubrum is the main pathogenic fungi for tinea pedis, having a prevalence as high as 80% among all tinea-pedis-associated pathogenic microbes [4] . Traditionally, to treat tinea pedis, synthetic fungicides such as fluconazole, itraconazole, echinocandins [5] , and miconazole nitrate, either by oral medication or external use [6] , have been used to treat this disease. Vermes et al. ( 2000 ) found that flucytosine and AMB (amphotericin B) were moderately effective in fighting against invasive fungal infections [7–9] . Similar studies on Itraconazole have demonstrated that it is effective against fungal infections [10] . However, due to side effects or the continuous drug resistance, some oral medications are unsafe for patients [11], and these chemicals also cause potential deleterious effects on the environment due to their residues [12–13] , which has prompted researchers to develop newer and safer antifungal agents. Generally speaking, natural products extracted from plants represent a rich resource for screening bioactive compounds [14] . Puffballs are widely distributed in many provinces of China, and are represented by more than 100 species [15] . Calvatia gigantean(Batsch ex Pers.)Lloyd, Calvatia lilacina (Mont.et Berk.) Lloyd, Lasiosphaera fenzlii Reich, Lycoperdon pyriforme Schaeff.:pers , Bovistella radicata (Mont.) Pat, Handkea utriformis (HU), H. excipuliformis (HE) , and Vascellum pratense (VP) are a ll common medicinal puffballs. Although no longer edible in their mature state (because of their powdery consistency), these puffballs have been shown to be a source of active compounds of various biological activities. Puffballs are believed to have several therapeutic properties when used medicinally: hemostasis [16] , cough relief [17] , suppression of cell division, and antitumor [18] and antimicrobial [19] properties. Petrović P, et al(2016) reported noticeable antimicrobial activity diversity for the methanol extracts obtained from Handkea utriformis (HU), H. excipuliformis (HE), and Vascellum pratense (VP) [20] . The spore powder of HFTU-PB1 has been used traditionally as a folk remedy for tinea pedis. Picked specimen (named HFUT-PB1) was dried and deposited at the herbarium of the Department of Biology, Hefei University of Technology (HFUT), China. The aim of the present study was to evaluate the antifungal activity of the spore powder active fraction (SPAF) extracted from Bovistella radicata (Mont.) Pat and its fractions against different species of tinea pedis pathogens, including T. rubrum and T. mentagrophytes . The differences of antifungal activities and GC fingerprints between spore and sporophore powder were determined. The antifungal activities were evaluated in terms of their minimum inhibitory concentration (MIC) values and zone of inhibition(ZOI) values [21] , and the chemical constituents responsible for this activity were identified. Results In Vitro Antifungal Activity Assay The puffballs ( B. radicata, C. gigantean, C. lilacina, L. fenzlii Reich , and L. pyriforme ) exhibited different antifungal activity against T. rubrum , T. mentagrophytes , E. floccosum , and C. albicans . The results were shown in Table 1. Alcohol extract of B. radicata spore powder showed good activity against the tested microorganisms (MIC was 62.5 and 125 µg/ml), while alcohol extract of B. radicata sopophore powder and other puffballs ( C. gigantean, C. lilacina, L. fenzlii Reich , and L. pyriforme ) showed weak activity against the fungus (MIC was about 250 and 500 µg/ml). Table 1 MIC volues of puffball spore or sporophore powder MIC(µg/ml) Test items T.rubrum T.mentagrophytes E.floccosum C.albicans T U T U T U T U B. radicata 62.5 250 125 250 125 250 125 250 C.lilacina Lioyd 250 500 250 500 250 500 500 500 L.fenzlii Reich 500 ༞500 500 500 500 ༞500 500 ༞500 L.pyriforme 500 ༞500 500 ༞500 250 500 500 500 C.gigantean 250 500 500 ༞500 500 ༞500 125 250 Terbinafine 62.5 31.2 62.5 31.2 Note T spore powder groups treated with alcohol, U sporophore powder groups treated with alcohol, Terbinafine is positive control. As shown in Table 1, antifungal effect of B. radicata sporophore powder and other puffball alcohol extract performed poor antifungal activity. The zone of inhibition (ZOI) was observed in PDA medium inoculated with T. rubrum , after co-cultivation with different alcohol extracts from B. radicata and L.fenzlii Reich , The results were showed in Fig. 1. Figure1. zone of inhibition(ZOI) of spore and sporophore powder from B.radicata From Fig. 1, the inhibitory effects on the growth of T. rubrum suggested that antifungal compound maybe was from B. radicata spore powder. SPAF-1 preparation from B. radicata alcohol extract by macroporous resin D-101 We focused on B. radicata spore powder alcohol extract for further extraction and purification. This portion was separated by column chromatography using macroporous resin D-101 with 90% ethanol as eluent. The antifungal effectiveness of alcohol extract from B. radicata , eluent fractions with distilled water(EF) and 90% ethanol (SPAF-1) were assessed against T. rubrum (the main pathogenic fungi of tinea pedis). The antifungal assay confirmed that the alcohol extract and SPAF-1 exhibited high antifungal effectiveness, while EF exhibited weak antifungal activity (MIC = 250 mg/l). MIC of alcohol extract was 62.5 mg/l. While SPAF-1( 90% ethanol eluent fraction) exhibited the most complete activity with a 100% inhibition rate at a concentration of 31.2 mg/l. HPLC analysis of alcohol extract and SPAF-1 from B. radicata As stated above, alcohol extract and SPAF-1 from B. radicata have obvious antifungal activity, while the eluent fractions(EF) has weak antifungal activity. It meaned that the active ingredient(SPAF-1) could be purified by macroporous resin D-101. Alcohol extract and SPAF-1 were analysed by HPLC. The characteristic peak1(2.42 min) and peak2(16.04 min) were shown in Fig. 2B (SPAF-1), the results also demonstrated macroporous resin D-101 was a good selectivity for purification of SPAF-1. Figure 2 . HPLC chromatograms of SPAF-1 and alcohol extract from B. radicata spore powder Chemical constituent analysis of SPAF-1 by GC-MS and HPLC The antifungal substances in B. radicata were also analyzed by GC-MS, among the GC-MS chromatograms of alcohol extract of spore powder, sporophore powder and SPAF-1. More than 50 compounds were identified from spore and sporophore powder of B. radicata by GC-MS. Fitting analysis results of spore and sporophore GC chromatograms showed that spore chromatograms presented characteristic peak P1(8.791 min) and P2(17.825 min) (Fig. 3 ), the characteristic peak time was consistent with SPAF-1. Figure 3 . Fitting gas chromatogram of B. radicata spore and sporophore powder The test results were submitted to the National Institute of Standards and Technology (NIST) library, we analyzed the mass chromatograms of SPAF-1, the results were showed in Table 2 . Peak1 revealed a peak of m/z 116 (M + H), by comparing in NIST, the substance were 2-propyl-1-pentanol. Peak2 revealed peaks of m/z 158,145,130,141 (M + H), by comparing in NIST, the substances may include decanal, n-decanol and 2E-decanol, The results are showed in Table 2 . Table 2 chemical composition and antifungal activity of alcohol extract from B. radicata name time MIC content(%) chemical name molecular molecular (min) (µg/ml) formula weight peak1 8.791 250 13.6 2-propyl-1-pentanol C 7 H 16 116 peak2 17.83 62.5 24.3 decanal C 10 H 19 O 156 31.2 27.9 n-decanol C 10 H 21 O 158 31.2 21.2 2E-decanol C 10 H 21 O 158 Among them, decanol, n-decanol and 2E-decanol can inhibite the growth of T. rubrum at minimum inhibitory concentration of 62.5, 31.2 and 31.2 µg/ml. FT-IR analysis of SPAF-1 The FT-IR spectral results of alcohol extract, SPAF-1 and EF are shown in Fig. 