不同发酵方式对海南粗榧内生真菌CH1307c合成次级代谢产物的影响

为研究不同发酵方式对内生真菌次级代谢产物产量、成分类别及其对3种常见白血病细胞株的细胞毒活性影响。分别采用大米固态发酵、麦麸固态发酵、马铃薯液态静置发酵、马铃薯动态发酵4种发酵方式对分离自海南粗榧韧皮部内生真菌CH1307c进行发酵,并采用乙酸乙酯进行萃取,通过试管定性试验和MTT试验测定其粗提物的成分类别及其对细胞株K562、NB4、HL60的细胞毒活性。结果表明:大米固态发酵、麦麸固态发酵的次级代谢粗提物产量相对于马铃薯动态发酵分别增加了45.9倍和28倍;马铃薯动态发酵较其静置发酵产量高9.1倍。试管定性试验结果表明,大米固态发酵获得的粗提物,化合物种类最多,麦麸发酵和动态发酵次之,马铃薯液态静置发酵最少。此外,4种发酵方式获得的代谢粗提物对3株细胞株的抗肿瘤活性(IC50值)并未呈现明显差异。研究结果为该菌株的大规模发酵提供理论依据。     

海南粗榧树皮内生真菌的初步鉴定

从采自中国海南和泰国的海南粗榧(Cephalataxus mannii Hook.f)树皮样品中分离得到213株内生真菌,依据菌落形态和显微特征对其中142株进行初步的种属鉴定。结果显示,它们分属34个属,未知属37株。其中,采自海南的样品中内生真菌分属13属,未知属13株;采自泰国的样品中内生真菌分属27属,未知属24株,表明不同地区的海南粗榧内生真菌类群存在一定差异。海南产海南粗榧内生真菌的优势种群为根霉属(Rhizopusspp.)和青霉属(Penicillium spp.),泰国产海南粗榧内生真菌的优势种群为酵母属(Saccharomyces spp.)和毛霉属(Mucor spp.)。     

海南粗榧内生真菌对罗非鱼无乳链球菌抑菌活性研究

利用活菌菌饼法和滤纸片法研究筛选64株海南粗榧内生真菌对罗非鱼源无乳链球菌的抑菌活性,并对活性菌株的代谢产物化学成分进行初步测试。菌饼试验结果显示,11株海南粗榧内生真菌对无乳链球菌具有明显抑制作用,占总菌株数的17.2％,抑菌圈在Φ(8.7±0.3～16.2±0.2)mm间。滤纸片试验结果显示,12株具有明显抑制作用,占总菌数的18.8％,抑菌圈在Φ（8.7±0.3～28.2±0.4）mm间。2种方法的最高活性菌株不一致,但最高活性菌株（B30、F52）和同时显示较高活性的菌株（B14、B30、B32、F7、F52）被测出都含有甾体及三萜类化合物。根据结果推测,对无乳链球菌有显著抑制作用的菌株可能含有甾体及三萜类物质。结果为深入研究内生真菌对罗非鱼无乳链球菌的抑制奠定基础,也为生物防治罗非鱼病害提供有益借鉴。     

沉香样品中曲霉属真菌菌株 HNWSW-20 的分离鉴定及其次生代谢产物的研究

本文采用平板分离法从沉香样品中分离得到一株真菌 HNWSW-20,从形态学和分子生物学上鉴定其为曲霉菌（Aspergillus sp.）,并利用多种色谱技术对其次生代谢产物进行分离纯化,根据波谱数据和理化性质鉴定其化合物结构为：2?,3?-dihydrosorbicillin（1）,sorbicillin（2）,2,3-二氢-2-(1-丙烯)-6,8-二甲基-7-羟基-色酮（3）,邻苯二甲酸二丁酯（4）,7-羟基-异苯并呋喃-1(3H)-酮（5）二十烷酸甲酯（6）;分别采用 Ellman 比色法和 pNPG 法测定化合物的乙酰胆碱酯酶抑制活性和 α-葡萄糖苷酶抑制活性,结果显示上述化合物 1、2、4 具有乙酰胆碱酯酶抑制活性,而化合物 1、3、5 具有 α-葡萄糖苷酶抑制活性。本文中化合物 1~2 为首次从曲霉属中分离得到,并首次报道具有乙酰胆碱酯酶和 α-葡萄糖苷酶抑制活性。     

