Integrative Extraction of Ergosterol, （1→3）-α-D-Glucan and Chitosan from Penicillium chrysogenum Mycelia

Abstract Ergosterol,（1→3）-α-D-glucan and chitosan are important biomaterials. In this research, a process has been developed to integratively extract ergosterol, （1→3）-α-D-glucan, and chitosan from Penicillium chrysongenum mycelium. First the mycelia are pretreated with 0.1mol·L^-1 of NaOH. After recovery by centrifugation the solid portion is made to undergo saponification and deacetylation reactaons by addition of 2mol·L^-1 NaOH and et anol.After reaction, extraction is carried out by addition of petroleum ether, which separates the reaction mixture into two phases. The upper layer of petroleum ether contains extracted ergosterol, and the .bottom layer of NaOH solution contains （1→3）-α-DEglucan; the chitosan is on the mycelia residuum. After isolation, the recovery yield of ergosterol is 0.52% of dry mycelium. That of （1→3）-α-D-glucan is about 8.2%; and chitosan is 5.7% with 86% deacetylation. The compositions have been characterized by 1R, HPLC analyses.

Regulatory effect of salicylic acid and methyl jasmonate supplementation on ergosterol production in Hericium erinaceus mycelia

We evaluated the biomass and ergosterol content of Hericium erinaceus mycelium, and extracellular enzyme activities in H. erinaceus liquid culture following salicylic acid（SA） and methyl jasmonic acid（Me JA）supplementation. The optimal SA concentration was100 lmoláL-1, where the highest ergosterol content of 2.33 mgág-1was obtained following 6-day cultivation with100 lmoláL-1SA supplementation, and which was significantly higher than the unsupplemented control（p ／ 0.01）. Following 4-day supplementation with50 lmoláL-1Me JA, the highest ergosterol content obtained was 1.988 mgág-1, which was 25.8 % higher than the unsupplemented control. Our data indicate that SA and Me JA supplementation improves ergosterol content in H.erinaceus mycelium.

Two novel compounds, ergosterol and ergosta-5,8-dien-3-ol, from Termitomyces heimii Natarajan demonstrate promising antihepatocarcinoma activity

Background:Mushroom-derived components have immense potential to become a safe alternative in identifying lead anti-cancer molecules.Termitomyces heimii Natarajan(T.heimii)is a traditionally used edible mushroom with no previous record of anti-hepatocarcinoma activity.Methods:The anti-proliferative efficacy of the mushroom ethyl acetate extract was screened against a panel of seven cancer cell lines,namely Hep G2,MCF-7,MDA-MB-231,MAD-MB-436,MOLT-4,Reh,and K-562,and against peripheral blood mononuclear cells isolated from normal healthy donors by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay.The impact of the extract on nuclear morphology was examined by 40,6-diamidino-2-phenylindole staining and the apoptotic potential of the extract was evaluated through flow cytometry and Annexin V-PI dual staining,followed by an in vitro scratch assay to elucidate the anti-migratory potential of the extract.The apoptotic and antimigratory effects were further validated using in silico molecular docking with four compounds,ergosterol,ergosta-5,8-dien-3-ol,lanosterol,and eburicol,against two anti-apoptotic proteins,Bcl-2 and Bcl-XL,and two angiogenic receptors,VEGFR-1 and VEGFR-2.Results:The screening data revealed that ethyl acetate extract exhibited remarkable anti-proliferative efficacy against Hep G2 cells,with a half maximal inhibitory concentration(IC50)value of 263.53(8.09)mg/mL,followed by MCF-7 cell lines and showed a negligible effect on peripheral blood mononuclear cells.A clear alteration of the cellular and nuclear morphology was concentration-dependently observed in Hep G2 cells.The extract induced robust apoptosis and a significant concentrationdependent increase in the scratch area.The results of in silico docking revealed that compared to standard drug sunitinib,both ergosterol and ergosta-5,8-dien-3-ol displayed lower binding energy,and satisfactory drugability and absorption,distribution,metabolism,excretion,and toxicity properties.Conclusions:T.heimii is a potential source for isolating lead anticancer molecules in the future.Ergosterol and ergosta-5,8-dien-3-ol hold great promise as new drugs against hepatocarcinoma.

Investigation of Ergosterol in situ Circular Dichroic Spectroelectrochemistry

In the present paper, the electrochemical behavior of ergosterol has been investigated by in situ circular dichroism (CD) spectroelectrochemistry with long path-length thin layer cell. E-0 (1.02V), alpha n(alpha) (0.302) of the electroxidation process of ergosterol were obtained from the CD spectroelectrochemical data. The mechanism of the electroxidation process of ergosterol is suggested.