4 , the differences range among them are from 1100 cm − 1 to 1800 cm − 1 and 720 cm − 1 to 900 cm − 1 . The broad stretch of frequency from 3500 -3200cm − 1 was assigned to the hydroxyl group. There are no stretching vibrations at 1600,1580, 1500 and 1450 cm − 1 which is considered to no aromatics in alcohol extract, EF and SPAF-1. In addition, the band obtained at ~ 1433 cm − 1 (SPAF-1) and 1652 cm − 1 (alcohol) is assigned to the stretching vibrations of unsaturated –C = O and the stretch from 1300 cm − 1 to 1000 cm − 1 was attributed to -C-O. The –C = O and -C-O stretching vibrations could be due to aldehyde group and hydroxyl group. The absorbance signals at 2930, 2932, and from 2300 to 2500 cm − 1 could be explained by a long fat chain existing in the compounds. Decanal, n-decanol, 2E-decanol and 2-Propyl-1-pentanol as components of SPAF-1 were verified by FT-IR. Figure 4 . FT-IR spectral of SPAF-1, alcohol extract and EF from HFUT-PB1 Ultrastructural changes in T.rubrum with SPAF-1 To investigate the changes in shape and ultrastructure of T.rubrum cells, cells were examined after co-cultivation with SPAF-1 by transmission electron microcopy. As shown in Fig. 5A. The membrane of the non-inoculated control cells was intact with uniformly distributed cytoplasm and electron density inside the cells. In contrast, more than 30% of T. rubrum cells showed concentrated cytoplasm and altered cell morphology after co-cultivation with SPAF-1 (Fig. 5B).As indicated by the red arrow 1, the cell membrane is badly shrunk out of wall. As indicated by the red arrow 2, the cell wall is obviously thinner and the edge is blurred comparing with the control. which may increase membrane permeability and cause leakage of intracellular substances. Figure 5. Transmission electron microscope images of T. rubrum co-cultured with SPAF-1 Conclusions In this paper, we successfully separate SPAF from alcohol extract of B. radicata . The antifungal effect of SPAF is similar to positive control. The compound SPAF was characterized as decanal(24.3%) n-decanol(27.9%), 2E-decenol (21.2%) and 2-Propyl-1-pentanol (13.6%) by GC-MS, FT-IR and HPLC analysis. They could inhibite the growth of T. rubrum at minimum inhibitory concentration of 62.5 µg/ml, 31.2 µg/ml, 31.2 µg/ml and 250 µg/ml respectively. TEM (transmission electron microscope) analysis showed altered surface morphology in the majority of T. rubrum cells after treatment with SPAF. Methods Tested sample and pathogenic microbiology Tested puffball was isolated from pine forest of Jiangxi province, China and identified as Bovistella radicata (Mont.) Pat . The other tested puffballs included Calvatia gigantea, Calvatia lilacina Lloyd, Lasiosphaera fenzlii , and Lycoperdon pyriforme purchased from Anhui Bozhou traditional Chinese medicine commodity trading center. All of these puffball specimens were dried and deposited at herbarium of Department of Biology, Hefei University of Technology. The four tested pathogenic bacterias included Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6051), Escherichia coli (ATCC 8739) and Pseudomonas aeruginosa (ATCC 15442), four fungi Trichophyton rubrum (ATCC 28188), Trichophyton mentagrophytes (ATCC 9533), Epidermophyton floccosum (ATCC 52066), and Candida albicans (ATCC 10231). In Vitro Antagonistic Activity of Puffball The examined methods were the minimum inhibitory concentrations (MICs)(Negi et al.2003)and zone of inhibitions (ZOIs). MIC value was determined in the 96-well plates by the double micro dilution method(7.8 ~ 250 µg/mL) against pathogens. ZOI (100 µg/ml) was also evaluated [33] , Terbinafine and Gentamicin sulfate as the positive control. Alcohol extraction by Soxhlet system Puffball samples include spore and sporophore powder. The Soxhlet system includes extraction bottle, extraction tube and condenser. When extracting, the samples were wrapped in a degreased filter paper bag and put into the extraction tube. Alcohol was added into the extraction bottle, the extraction bottle was heated, alcohol was gasified, risen, condensed, dripped into the extraction tube, the bioactive components were extracted into the extraction bottle. so that the cycle reciprocates untile the extraction is complete. The chemical constituents of alcohol extraction by GC–MS The alcohol extracts were recorded on a GC–MS(Gas Chromatography-Mass Spectrometer) system. One microliter of alcohol extract of spore and sporophore powder was injected into a DB-5MS capillary column coated with 5% diphenyl cross-linked 95% dimethylpolysiloxane (30 m × 250 µm inner diameter, 0.25 µm film thickness; J&W Scientific, Folsom, CA, USA). The alcohol extract were injected in the splitless mode. Helium was used as the carrier gas. The front inlet purge flow was 1 mL/min, and the gas flow rate through the column was 20 mL/min. The initial temperature was kept at 40 ℃, held for 3.0 min, then raised to 150 ℃ at a rate of 10 ℃/min. The temperature was kept for 10 min at 150 ℃. The mass spectrometer was operated in electronionization (EI) mode at 70 eV. The injection, transfer line, and ion source temperatures were 150, 250, and 230 ℃, respectively. The mass spectrometry data were acquired in full-scan mode with an m/z range of 10–500 u at a rate of 20 u spectra per second after a solvent delay of 210 s. Peaks were identified by comparing with the mass spectra data from the National Institute of Standards and Technology (NIST) spectral library. HPLC analysis of alcohol extract and SPAF-1 from Bovistella radicata (Mont.) Pat Alcohol extract from B. radicata was also analyzed via high-performance liquid chromatography (HPLC) coupled with an ultraviolet (UV) detector (Agilent, 1260 Infinity II Prime, USA). The aim was to identify the most active compounds in the fractions of the puffballs. A C18 reversed-phase column (Hypersil Gold 25 mm × 2.1 mm,1.8 µm, Thermo Scientific, Massachusetts, USA) was used with the following solvent system: A = acetonitrile, B = 0.15% ammonium acetate–water. The gradient elution was 7% A in 5 min, 7–10% A in 3 min, 10% A in 2 min, 10–15% A in 5 min, 15% A in 3 min, 5–15% A in 2 min. The LC system was operated at a flow rate of 1.0 mL/min for 20 min. The injection volume was 5 µL, and the detection was at 220 nm. FT-IR analysis of SPAF-1 The FT-IR(Fourier transform infrared spectroscopy) spectra of samples were recorded using a NICOLET 5700 Fourier Transform Infrared Spectrometer (provided by Nicolet Instrument Co., U.S.A) using potassium bromide (KBr) pellets. The pellets were designed by blending the sample and KBr at a ratio of 1:100 and were ground into particles smaller than 0.1 mm. The FT-IR measurement scanned the range from 400 to 4000 cm − 1 . A He-Ne laser source operating at 0.5 W was utilized for sample excitation. Transmission Electron Microscopy Overnight cultures of T. rubrum . were on PDA. The wells (5.0 mm in diameter) were cut from PDA medium, The tested compounds (SPAF-1) were added to the wells, the wells added normal saline were used as controls, then incubated at 28℃ for 48 hours. Following the incubation period samples were pick up from PDA and fixed overnight in 2.5% glutaraldehydein PBS (phosphate-buffered saline), washed three times in PBS and postfixed overnight in 1% osmium tetroxide. Following ethanol dehydration, probes were embedded in Epon resin (Sigma-Aldrich), cut on a ultramicrotome Leica UC7 and contrasted in uranyl acetate and lead citrate. Transmission electron microscopy studies were performed using a Philips CM100 electron microscope [34] . Discussion In the present study, B. radicata from Jiangxi province, China, shows remarkable antifungal activities. These data are consistent with previous findings on the minimum inhibitory concentrations (MICs) of B. radicata [19] . According to the Chinese Pharmacopeia, the