内生真菌HND5挥发性物质组分分析及其抑菌作用测定

HND5是从健康的臂形草叶片中分离得到的一株顶孢霉属(Acremonium sp.)内生真菌,可通过产生挥发性物质抑制病原菌生长。采用顶空固相微萃取技术(HS-SPME)提取富集了内生真菌HND5的挥发性有机物(VOCs),利用气相色谱-质谱联用法(GC-MS)进行了挥发性物质的组分分析,共分离鉴定得到14个单体化合物。活性分析发现,石竹烯(Caryophyllene)和4-乙烯基-1,2-二甲氧基苯(Benzene,4-ethenyl-1,2-dimethoxy-)具有抑菌活性,对峙试验表明2种物质均对尖孢镰刀菌(Fusarium oxysporum)、胶胞炭疽菌(Colletotrichum gloeosporioides)和多主棒孢菌(Corynespora cassiicola)具良好的抑制作用,其中石竹烯的有效中浓度(EC50)分别为2 916.07、729.54和404.23μL/L,4-乙烯基-1,2二甲氧基苯的有效中浓度(EC50)分别为62.72、27.60和23.54μL/L。     

一种国外沉香中2-(2-苯乙基)色酮类化合物研究

为了了解一种国外沉香中的2-(2-苯乙基)色酮类化合物,采用多种色谱分离技术从该沉香的乙醇提取物中分离得到6个化合物,通过波谱学方法分别鉴定为：6-羟基-8-氯-2-[2-(4°-羟基苯基)乙基]色酮（1）,6-羟基-2-[2-(3°-甲氧基-4°-羟基苯基)乙基]色酮（2）,6-羟基-7-甲氧基-2-[2-(4°-羟基苯基)乙基]色酮（3）,沉香四醇（4）,5α,6β,7β,8α-四羟基-2-[2-(4°-甲氧基苯基)乙基]-5,6,7,8-四氢色酮（5）和6-羟基-2-[2-(3°-甲氧基-4°-羟基苯基)乙烯基]色酮（6）。化合物1为新的2-(2-苯乙基)色酮。对化合物2~6的细胞毒活性测试结果表明,化合物6对5株人体肿瘤细胞均表现出一定活性,其中对人慢性髓原白血病细胞K562和人肝癌细胞BEL-7402具有显著抑制活性,半数抑制浓度（IC₅₀）为2.87和 4.75 μg/mL,化合物2、3、5对5株人体肿瘤细胞表现出中等活性,IC₅₀范围为9.91~45.38 μg/mL。     

海洋放线菌Streptomyces sp. HNWSW-49的次生代谢产物研究

对1株具有抗芒果炭疽病菌活性的海口湾海泥来源放线菌Streptomyces sp.HNWSW-49发酵液的化学成分进行研究。采用多种色谱技术对化合物进行分离纯化,以波谱数据确定化合物的结构。从来源于海口湾海泥的放线菌Streptomyces sp.HNWSW-49发酵液的乙酸乙酯提取物中分离鉴定了7个含氮化合物,分别为:环-(苯丙-丙)二肽(1),环-(亮-甘)二肽(2),环-(苯丙-缬)二肽(3),环-(羟脯-苯丙)二肽(4),N-(2-苯基)乙酰胺(5),β-腺苷(6),和2-哌啶酮(7)。所有化合物均为首次从该放线菌中分离得到,其中化合物6为首次从微生物中分离得到。      

红树林来源真菌Xylaria sp. HNWSW-2异香豆素类次生代谢产物及其生物活性的研究

为了研究红树林来源真菌Xylaria sp. HNWSW-2的次生代谢产物及其生物活性,综合利用多种色谱技术对该菌株发酵产物进行分离纯化,结合波谱学与理化常数分析进行化合物结构鉴定,分别采用液体浸泡法和Ellman比色法对化合物的全齿复活线虫致死活性和乙酰胆碱酯酶抑制活性进行测试。从Xylaria sp. HNWSW-2发酵产物乙酸乙酯萃取物中分离鉴定了7个异香豆素类化合物,分别为 (S)-(+)-8-O-methylmellein (1),(3S,4S)-(+)-4-hydroxy-8-O- methylmellein (2),(3S,4R)-(+)-4-hydroxy-8-O-methylmellein (3),(3S,4S)-(+)-4-hydroxymellein (4),(3S,4R)-(+)-4- hydroxymellein (5),(3R,4R)-(-)-4-hydroxy-5-methylmellein (6)和(3R,4S)-(+)-4-hydroxy-5-methylmellein (7)。其中,化合物1具有较强全齿复活线虫致死活性,化合物1~3、6和7具有一定的乙酰胆碱酯酶抑制活性。本研究首次发现化合物(S)-(+)-8-O-methylmellein具有较强的抗线虫活性,为相关杀线虫药物的研发提供理论依据。     