Ten ring-B aromatized ergosterols from Aspergillus spectabilis

Spectasterols F−O(1−10),ten interesting ergosterols with an aromatized B ring,were obtained from Aspergillus spectabilis.Their structures and absolute configurations were determined using a combination of high-resolution electrospray ionization mass spectrometry(HR-ESI-MS),nuclear magnetic resonance(NMR)spectroscopy,single-crystal X-ray diffraction analyses,and electronic circular dichroism(ECD)calculations.Structurally,these aromatic ergosterols feature versatile side chains.Notably,compound aromatic ergosterols featured versatile side chains,and compound 4 is an unusual C23 ergosterol characterized by a shorter side chain due to oxidative cleavage between C-23 and C-24.All compounds were evaluated for their neuroprotective activities,with compound 8 showing a dose-dependent ability to reduce apoptosis and protect mitochondrial function in glutamate-induced SH-SY5Y cells.

Antifungal Activity of Aspidin BB from Dryopteris fragrans against Trichophyton rubrum Involved Inhibition of Ergosterol Biosynthesis

Objectives Aspidin BB, a typical phloroglucinol derivative from Dryopteris fragrans, possesses significant antifungal property. This study aimed to investigate potential mechanism of antifungal activity of Aspidin BB against Trichophyton rubrum which is the most common pathogens responsible for chronic dermatophytosis. Methods The minimum inhibitory concentration （MIC） ofAspidin BB against strains was determined by broth microdilution. The effects of Aspidin BB on ergosterol biosynthesis were investigated by content determination based on UPLC method. Besides, the effects of drugs on squalene epoxidase （SE） in T. rubrum cell membrane were analyzed. Results MIC value of Aspidin BB against T. rubrum was 25.0 IJg/mL. Aspidin BB reduced ergosterol content significantly, but no notable effect on squalene epoxidase activity. Conclusion The results suggested that Aspidin BB inhibited ergosterol biosynthesis. However, it was not squalene epoxidase but other components may sever as possible targets in ergosterol biosynthesis pathway.

Isolation, characterization and modulatory potentials of β-stigmasterol, ergosterol and xylopic acid from Anchomanes difformis on mitochondrial permeability transition pore in vitro

Objective: Secondary metabolites and polyphenolic compounds from medicinal plants have been demonstrated to have multiple biological functions with promising research and development prospects. This study examined the effect of β-stigmasterol(with ergosterol) and xylopic acid isolated from Anchomanes difformis on liver mitochondrial permeability transition pore(mPTP).Methods: The compounds were isolated by vacuum liquid chromatography. Mitochondrial swelling was assessed as changes in absorbance under succinate-energized conditions.Results:1H and13C NMR spectroscopic elucidation of the isolates affirmed the presence of β-stigmasterol with ergosterol(1:0.3) and xylopic acid. The isolates reversed the increase in lipid peroxidation and inhibited the opening of mitochondrial permeability transition pores caused by calcium and glucose.Pharmacological inhibition of m PTP offers a promising therapeutic target for the treatment of mitochondrial-associated disorders.Conclusion: Reduction in the activity of calcium ATPase and the expression of Caspase-3 and-9 were observed, suggesting that they could play a role in protecting physicochemical properties of membrane bilayers from free radical-induced severe cellular damage and be useful in the management of diseases where much apoptosis occurs.

The effect of ergosterol on the allogrooming behavior of termites in response to the entomopathogenic fungus Metarhiziumanisopliae

Termites have physiological and behavioral immunities that make them highly resistant to pathogen infections,which complicates biocontrol efforts.However,the stimuli that trigger the pathogen-avoidance behaviors of termites are still unclear.Our study shows that workers of Coptotermes formosanus exposed to the conidia of Metarhizium anisopliae exhibited a significantly higher frequency and longer duration of allogrooming behaviors compared with untreated termites.Volatile compounds in the cuticle of control termites and termites previously exposed to a suspension of M.anisopliae conidia were analyzed and compared using a gas chromatography-mass spectrometer(GC-MS).Our results showed that the amount of ergosterol differed between the fungus-exposed and control termites.Choice tests showed that termites significantly preferred to stay on filter paper treated with ergosterol(0.05,0.1,or 1.0 mg/mL)compared with control filter paper.In addition,termites exposed to ergosterol followed by M.anisopliae conidia were allogroomed at a significantly higher frequency and for a longer duration than termites exposed to alcohol(the solvent used with the ergosterol in the ergosterol trials)alone followed by M.anisopliae conidia.These results showed that ergosterol may enhance the allogrooming behavior of termites in the presence of entomopathogenic fungi.