main anti-microorganism activity of the puffball is against S. aureus and P.aeruginosa . The antifungal function of puffballs has not been reported previously. The novel antimicrobial activities of B. radicata might be due to different geographic sources of the material used and different strains used [22] . I n vitro assays demonstrated that alcohol extract and SPAF-1 from B. radicata were able to suppress the mycelial growth of T. rubrum and T. mentagrophytes. The MICs of SPAF-1 from alcohol extract are 31.2 µg/ml and 31.2 µg/ml against T. rubrum and T. mentagrophytes , respectively. Terbinafine was used as a positive control in this study with MIC values 62.5 and 31.2 µg/ml. The antifungal effect of SPAF-1 was similar with that of positive control. The SPAF-1 were purified by macroporous resin D-101 and submitted to GC‒MS analysis. The main constituents were identified alcohols(62.7%) and aldehydes(30.2%), the main alcohols were n-decanol(27.9%), 2E-decenol(21.2%) and 2-ethyl-1-Hexanol(13.6%), while the major aldehydes were decanal(24.3%). alcohols, aldehydes and 2-Propyl-1-pentanol have a long alphatic chain. Alphatic alcohols are the most widespread compounds found in plants, and are also known to cause a variety of biochemical responses [23] . Long-chain aliphatic alcohols ranging in chain length from 6 to greater than 20 carbon atoms can inhibit the growth of various types of bacteria and fungi [24–26] . n-decanol, 2E-decenol are volatile organic compounds (VOC) and have been reported to possess various bioactivities [27] . Previous studies reported that 1-Decanol exhibited antibacterial [28] and antimicrobial [29] activities. Begnami et al ( 2010 ) found that 1-decanol, 2E-decenol, 2Z-dodecenol, and aldehydes from Coriandrum sativum L(Apiaceae) exhibited effective growth-inhibitory activities against five species of Candida albicans [30] , while activities against T. rubrum and T. mentagrophytes , the main pathogens of tinea pedis have not been reported. All these aliphatic alcohols and aldehydes obtained from B. radicata alcohol extracts increase the fluidity of the membrane and are known to inhibit the key enzyme of saturated fatty acids [31] . The aliphatic alcohols also have one hydroxyl group. The hydroxyl group mainly determines the hydrophilicity of the molecule, resulting in much easier interaction with its target in a living organism [32] . Future work concentrating on determining the antifungal mechanisms of aliphatic alcohols and aldehydes will be performed, which will be helpful in laying a foundation for overcoming the drug resistance that pathogens quickly develop against antibiotic drugs. Abbreviations AMB Amphotericin B PDA Potato Dextrose Agar EI electronionization HPLC High Performance Liquid Chromatography UV ultraviolet MIC Minimum Inhibitory Concentration NIST National Institute of Standards and Technology GC–MS Gas Chromatography-Mass Spectrometer FT-IR Fourier-Transform Infrared VOC Volatile Organic Compound Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and material Not applicable Competing interests Not applicable Funding This study was supported by the National Natural Science Foundation of China (Grant No. 31371844; 31071556) and the Science and Technology Department of Anhui province, PR of China (Grant No. 130103215) Authors' contributions Yong ye conceived and designed the experiments. Yong ye performed the experiments and analyzed the data. Qinghua zeng analyzed the data. Yong ye wrote and edited the manuscript. 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Aghayeva DN, Lutz M, Piatek M. Transmission electron microscopy of Tuberculina species (Helicobasidiales) reveals an unique mode of conidiogenesis within Basidiomycota. Fungal Biology. 2016;120(8):1010–6. 238125 - 47625Figure1 . zone of inhibition ( ZOI ) of spore and sporophore powder from B.radicata is ZOI of. alcohol extract from B. radicata spore powder; 1 is alcohol extract from B. radicata sporophore powder; and 2 is alcohol extract from L.fenzlii Reich. “+” is positive control and “-“ is negative control. -190500-304800 Figure . 2 . HPLC chromatograms of SPAF-1 and alcohol extract from B. radicata spore powder. A:; HPLC chromatograms of alcohol extract from B. radicata spore powder . B . HPLC chromatograms of SPAF-1 . Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-33942","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":650778,"identity":"38b1492b-3c8c-4bf8-b7b6-cc843586ff0b","order_by":0,"name":"Yong Ye","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Ye","suffix":""},{"id":650779,"identity":"25b989a2-4982-4253-b334-e483f0ae670e","order_by":1,"name":"Qinghua Zeng","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qinghua","middleName":"","lastName":"Zeng","suffix":""},{"id":650780,"identity":"16eab430-2810-48b2-a937-3d2859194f5d","order_by":2,"name":"Kun Liu","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Liu","suffix":""},{"id":650781,"identity":"143c77ef-2aca-489d-b4fd-7f60905b8e94","order_by":3,"name":"zeng qingmei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYNACAxDB2PiYwQbBJUpLszFDGoMEkVoggE2aKC3y7b2HX7wpuGO3tr25rbog4XAdA3vzNgmGmjs4tTD2nEuznGPwLHnbmYNtt2ckHJZg4DlWJsFw7BlOLcwSOWbGPAaHk81uJLbd5v0B1AIUkWBsOIzb/fJvoFruP2wr5gHZAhTBq4VHgsf4MVCLndkNxjZmsBYJHvxaJHhyzBjnGBxOMDuT2CzNk5Au2caTVmyRcAy3Fvn2M8Yf3vw5bG92/PjDzzwJ1vz87Ic33vhQg1sLyDsSPAwMiQ1wLohIwKcBGGgfgFrs8asZBaNgFIyCEQ0AKhJR4KMUkroAAAAASUVORK5CYII=","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"zeng","middleName":"","lastName":"qingmei","suffix":""}],"badges":[],"createdAt":"2020-06-05 18:32:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-33942/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-33942/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":1299858,"identity":"2eec7172-59f0-47a5-ae58-ff763b0df4fe","added_by":"auto","created_at":"2020-06-10 17:49:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52552,"visible":true,"origin":"","legend":"zone of inhibition(ZOI) of spore and sporophore powder from B.radicata \n0 is ZOI of alcohol extract from B. radicata spore powder; 1 is alcohol extract from B. radicata sporophore powder; and 2 is alcohol extract from L.fenzlii Reich. “+” is positive control and “-“ is negative control.","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-33942/v1/Figure1.jpg"},{"id":1299859,"identity":"b35c56e0-6a0c-4fdd-aad5-7a83f03fdc11","added_by":"auto","created_at":"2020-06-10 17:49:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":36333,"visible":true,"origin":"","legend":"HPLC chromatograms of SPAF-1 and alcohol extract from B. radicata spore powder\nA :; HPLC chromatograms of alcohol extract from B. radicata spore powder\nB: HPLC chromatograms of SPAF-1","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-33942/v1/Figure2.jpg"},{"id":1299860,"identity":"98a832da-c722-458a-a08f-d5a2d9849a64","added_by":"auto","created_at":"2020-06-10 17:49:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73976,"visible":true,"origin":"","legend":"Fitting gas chromatogram of B. radicata spore and sporophore powder","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-33942/v1/Figure3.jpg"},{"id":1299861,"identity":"4f384acc-1b1a-4ddd-8cae-ba0791b679ca","added_by":"auto","created_at":"2020-06-10 17:49:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":75647,"visible":true,"origin":"","legend":"FT-IR spectral of SPAF-1, alcohol extract and EF from HFUT-PB1","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-33942/v1/Figure4.jpg"},{"id":1299862,"identity":"61376508-9aee-4dfe-92b7-64ccab623c6f","added_by":"auto","created_at":"2020-06-10 17:49:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":84974,"visible":true,"origin":"","legend":"Transmission electron microscope images of T. rubrum co-cultured with SPAF-1.