Controlling antifungal activity with light:Optical regulation of fungal ergosterol biosynthetic pathway with photo-responsive CYP51 inhibitors

Invasive fungal infections(IFIs)have been associated with high mortality,highlighting the urgent need for developing novel antifungal strategies.Herein the first light-responsive antifungal agents were designed by optical control of fungal ergosterol biosynthesis pathway with photocaged triazole lanosterol 14a-demethylase(CYP51)inhibitors.The photocaged triazoles completely shielded the CYP51inhibition.The content of ergosterol in fungi before photoactivation and after photoactivation was 4.4%and 83.7%,respectively.Importantly,the shielded antifungal activity(MIC80≥64μg/m L)could be efficiently recovered(MIC80=0.5—8μg/m L)by light irradiation.The new chemical tools enable optical control of fungal growth arrest,morphological conversion and biofilm formation.The ability for highprecision antifungal treatment was validated by in vivo models.The light-activated compound A1 was comparable to fluconazole in prolonging survival in Galleria mellonella larvae with a median survival of 14 days and reducing fungal burden in the mouse skin infection model.Overall,this study paves the way for precise regulation of antifungal therapy with improved efficacy and safety.

麦角甾醇酯的酶促合成及对非酒精性脂肪肝的预防作用

探究了麦角甾醇亚油酸酯(简称麦角甾醇酯)的酶促合成及对非酒精性脂肪肝的预防作用。将30只雄性昆明小鼠分为四组:空白组(n=6)、高脂组(n=8)、麦角甾醇组(n=8)和麦角甾醇亚油酸酯组(n=8),持续喂养6周。测定血浆总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)水平,计算LDL-C/HDL-C比值,检测肝脏和粪便脂质水平,并分析肝脏脂肪变性程度。结果显示,以Candida sp.99-125脂肪酶为催化剂可以成功合成麦角甾醇亚油酸酯。麦角甾醇酯使得肝脏总脂肪酸含量降低35.3%(P<0.05),可以有效预防高脂膳食诱导肝脏脂肪变性,并导致粪便总脂肪酸水平增加74.8%(P<0.05)。同时,麦角甾醇酯降低了血浆LDL-C水平和LDL-C/HDL-C比值分别为24.3%(P<0.05)和36.5%(P<0.05),引起血浆HDL-C水平提高52.0%(P<0.05)。以上结果表明,麦角甾醇酯能够有效预防高脂膳食诱导的非酒精性脂肪肝。该文为非酒精性脂肪肝的预防提供了一种新的干预策略。

粗毛纤孔菌不同提取物对免疫抑制小鼠免疫功能的影响

采用粗毛纤孔菌(Inonotus hispidus)石油醚提取物(高、低剂量分别为12、6 mg·kg^(-1))、乙酸乙酯提取物(高、低剂量组分别为17、8.5 mg·kg^(-1))、正丁醇提取物(高、低剂量分别为45、22.5 mg·kg^(-1))、水提取物(高、低剂量分别为600、300 mg·kg^(-1))、麦角甾醇(高、低剂量分别为10、5 mg·kg^(-1))灌胃环磷酰胺所致免疫抑制小鼠,测定免疫器官指数、吞噬指数、吞噬系数、耳肿胀度、耳肿胀率和血清细胞因子、免疫球蛋白含量,采用苏木素-伊红染色小鼠脾脏、胸腺、胰腺、回肠后观察组织形态,研究粗毛纤孔菌不同提取物对免疫抑制小鼠免疫功能的影响。结果表明:与模型组相比,石油醚、水提取物高剂量组和麦角甾醇高、低剂量组的胸腺指数显著升高;麦角甾醇高、低剂量组的吞噬指数和吞噬系数均显著升高,水提取物高、低剂量组的吞噬系数显著升高;水提取物和麦角甾醇高、低剂量组的耳肿胀度和耳肿胀率均极显著升高;石油醚、水提取物和麦角甾醇高、低剂量组,乙酸乙酯提取物高剂量组的IL-1β含量显著增加;石油醚、乙酸乙酯、水提取物高、低剂量组和麦角甾醇高、低剂量组的IL-2含量极显著增加;除正丁醇提取物低剂量组外,其余提取物组和麦角甾醇高、低剂量组的IFN-γ和IL-10含量均极显著增加;石油醚、水提取物和麦角甾醇高、低剂量组,乙酸乙酯提取物高剂量组的IL-4含量极显著增加;石油醚、水提取物和麦角甾醇高、低剂量组的IgG含量显著增加。研究结果表明,粗毛纤孔菌提取物对环磷酰胺所致免疫抑制小鼠具有良好的免疫保护作用,可为粗毛纤孔菌进一步开发提供参考。