\n(A) Untreated control T. rubrum. (B) T. rubrum co-cultured with SPAF-1","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-33942/v1/Figure5.jpg"},{"id":13538650,"identity":"47317a3d-e95b-4c42-9175-8ddb18ebfc49","added_by":"auto","created_at":"2021-09-17 01:40:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":675652,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-33942/v1/27b03f06-6867-4790-973d-b320799b66f3.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eScreening of Antifungal Substances from Bovistella Radicata (Mont.) Pat and Their Antifungal Effect\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eTinea pedis is a chronic fungal infection of the feet\u003csup\u003e[1]\u003c/sup\u003e. Patients that have tinea pedis may be affected by several pathogens, including filamentous fungi named \u003cem\u003eTrichophyton rubrum\u003c/em\u003e and \u003cem\u003eTrichophyton mentagrophytes\u003c/em\u003e \u003csup\u003e[2]\u003c/sup\u003e, as well as a yeast named \u003cem\u003eCandida albicans\u003c/em\u003e \u003csup\u003e[3]\u003c/sup\u003e. \u003cem\u003eT. rubrum\u003c/em\u003e is the main pathogenic fungi for tinea pedis, having a prevalence as high as 80% among all tinea-pedis-associated pathogenic microbes\u003csup\u003e[4]\u003c/sup\u003e. Traditionally, to treat tinea pedis, synthetic fungicides such as fluconazole, itraconazole, echinocandins \u003csup\u003e[5]\u003c/sup\u003e, and miconazole nitrate, either by oral medication or external use\u003csup\u003e[6]\u003c/sup\u003e, have been used to treat this disease. Vermes et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) found that flucytosine and AMB (amphotericin B) were moderately effective in fighting against invasive fungal infections\u003csup\u003e[7\u0026ndash;9]\u003c/sup\u003e. Similar studies on Itraconazole have demonstrated that it is effective against fungal infections\u003csup\u003e[10]\u003c/sup\u003e. However, due to side effects or the continuous drug resistance, some oral medications are unsafe for patients \u003csup\u003e[11],\u003c/sup\u003e and these chemicals also cause potential deleterious effects on the environment due to their residues\u003csup\u003e[12\u0026ndash;13]\u003c/sup\u003e, which has prompted researchers to develop newer and safer antifungal agents. Generally speaking, natural products extracted from plants represent a rich resource for screening bioactive compounds\u003csup\u003e[14]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePuffballs are widely distributed in many provinces of China, and are represented by more than 100 species\u003csup\u003e[15]\u003c/sup\u003e. \u003cem\u003eCalvatia gigantean(Batsch ex Pers.)Lloyd, Calvatia lilacina (Mont.et Berk.) Lloyd, Lasiosphaera fenzlii Reich, Lycoperdon pyriforme Schaeff.:pers\u003c/em\u003e, \u003cem\u003eBovistella radicata (Mont.) Pat, Handkea utriformis (HU), H. excipuliformis (HE)\u003c/em\u003e, and \u003cem\u003eVascellum pratense (VP)\u003c/em\u003e are \u003cem\u003ea\u003c/em\u003ell common medicinal puffballs. Although no longer edible in their mature state (because of their powdery consistency), these puffballs have been shown to be a source of active compounds of various biological activities. Puffballs are believed to have several therapeutic properties when used medicinally: hemostasis\u003csup\u003e[16]\u003c/sup\u003e, cough relief\u003csup\u003e[17]\u003c/sup\u003e, suppression of cell division, and antitumor\u003csup\u003e[18]\u003c/sup\u003e and antimicrobial\u003csup\u003e[19]\u003c/sup\u003e properties. Petrović P, et al(2016) reported noticeable antimicrobial activity diversity for the methanol extracts obtained from Handkea utriformis (HU), H. \u003cem\u003eexcipuliformis\u003c/em\u003e (HE), and \u003cem\u003eVascellum pratense\u003c/em\u003e (VP)\u003csup\u003e[20]\u003c/sup\u003e. The spore powder of HFTU-PB1 has been used traditionally as a folk remedy for tinea pedis. Picked specimen (named HFUT-PB1) was dried and deposited at the herbarium of the Department of Biology, Hefei University of Technology (HFUT), China.\u003c/p\u003e \u003cp\u003eThe aim of the present study was to evaluate the antifungal activity of the spore powder active fraction (SPAF) extracted from \u003cem\u003eBovistella radicata (Mont.) Pat\u003c/em\u003e and its fractions against different species of tinea pedis pathogens, including \u003cem\u003eT. rubrum\u003c/em\u003e and \u003cem\u003eT. mentagrophytes\u003c/em\u003e. The differences of antifungal activities and GC fingerprints between spore and sporophore powder were determined. The antifungal activities were evaluated in terms of their minimum inhibitory concentration (MIC) values and zone of inhibition(ZOI) values\u003csup\u003e[21]\u003c/sup\u003e, and the chemical constituents responsible for this activity were identified.\u003c/p\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIn Vitro Antifungal Activity Assay\u003c/h2\u003e \u003cp\u003eThe puffballs (\u003cem\u003eB. radicata, C. gigantean, C. lilacina, L. fenzlii Reich\u003c/em\u003e, and \u003cem\u003eL. pyriforme\u003c/em\u003e) exhibited different antifungal activity against \u003cem\u003eT. rubrum\u003c/em\u003e, \u003cem\u003eT. mentagrophytes\u003c/em\u003e, \u003cem\u003eE. floccosum\u003c/em\u003e, and \u003cem\u003eC. albicans\u003c/em\u003e. The results were shown in Table\u0026nbsp;1. Alcohol extract of \u003cem\u003eB. radicata\u003c/em\u003e spore powder showed good activity against the tested microorganisms (MIC was 62.5 and 125\u0026nbsp;\u0026micro;g/ml), while alcohol extract of \u003cem\u003eB. radicata\u003c/em\u003e sopophore powder and other puffballs (\u003cem\u003eC. gigantean, C. lilacina, L. fenzlii Reich\u003c/em\u003e, and \u003cem\u003eL. pyriforme\u003c/em\u003e) showed weak activity against the fungus (MIC was about 250 and 500\u0026nbsp;\u0026micro;g/ml).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable\u0026nbsp;1 MIC volues of puffball spore or sporophore powder\u003c/b\u003e \u003c/p\u003e \u003cp\u003e MIC(\u0026micro;g/ml)\u003c/p\u003e \u003cp\u003e Test items \u003cem\u003eT.rubrum T.mentagrophytes E.floccosum C.albicans\u003c/em\u003e\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eT U T U T U T U\u003c/h2\u003e \u003cp\u003e \u003cem\u003eB. radicata\u003c/em\u003e 62.5 250 125 250 125 250 125 250\u003c/p\u003e \u003cp\u003e \u003cem\u003eC.lilacina Lioyd\u003c/em\u003e 250 500 250 500 250 500 500 500\u003c/p\u003e \u003cp\u003e \u003cem\u003eL.fenzlii Reich\u003c/em\u003e 500 ༞500 500 500 500 ༞500 500 ༞500\u003c/p\u003e \u003cp\u003e \u003cem\u003eL.pyriforme\u003c/em\u003e 500 ༞500 500 ༞500 250 500 500 500\u003c/p\u003e \u003cp\u003e \u003cem\u003eC.gigantean\u003c/em\u003e 250 500 500 ༞500 500 ༞500 125 250\u003c/p\u003e \u003cp\u003e Terbinafine 62.5 31.2 62.5 31.2\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNote\u003c/strong\u003e \u003cp\u003eT spore powder groups treated with alcohol, U sporophore powder groups treated with alcohol, Terbinafine is positive control.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;1, antifungal effect of \u003cem\u003eB. radicata\u003c/em\u003e sporophore powder and other puffball alcohol extract performed poor antifungal activity. The zone of inhibition (ZOI) was observed in PDA medium inoculated with \u003cem\u003eT. rubrum\u003c/em\u003e, after co-cultivation with different alcohol extracts from \u003cem\u003eB. radicata\u003c/em\u003e and \u003cem\u003eL.fenzlii Reich\u003c/em\u003e, The results were showed in Fig.\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure1. zone of inhibition(ZOI) of spore and sporophore powder from\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eB.radicata\u003c/span\u003e\u003c/p\u003e \u003cp\u003eFrom Fig.\u0026nbsp;1, the inhibitory effects on the growth of \u003cem\u003eT. rubrum\u003c/em\u003e suggested that antifungal compound maybe was from \u003cem\u003eB. radicata\u003c/em\u003e spore powder.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSPAF-1 preparation from \u003cem\u003eB. radicata\u003c/em\u003e alcohol extract by macroporous resin D-101\u003c/h2\u003e \u003cp\u003eWe focused on \u003cem\u003eB. radicata\u003c/em\u003e spore powder alcohol extract for further extraction and purification. This portion was separated by column chromatography using macroporous resin D-101 with 90% ethanol as eluent. The antifungal effectiveness of alcohol extract from \u003cem\u003eB. radicata\u003c/em\u003e, eluent fractions with distilled water(EF) and 90% ethanol (SPAF-1) were assessed against \u003cem\u003eT. rubrum\u003c/em\u003e (the main pathogenic fungi of tinea pedis). The antifungal assay confirmed that the alcohol extract and SPAF-1 exhibited high antifungal effectiveness, while EF exhibited weak antifungal activity (MIC\u0026thinsp;=\u0026thinsp;250\u0026nbsp;mg/l). MIC of alcohol extract was 62.5\u0026nbsp;mg/l. While SPAF-1( 90% ethanol eluent fraction) exhibited the most complete activity with a 100% inhibition rate at a concentration of 31.2\u0026nbsp;mg/l.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHPLC analysis of alcohol extract and SPAF-1 from \u003cem\u003eB. radicata\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eAs stated above, alcohol extract and SPAF-1 from \u003cem\u003eB. radicata\u003c/em\u003e have obvious antifungal activity, while the eluent fractions(EF) has weak antifungal activity. It meaned that the active ingredient(SPAF-1) could be purified by macroporous resin D-101. Alcohol extract and SPAF-1 were analysed by HPLC. The characteristic peak1(2.42\u0026nbsp;min) and peak2(16.04\u0026nbsp;min) were shown in Fig.\u0026nbsp;2B (SPAF-1), the results also demonstrated macroporous resin D-101 was a good selectivity for purification of SPAF-1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2\u003c/b\u003e. HPLC chromatograms of SPAF-1 and alcohol extract from B. radicata spore powder\u003c/p\u003e \u003cp\u003e \u003cb\u003eChemical constituent analysis of SPAF-1 by GC-MS and HPLC\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe antifungal substances in \u003cem\u003eB. radicata\u003c/em\u003e were also analyzed by GC-MS, among the GC-MS chromatograms of alcohol extract of spore powder, sporophore powder and SPAF-1. More than 50 compounds were identified from spore and sporophore powder of \u003cem\u003eB. radicata\u003c/em\u003e by GC-MS. Fitting analysis results of spore and sporophore GC chromatograms showed that spore chromatograms presented characteristic peak P1(8.791\u0026nbsp;min) and P2(17.825\u0026nbsp;min) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e), the characteristic peak time was consistent with SPAF-1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e. \u003cb\u003eFitting gas chromatogram of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eB. radicata\u003c/span\u003e \u003cb\u003espore and sporophore powder\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe test results were submitted to the National Institute of Standards and Technology (NIST) library, we analyzed the mass chromatograms of SPAF-1, the results were showed in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Peak1 revealed a peak of m/z 116 (M\u0026thinsp;+\u0026thinsp;H), by comparing in NIST, the substance were 2-propyl-1-pentanol. Peak2 revealed peaks of m/z 158,145,130,141 (M\u0026thinsp;+\u0026thinsp;H), by comparing in NIST, the substances may include decanal, n-decanol and 2E-decanol, The results are showed in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003echemical composition and antifungal activity of alcohol extract from \u003cem\u003eB. radicata\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ename\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003etime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003econtent(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003echemical name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003emolecular\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003emolecular\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eformula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eweight\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epeak1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.791\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2-propyl-1-pentanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e116\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003epeak2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e17.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003edecanal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e156\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en-decanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e158\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2E-decanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e158\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eAmong them, decanol, n-decanol and 2E-decanol can inhibite the growth of \u003cem\u003eT. rubrum\u003c/em\u003e at minimum inhibitory concentration of 62.5, 31.2 and 31.2\u0026nbsp;\u0026micro;g/ml.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cb\u003eFT-IR analysis of SPAF-1\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eThe FT-IR spectral results of alcohol extract, SPAF-1 and EF are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the differences range among them are from 1100\u0026nbsp;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 1800\u0026nbsp;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 720\u0026nbsp;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 900\u0026nbsp;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The broad stretch of frequency from 3500 -3200cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was assigned to the hydroxyl group. There are no stretching vibrations at 1600,1580, 1500 and 1450\u0026nbsp;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e which is considered to no aromatics in alcohol extract, EF and SPAF-1. In addition, the band obtained at ~\u0026thinsp;1433\u0026nbsp;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (SPAF-1) and 1652\u0026nbsp;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (alcohol) is assigned to the stretching vibrations of unsaturated \u0026ndash;C\u0026thinsp;=\u0026thinsp;O and the stretch from 1300\u0026nbsp;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 1000\u0026nbsp;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was attributed to -C-O. The \u0026ndash;C\u0026thinsp;=\u0026thinsp;O and -C-O stretching vibrations could be due to aldehyde group and hydroxyl group. The absorbance signals at 2930, 2932, and from 2300 to 2500\u0026nbsp;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e could be explained by a long fat chain existing in the compounds. Decanal, n-decanol, 2E-decanol and 2-Propyl-1-pentanol as components of SPAF-1 were verified by FT-IR.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e. \u003cb\u003eFT-IR spectral of SPAF-1, alcohol extract and EF from HFUT-PB1\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eUltrastructural changes in\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eT.rubrum\u003c/span\u003e \u003cb\u003ewith SPAF-1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the changes in shape and ultrastructure of \u003cem\u003eT.rubrum\u003c/em\u003e cells, cells were examined after co-cultivation with SPAF-1 by transmission electron microcopy. As shown in Fig.