林分改变驱动的优势菌根真菌类型变化影响土壤有机碳积累

马尾松林(PinusmassonianaLamb.)是典型的外生菌根(Ectomycorrhiza,ECM)优势林,但是近年来受到松材线虫病的影响,生态服务功能下降,逐渐被丛枝菌根(Arbuscularmycorrhiza,AM)占优势的阔叶林所替代,但亚热带地区马尾松林转变为阔叶林过程中,优势菌根类型的改变对土壤有机碳积累的影响仍不清楚。以建德市马尾松林和阔叶林为研究对象,通过高效液相色谱和中性脂肪酸、磷脂脂肪酸等技术,测定优势菌根真菌生物量、球囊霉素相关土壤蛋白(GRSP)含量以及土壤胞外酶活性和微生物群落特征。结果表明:AM真菌占优势的阔叶林(AMD)取代ECM真菌占优势的马尾松林(ECMD),土壤有机碳显著提高了36.81%,微生物碳利用效率(CUE)显著提高了53.85%,AM真菌生物量提高了25.57%,ECM真菌生物量下降45.04%,并且ECM真菌占优势的马尾松林受到更严重的氮限制。磷脂脂肪酸分析显示,相比于AM真菌占优势的阔叶林,ECM真菌占优势的马尾松林革兰氏阳性细菌(G^(+))以及革兰氏阳性与阴性细菌之比(G^(+)/G^(–))分别显著下降了21.47%和6.46%。冗余分析(Redundancy analysis,RDA)结果表明,AM真菌占优势和ECM真菌占优势的森林之间土壤微生物群落结构存在显著差异(P<0.05),其中AM真菌生物量和土壤有机碳与微生物群落结构变异显著相关。GRSP含量下降以及不同类型菌根真菌招募微生物类群不同是导致ECM真菌占优势森林土壤有机碳下降的重要原因。因此,亚热带地区马尾松林被阔叶林替代后增加森林土壤有机碳含量,提高森林碳汇功能。

香菇中麦角甾醇的提取纯化、表征及其抗氧化活性

为建立一种香菇中麦角甾醇检测的三波长分光光度法,通过单因素试验和响应面试验获得最佳提取条件,并利用溶剂提取法、重结晶法及制备型高效液相法纯化香菇麦角甾醇,对其进行表征及抗氧化活性研究。结果表明,三波长分光光度法可以有效消除杂质和其他背景因素带来的影响,精确测定香菇中麦角甾醇的含量。香菇麦角甾醇的最佳提取条件为超声时间90 min、料液比1∶59(g/mL)、提取温度69℃,麦角甾醇含量的预测值为3.868 78 mg/g,实测值为3.781 4 mg/g。纯化后单体麦角甾醇的纯度可达96%以上,清除DPPH自由基、ABTS+自由基的半数抑制浓度IC_(50)分别为(0.72±0.06)、(0.17±0.01)mg/mL。所建立的方法重复性、稳定性、精密度良好,是一种快速准确测定香菇麦角甾醇的检测方法,且纯化的麦角甾醇具有良好的抗氧化活性。

黄精霉变过程中麦角甾醇变化规律及基于电子鼻技术的快速预测模型研究

目的:麦角甾醇是霉变相关的重要指标之一,探索建立一种麦角甾醇快速检测方法。方法:采用人工模拟培养获取不同霉变程度的黄精样品;采用传统性状鉴定法开展黄精外观性状的观察、鉴定与分类;使用超高效液相色谱法进行麦角甾醇含量测定;通过电子鼻技术实现黄精气味的数字化检测。结果:共制备得到35份不同霉变程度的黄精样品;感官评价将黄精样品分为未霉变、轻微霉变和严重霉变3个阶段;35份黄精样品中麦角甾醇质量分数为0~90.4356 mg·kg^(–1),随着黄精霉变程度增加而上升;电子鼻的18根传感器中,S1~S16均表现出随着黄精霉变程度加深其最大响应值的绝对值降低,S18则相反,其最大响应值随着黄精霉变程度增加而升高。相关性分析显示,麦角甾醇含量与S1、S7~S16响应值呈极显著负相关,与S2~S6、S17、S18响应值呈极显著正相关,并且建立了回归方程Y=67.577–78.453X(r=0.634),其中Y为麦角甾醇含量、X为传感器PA/2最大响应值。结论:黄精的气味与其内在的化学成分麦角甾醇密切相关,电子鼻技术联合回归分析能为初步预测黄精中麦角甾醇的含量提供一种可行、有效的方法。