\u0026nbsp;5A. The membrane of the non-inoculated control cells was intact with uniformly distributed cytoplasm and electron density inside the cells. In contrast, more than 30% of \u003cem\u003eT. rubrum\u003c/em\u003e cells showed concentrated cytoplasm and altered cell morphology after co-cultivation with SPAF-1 (Fig.\u0026nbsp;5B).As indicated by the red arrow 1, the cell membrane is badly shrunk out of wall. As indicated by the red arrow 2, the cell wall is obviously thinner and the edge is blurred comparing with the control. which may increase membrane permeability and cause leakage of intracellular substances.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 5. Transmission electron microscope images of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eT. rubrum\u003c/span\u003e \u003cb\u003eco-cultured with SPAF-1\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e "},{"header":"Conclusions","content":"\u003cp\u003eIn this paper, we successfully separate SPAF from alcohol extract of \u003cem\u003eB. radicata\u003c/em\u003e. The antifungal effect of SPAF is similar to positive control. The compound SPAF was characterized as decanal(24.3%) n-decanol(27.9%), 2E-decenol (21.2%) and 2-Propyl-1-pentanol (13.6%) by GC-MS, FT-IR and HPLC analysis. They could inhibite the growth of \u003cem\u003eT. rubrum\u003c/em\u003e at minimum inhibitory concentration of 62.5\u0026nbsp;\u0026micro;g/ml, 31.2\u0026nbsp;\u0026micro;g/ml, 31.2\u0026nbsp;\u0026micro;g/ml and 250\u0026nbsp;\u0026micro;g/ml respectively. TEM (transmission electron microscope) analysis showed altered surface morphology in the majority of T. rubrum cells after treatment with SPAF.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eTested sample and pathogenic microbiology\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTested puffball was isolated from pine forest of Jiangxi province, China and identified as \u003cem\u003eBovistella radicata (Mont.) Pat\u003c/em\u003e. The other tested puffballs included \u003cem\u003eCalvatia gigantea, Calvatia lilacina Lloyd, Lasiosphaera fenzlii\u003c/em\u003e, and \u003cem\u003eLycoperdon pyriforme\u003c/em\u003e purchased from Anhui Bozhou traditional Chinese medicine commodity trading center. All of these puffball specimens were dried and deposited at herbarium of Department of Biology, Hefei University of Technology.\u003c/p\u003e \u003cp\u003eThe four tested pathogenic bacterias included Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6051), Escherichia coli (ATCC 8739) and Pseudomonas aeruginosa (ATCC 15442), four fungi \u003cem\u003eTrichophyton rubrum\u003c/em\u003e(ATCC 28188), \u003cem\u003eTrichophyton mentagrophytes\u003c/em\u003e(ATCC 9533), \u003cem\u003eEpidermophyton floccosum\u003c/em\u003e(ATCC 52066), and \u003cem\u003eCandida albicans\u003c/em\u003e (ATCC 10231).\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIn Vitro Antagonistic Activity of Puffball\u003c/h2\u003e \u003cp\u003eThe examined methods were the minimum inhibitory concentrations (MICs)(Negi et al.2003)and zone of inhibitions (ZOIs). MIC value was determined in the 96-well plates by the double micro dilution method(7.8\u0026thinsp;~\u0026thinsp;250\u0026nbsp;\u0026micro;g/mL) against pathogens. ZOI (100\u0026nbsp;\u0026micro;g/ml) was also evaluated \u003csup\u003e[33]\u003c/sup\u003e, Terbinafine and Gentamicin sulfate as the positive control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAlcohol extraction by Soxhlet system\u003c/h2\u003e \u003cp\u003ePuffball samples include spore and sporophore powder. The Soxhlet system includes extraction bottle, extraction tube and condenser. When extracting, the samples were wrapped in a degreased filter paper bag and put into the extraction tube. Alcohol was added into the extraction bottle, the extraction bottle was heated, alcohol was gasified, risen, condensed, dripped into the extraction tube, the bioactive components were extracted into the extraction bottle. so that the cycle reciprocates untile the extraction is complete.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eThe chemical constituents of alcohol extraction by GC\u0026ndash;MS\u003c/h2\u003e \u003cp\u003eThe alcohol extracts were recorded on a GC\u0026ndash;MS(Gas Chromatography-Mass Spectrometer) system. One microliter of alcohol extract of spore and sporophore powder was injected into a DB-5MS capillary column coated with 5% diphenyl cross-linked 95% dimethylpolysiloxane (30\u0026nbsp;m\u0026thinsp;\u0026times;\u0026thinsp;250\u0026nbsp;\u0026micro;m inner diameter, 0.25\u0026nbsp;\u0026micro;m film thickness; J\u0026amp;W Scientific, Folsom, CA, USA). The alcohol extract were injected in the splitless mode. Helium was used as the carrier gas. The front inlet purge flow was 1\u0026nbsp;mL/min, and the gas flow rate through the column was 20\u0026nbsp;mL/min. The initial temperature was kept at 40 ℃, held for 3.0\u0026nbsp;min, then raised to 150 ℃ at a rate of 10 ℃/min. The temperature was kept for 10\u0026nbsp;min at 150 ℃. The mass spectrometer was operated in electronionization (EI) mode at 70\u0026nbsp;eV. The injection, transfer line, and ion source temperatures were 150, 250, and 230 ℃, respectively. The mass spectrometry data were acquired in full-scan mode with an m/z range of 10\u0026ndash;500 u at a rate of 20 u spectra per second after a solvent delay of 210\u0026nbsp;s. Peaks were identified by comparing with the mass spectra data from the National Institute of Standards and Technology (NIST) spectral library.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHPLC analysis of alcohol extract and SPAF-1 from \u003cem\u003eBovistella radicata (Mont.) Pat\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eAlcohol extract from \u003cem\u003eB. radicata\u003c/em\u003e was also analyzed via high-performance liquid chromatography (HPLC) coupled with an ultraviolet (UV) detector (Agilent, 1260 Infinity II Prime, USA). The aim was to identify the most active compounds in the fractions of the puffballs. A C18 reversed-phase column (Hypersil Gold 25 mm\u0026thinsp;\u0026times;\u0026thinsp;2.1\u0026nbsp;mm,1.8\u0026nbsp;\u0026micro;m, Thermo Scientific, Massachusetts, USA) was used with the following solvent system: A\u0026thinsp;=\u0026thinsp;acetonitrile, B\u0026thinsp;=\u0026thinsp;0.15% ammonium acetate\u0026ndash;water. The gradient elution was 7% A in 5\u0026nbsp;min, 7\u0026ndash;10% A in 3\u0026nbsp;min, 10% A in 2\u0026nbsp;min, 10\u0026ndash;15% A in 5\u0026nbsp;min, 15% A in 3\u0026nbsp;min, 5\u0026ndash;15% A in 2\u0026nbsp;min. The LC system was operated at a flow rate of 1.0\u0026nbsp;mL/min for 20\u0026nbsp;min. The injection volume was 5\u0026nbsp;\u0026micro;L, and the detection was at 220\u0026nbsp;nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFT-IR analysis of SPAF-1\u003c/h2\u003e \u003cp\u003eThe FT-IR(Fourier transform infrared spectroscopy) spectra of samples were recorded using a NICOLET 5700 Fourier Transform Infrared Spectrometer (provided by Nicolet Instrument Co., U.S.A) using potassium bromide (KBr) pellets. The pellets were designed by blending the sample and KBr at a ratio of 1:100 and were ground into particles smaller than 0.1\u0026nbsp;mm. The FT-IR measurement scanned the range from 400 to 4000\u0026nbsp;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. A He-Ne laser source operating at 0.5\u0026nbsp;W was utilized for sample excitation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransmission Electron Microscopy\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOvernight cultures of \u003cem\u003eT. rubrum\u003c/em\u003e. were on PDA. The wells (5.0\u0026nbsp;mm in diameter) were cut from PDA medium, The tested compounds (SPAF-1) were added to the wells, the wells added normal saline were used as controls, then incubated at 28℃ for 48 hours. Following the incubation period samples were pick up from PDA and fixed overnight in 