彩色豆马勃子实体的化感作用及其化感物质的分离鉴定

彩色豆马勃能与松树、桉树形成外生菌根，研究其次生代谢产物对植物的影响具有重要意义．用水、乙醇和丙酮抽提彩色豆马勃的子实体，发现这些抽提物对稗草和水稻幼苗生长有极显著的抑制作用．丙酮抽提物对狼尾草和油菜幼苗生长有抑制作用．子实体的丙酮抽提物用硅胶柱色谱分离得到２个纯化合物，可鉴定为豆马勃内酯（Ｐｉｓｏｌａｃｔｏｎｅ）和麦角甾醇．该２个化合物在４００μｇ·ｍｌ－１浓度下均显著抑制稗草幼苗根生长．

蛹虫草化学成分研究

从蛹虫草菌丝中分离出六种化学成分。经结构鉴定为β-谷甾醇,麦角甾醇,D-甘露醇,腺嘌呤,腺苷及虫草素(3′-脱氧腺苷)。从蛹虫草菌丝中分离出虫草素、腺嘌呤及麦角甾醇尚属首次。

苍耳茎化学成分的研究

目的 :研究苍耳茎的化学成分。方法 :应用多种色谱方法进行分离和纯化 ,并用NMR和MS等方法解析其化学结构。结果 :从苍耳茎的乙醇提取物中分离得到 6个化合物 ,它们的结构分别被鉴定为 :β 豆甾醇(1)、豆甾醇 3 O β D 吡喃葡萄糖苷 (2 )、羽扇豆醇十六酸酯 (3)、麦角甾醇过氧化物 (4)、东莨菪内酯苷 (5 )及二十七酸 (6 )。结论 :化合物 3～ 6为从本植物中首次分离得到。

蒙古口蘑子实体的抗肿瘤活性

对蒙古口蘑子实体石油醚提取物及从中得到的麦角甾醇和麦角甾醇过氧化物的抗肿瘤活性进行研究,测定其对H22荷瘤小鼠抑瘤率、免疫器官指数及免疫因子含量的影响。同时探讨麦角甾醇和麦角甾醇过氧化物的细胞凋亡影响。结果表明:除了石油醚提取物上清低剂量组(50mg/(kg.d))外其他组均对肿瘤具有一定的抑制作用,其中石油醚提取物沉淀低剂量组(35mg/(kg.d))和麦角甾醇过氧化物低剂量组(5mg/(kg.d))抑瘤率分别达到69.61%和67.15%;各受试药组的胸腺指数与阳性对照组相比均有所增加,其中石油醚提取物沉淀高剂量组和麦角甾醇过氧化物的高剂量具有极显著性差异(P≤0.01);各组的脾指数没有规律性;所有石油醚提取物组的VD含量与正常对照组相近,麦角甾醇低剂量组和麦角甾醇过氧化物组与正常组对比具有极显著性差异(P≤0.01);所有受试药组的白介素-2(IL-2)含量明显增加,与阳性对照组和阴性对照组相比有极显著性差异(P≤0.01)。细胞凋亡实验结果表明麦角甾醇和麦角甾醇过氧化物均能诱导肝癌细胞(HepG2)凋亡,能够抑制HepG2细胞分裂,对HepG2细胞的早期细胞凋亡率分别为41.2%和42.33%。

RP-HPLC测定天然与人工冬虫夏草中游离麦角甾醇的含量

目的 :比较天然与人工冬虫夏草中游离麦角甾醇的含量 ,探讨麦角甾醇作为冬虫夏草质控指标的可行性。方法 :采用HP110 0高效液相色谱仪 ,Allsphere C18色谱柱 ,甲醇 -水 (95∶ 5 )为流动相 ,检测波长为 2 75 nm,流速 1ml/ m in。结果 :不同来源的天然冬虫夏草游离麦角甾醇含量差异显著 ,但均明显高于人工虫草菌丝。结论

麦角固醇高产菌株的构建及其培养优化条件的研究

通过初筛、单倍体分离、诱变及酵母菌种间原生质体融合技术构建了２株麦角固醇产量明显提高的优良菌株ＹＥＦ２１和ＹＥＦ２９。并对ＹＥＦ２１的培养优化条件进行了研究。结果表明在优化的实验条件下ＹＥＦ２１的生物量及麦角固醇含量的综合值分别为原始亲株ＹＥ２２７和ＹＥ１８０的１５４和１５５倍。经遗传稳定性分析，没有发现标记基因的分离现象，从而证明获得的融合菌株是遗传稳定的，是具有一定实际应用价值的麦角固醇高产菌株。