2.5% glutaraldehydein PBS (phosphate-buffered saline), washed three times in PBS and postfixed overnight in 1% osmium tetroxide. Following ethanol dehydration, probes were embedded in Epon resin (Sigma-Aldrich), cut on a ultramicrotome Leica UC7 and contrasted in uranyl acetate and lead citrate. Transmission electron microscopy studies were performed using a Philips CM100 electron microscope \u003csup\u003e[34]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eIn the present study, \u003cem\u003eB. radicata\u003c/em\u003e from Jiangxi province, China, shows remarkable antifungal activities. These data are consistent with previous findings on the minimum inhibitory concentrations (MICs) of \u003cem\u003eB. radicata\u003c/em\u003e \u003csup\u003e[19]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAccording to the Chinese Pharmacopeia, the main anti-microorganism activity of the puffball is against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP.aeruginosa\u003c/em\u003e. The antifungal function of puffballs has not been reported previously. The novel antimicrobial activities of \u003cem\u003eB. radicata\u003c/em\u003e might be due to different geographic sources of the material used and different strains used\u003csup\u003e[22]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eI\u003cem\u003en vitro\u003c/em\u003e assays demonstrated that alcohol extract and SPAF-1 from \u003cem\u003eB. radicata\u003c/em\u003e were able to suppress the mycelial growth of \u003cem\u003eT. rubrum\u003c/em\u003e and \u003cem\u003eT. mentagrophytes.\u003c/em\u003e The MICs of SPAF-1 from alcohol extract are 31.2\u0026nbsp;\u0026micro;g/ml and 31.2\u0026nbsp;\u0026micro;g/ml against \u003cem\u003eT. rubrum\u003c/em\u003e and \u003cem\u003eT. mentagrophytes\u003c/em\u003e, respectively. Terbinafine was used as a positive control in this study with MIC values 62.5 and 31.2\u0026nbsp;\u0026micro;g/ml. The antifungal effect of SPAF-1 was similar with that of positive control.\u003c/p\u003e \u003cp\u003eThe SPAF-1 were purified by macroporous resin D-101 and submitted to GC‒MS analysis. The main constituents were identified alcohols(62.7%) and aldehydes(30.2%), the main alcohols were n-decanol(27.9%), 2E-decenol(21.2%) and 2-ethyl-1-Hexanol(13.6%), while the major aldehydes were decanal(24.3%). alcohols, aldehydes and 2-Propyl-1-pentanol have a long alphatic chain. Alphatic alcohols are the most widespread compounds found in plants, and are also known to cause a variety of biochemical responses \u003csup\u003e[23]\u003c/sup\u003e. Long-chain aliphatic alcohols ranging in chain length from 6 to greater than 20 carbon atoms can inhibit the growth of various types of bacteria and fungi \u003csup\u003e[24\u0026ndash;26]\u003c/sup\u003e. n-decanol, 2E-decenol are volatile organic compounds (VOC) and have been reported to possess various bioactivities\u003csup\u003e[27]\u003c/sup\u003e. Previous studies reported that 1-Decanol exhibited antibacterial \u003csup\u003e[28]\u003c/sup\u003eand antimicrobial\u003csup\u003e[29]\u003c/sup\u003e activities. Begnami et al (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) found that 1-decanol, 2E-decenol, 2Z-dodecenol, and aldehydes from \u003cem\u003eCoriandrum sativum L(Apiaceae)\u003c/em\u003e exhibited effective growth-inhibitory activities against five species of Candida albicans\u003csup\u003e[30]\u003c/sup\u003e, while activities against \u003cem\u003eT. rubrum\u003c/em\u003e and \u003cem\u003eT. mentagrophytes\u003c/em\u003e, the main pathogens of tinea pedis have not been reported.\u003c/p\u003e \u003cp\u003eAll these aliphatic alcohols and aldehydes obtained from \u003cem\u003eB. radicata\u003c/em\u003e alcohol extracts increase the fluidity of the membrane and are known to inhibit the key enzyme of saturated fatty acids \u003csup\u003e[31]\u003c/sup\u003e. The aliphatic alcohols also have one hydroxyl group. The hydroxyl group mainly determines the hydrophilicity of the molecule, resulting in much easier interaction with its target in a living organism \u003csup\u003e[32]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFuture work concentrating on determining the antifungal mechanisms of aliphatic alcohols and aldehydes will be performed, which will be helpful in laying a foundation for overcoming the drug resistance that pathogens quickly develop against antibiotic drugs.\u003c/p\u003e "},{"header":"Abbreviations","content":" \u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAMB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAmphotericin B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePotato Dextrose Agar\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eelectronionization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh Performance Liquid Chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eultraviolet\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMinimum Inhibitory Concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNIST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNational Institute of Standards and Technology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGC\u0026ndash;MS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGas Chromatography-Mass Spectrometer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFT-IR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFourier-Transform Infrared\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVOC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVolatile Organic Compound\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eAvailability of data and material\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (Grant No. 31371844; 31071556) and the Science and Technology Department of Anhui province, PR of China (Grant No. 130103215)\u003c/p\u003e\n\u003cp\u003eAuthors' contributions\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYong ye conceived and designed the experiments. Yong ye performed the experiments and analyzed the data. Qinghua zeng analyzed the data. Yong ye wrote and edited the manuscript. The excellent technical support by Chuanxun Yuan and Qingmei Zeng\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e \u003cspan\u003eReferences.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eAlteras I, Cafri B, Feuerman EJ. The high incidence of Tinea pedis and unguium in patients with Kaposi's sarcoma. Mycopathologia. 1981;74(3):177\u0026ndash;9.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eKoltin Y, Hitchcock CA. The search for new triazole antifungal agents. Curr Opin Chem Biol. 1997;1(2):176\u0026ndash;82.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eErbagci Z, Tuncel A, Zer Y, Balci I. 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J Eur Acad Dermatol Venereol, 2017;31(9).\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eFrancis P, Walsh TJ. Evolving Role of Flucytosine in Immunocompromised Patients: New Insights into Safety, Pharmacokinetics, and Antifungal Therapy. Clin Infect Dis. 1992;15(6):1003\u0026ndash;18.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eStamm AM, Diasio RB, Dismukes WE, Shadomy SM, Cloud GA. Toxicity of amphotericin B plus flucytosine in 194 patients with cryptococcal meningitis. Am J Med. 1987;83(2):236\u0026ndash;42.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eVermes A, Guchelaar HJ, Dankert J. Flucytosine: a review of its pharmacology, clinical indications, pharmacokinetics, toxicity and drug interactions. J Antimicrob Chemother. 2000;46(2):171\u0026ndash;9.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eDenning DW, Venkateswarlu K, Oakley KL, Anderson MJ. NJ Manning. Itraconazole resistance in Aspergillus fumigatus. 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Beverage useful for relieving cough, moisturizing lungs and improving immunity, contains Asparagus, coltsfoot, Sonchus arvensis, maidenhair, lotus seed, corni fructus, puffball, galangal, boneset, club moss herb, sugar and preservative:2017; CN,CN106729277-A.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eLam YW, Ng TB, Wang HX. Antiproliferative and Antimitogenic Activities in a Peptide from Puffball Mushroom Calvatia caelata. Biochemical Biophysical Research Communications. 2001;289(3):744\u0026ndash;9.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eYe Y, Liu K, Zeng QH, Zeng QM. Antimicrobial activity of puffball(Bovistella radicata) and separation of bioactive compounds. Amb Express. 2017;7(1):99.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003ePetrović P, Vunduk J, Klaus A, Kozarski M, Nikšić M. Biological potential of puffballs: A comparative analysis. Journal of Functional Foods. 2016;21:36\u0026ndash;49.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eNegi PS, Anandharamakrishnan C, Jayaprakasha GK. Antibacterial activity of Aristolochia bracteata root extracts. J Med Food. 2003;6(4):401\u0026ndash;3.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eAli NAA, Mothana RAA, Lesnau A, Pilgrim H, Lindequist U. Antiviral activity of Inonotus hispidus. Fitoterapia. 2003;74(5):483\u0026ndash;5.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003ePeira E, Carlotti ME, Trotta C, Cavalli R, Trotta M. Positively charged microemulsions for topical application. Int J Pharm. 2008;346:119\u0026ndash;23.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eFujita KI, Fujita T, Kubo I. Antifungal activity of alkanols against Zygo saccharomyces bailii and their effects on fungal plasma membrane. Phytother Res. 2008;22:1349\u0026ndash;55.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eKubo I, Muroi H, Kubo A. Structural functions of antimicrobial long-chain alcohols and phenols. Bioorg Med Chem. 1995;3:873\u0026ndash;80.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eSuprapta DN, Arai K, Iwai H. Effects of volatile compounds on arthrospore germination and mycelial growth of Geotrichum candidum, citrus race. Mycoscience. 1997;38:31\u0026ndash;5.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eJing Chen W, Wang C, Shi J, Wan, Li Deng. Determination of Four Volatile Compounds with Anti-Inflammatory Biological Activity in Thunb. by Gas Chromatography and Gas Chromatography\u0026ndash;Mass Spectrometry. Anal Lett. 2014;47:730\u0026ndash;41.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eTogashi N, Shiraishi A, Nishizaka M, Matsuoka K, Endo K, Hamashima H. Inoue Y.Antibacterial activity of long-chain fatty alcohols against Staphylococcus aureus. Molecules. 2007;12:139.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eDing CH, Du XW, Xu Y, Xu XM, Mou JC. Screening for Differentially Expressed Genes in Endophytic Fungus Strain 39 During Co-culture with Herbal Extract of its Host Dioscorea nipponica, Makino. Curr Microbiol. 2014;69(4):517\u0026ndash;24.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eBegnami AF, Duarte MC, Furletti T, Rehder V. V L G. Antimicrobial potential of Coriandrum sativum L. against different Candida species in vitro. Food Chem. 2010;118:74\u0026ndash;7.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eMaguireboyle SJ, Barron AR. Organic compounds in produced waters from shale gas wells. Environmental Science Processes Impacts. 2014;16:2237.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eCalzada F, Alan\u0026atilde;S AD. Additional antiprotozoal flavonol glycosides of the aerial parts of Helianthemum glomeratum. Phytother Res. 2007;21(1):78\u0026ndash;80.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eGeetha R, Sathian CT, Prasad V, Gleeja VL. Efficacy of purified antimicrobial peptides from lactic acid bacteria against bovine mastitis pathogen. Asian J Dairy Food Res. 2015;34:259\u0026ndash;64.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eAghayeva DN, Lutz M, Piatek M. Transmission electron microscopy of Tuberculina species (Helicobasidiales) reveals an unique mode of conidiogenesis within Basidiomycota. Fungal Biology. 2016;120(8):1010\u0026ndash;6.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003e\u003cb\u003e238125\u003c/b\u003e-\u003cb\u003e47625Figure1\u003c/b\u003e. \u003cb\u003ezone of inhibition\u003c/b\u003e(\u003cb\u003eZOI\u003c/b\u003e) \u003cb\u003eof spore and sporophore powder from\u003c/b\u003e B.radicata\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eis ZOI of. alcohol extract from \u003cem\u003eB. radicata\u003c/em\u003e spore powder; 1 is alcohol extract from \u003cem\u003eB. radicata\u003c/em\u003e sporophore powder; and 2 is alcohol extract from \u003cem\u003eL.fenzlii Reich.\u003c/em\u003e \u0026ldquo;+\u0026rdquo; is positive control and \u0026ldquo;-\u0026ldquo; is negative control.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003e-190500-304800\u003cb\u003eFigure\u003c/b\u003e. \u003cb\u003e2\u003c/b\u003e. HPLC chromatograms of SPAF-1 and alcohol extract from B. radicata spore powder.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003e\u003cem\u003eA:; HPLC chromatograms of alcohol extract from B. radicata spore powder\u003c/em\u003e.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003e\u003cem\u003eB\u003c/em\u003e. \u003cem\u003eHPLC chromatograms of SPAF-1\u003c/em\u003e.\u003c/span\u003e \u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"antifungal activity, extracellular metabolites, HPLC, FT-IR, GC–MS, decanol","lastPublishedDoi":"10.21203/rs.3.rs-33942/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-33942/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground To analyse the antifungal active compounds in \u003cem\u003eB. radicata \u003c/em\u003ealcohol extract, the alcohol extract was purified by column chromatography (macroporous resinD-101) and the active compounds was named as SPAF-1(the spore powder active fraction). \u003c/p\u003e\u003cp\u003eResults Alcohol extracts and SPAF-1 were submitted to GC-MS analysis, there were two characteristic peaks(peak1 and peak2) in Gas chromatogram. By comparing in NIST, the compound were 2-propyl-1-pentanol corresponding to peak1 and decanal, n-decanol and 2E-decanol corresponding to peak2. The main constituents were decanal(24.3%) n-decanol(27.9%), 2E-decenol (21.2%) and 2-Propyl-1-pentanol (13.6%). Their MIC values were 62.5 µg/ml, 31.2 µg/ml, 31.2 µg/ml and 250 µg/ml against \u003cem\u003eT. rubrum\u003c/em\u003e respectively. Furthermore, transmission electron microscope (TEM) analysis showed altered surface morphology in the majority of \u003cem\u003eT. rubrum\u003c/em\u003e cells after treatment with SPAF-1. \u003c/p\u003e\u003cp\u003eConclusions In this paper, we successfully separate SPAF-1 from alcohol extract of\u003cem\u003e B. radicata\u003c/em\u003e. The antifungal effect of SPAF-1 is similar to positive control, the main component were decanal, n-decanol, 2E-decenol and 2-Propyl-1-pentanol, the anti-tinea pedis effect of them was obvious.\u003c/p\u003e","manuscriptTitle":"Screening of Antifungal Substances from Bovistella Radicata (Mont.) Pat and Their Antifungal Effect","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-06-10 17:49:16","doi":"10.21203/rs.3.rs-33942/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d709e22e-369c-4a44-827d-bd8148bb9f47","owner":[],"postedDate":"June 10th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":117091,"name":"General Microbiology"},{"id":117092,"name":"Applied \u0026 Industrial Microbiology"}],"tags":[],"updatedAt":"2020-09-11T16:01:20+00:00","versionOfRecord":[],"versionCreatedAt":"2020-06-10 17:49:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-33942","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-33942","identity":"rs-33942","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}