五指毛桃抗炎活性成分的分离及鉴定

以对5-LOX的抑制活性为抗炎活性评价指标,在活性跟踪下从五指毛桃中分离到6个化合物,经波谱数据分析后被鉴定为:羽扇豆醇棕榈酸酯(1)、β-香树脂醇乙酸酯(2)、补骨脂素(3)、芹菜素(4)、壬二酸(5)和胡萝卜苷(6),其中,化合物5、1对5-LOX表现出强的抑制活性,半数抑制浓度(IC50)分别为13.74μg/m L和13.96μg/m L;化合物1为首次从五指毛桃中分离得到。本研究结果可为五指毛桃的抗炎作用研究及其临床应用提供理论参考。     

海南粗榧外植体灭菌方法研究

以海南粗榧(Cephalotaxus mannii Hk.f.)的枝条、叶为外植体进行愈伤组织诱导,采用升汞、次氯酸钠与臭氧为消毒剂,探讨不同灭菌方法对海南粗榧外植体污染和细胞活性的影响。结果表明,以升汞与臭氧复合处理的灭菌方法最佳,最佳灭菌条件为:75%酒精灭菌1 min,臭氧灭菌30 min,再用0.1%HgCl2溶液灭菌10 min。在此条件下,外植体污染率5%,愈伤组织诱导率95%,外植体细胞活力比对照提高38%。     

Cytotoxic Minor Piericidin Derivatives from the Actinomycete Strain Streptomyces psammoticus SCSIO NS126

The mangrove-sediment-derived actinomycete strain Streptomyces psammoticus SCSIO NS126 was found to have productive piericidin metabolites featuring anti-renal cell carcinoma activities. In this study, in order to explore more diverse piericidin derivatives, and therefore to discover superior anti-tumor lead compounds, the NS126 strain was further fermented at a 300-L scale under optimized fermentation conditions. As a result, eight new minor piericidin derivatives (piericidins L-R (1–7) and 11-demethyl-glucopiericidin A (8)) were obtained, along with glucopiericidin B (9). The new structures including absolute configurations were determined by spectroscopic methods coupled with experimental and calculated electronic circular dichroism. We also proposed plausible biosynthetic pathways for these unusual post-modified piericidins. Compounds 1 and 6 showed selective cytotoxic activities against OS-RC-2 cells, and 2–5 exhibited potent cytotoxicity against HL-60 cells, with IC₅₀ values lower than 0.1 μM. The new piericidin glycoside 8 was cytotoxic against ACHN, HL-60 and K562, with IC₅₀ values of 2.3, 1.3 and 5.5 μM, respectively. The ability to arrest the cell cycle and cell apoptosis effects induced by 1 and 6 in OS-RC-2 cells, 2 in HL-60 cells, and 8 in ACHN cells were then further investigated. This study enriched the structural diversity of piericidin derivatives and confirmed that piericidins deserve further investigations as promising anti-tumor agents.     

4-Methylenesterols from a Sponge Theonella swinhoei

Three new 4-methylenesterols, theonellasterol K (1), acetyltheonellasterol (2) and acetyldehydroconicasterol (3), along with two known sterols, theonellasterol (4) and theonellasterone (5), were isolated from the sponge Theonella swinhoei. The structures of these compounds were elucidated on the basis of their spectroscopic data and comparison of the NMR data with those of known analogues. Compound 1 exhibited significant cytotoxic activity against HCT-116, K562 and Molt 4 cancer cell lines.     

Polyhydroxylated Steroids from the South China Sea Soft Coral Sarcophyton sp. and Their Cytotoxic and Antiviral Activities

Chemical investigation on the soft coral Sarcophyton sp. collected from the South China Sea yielded three new polyhydroxylated steroids, compounds (1–3), together with seven known ones (4–10). Their structures were established by extensive spectroscopic methods and comparison of their data with those of the related known compounds. All the isolates possessed the 3β,5α,6β-trihydroxylated steroidal nucleus. The cytotoxicities against selected HL-60, HeLa and K562 tumor cell lines and anti-H1N1 (Influenza A virus (IAV)) activities for the isolates were evaluated. Compounds 2, 3 and 5–8 exhibited potent activities against K562 cell lines with IC₅₀ values ranging from 6.4 to 10.3 μM. Compounds 1, 6–8 potently inhibited the growth of HL-60 tumor cell lines, and 6 also showed cytotoxicity towards HeLa cell lines. In addition, preliminary structure-activity relationships for the isolates are discussed. The OAc group at C-11 is proposed to be an important pharmacophore for their cytotoxicities in the 3β,5α,6β-triol steroids. Compounds 4 and 9 exhibited significant anti-H1N1 IAV activity with IC₅₀ values of 19.6 and 36.7 μg/mL, respectively.     

Four New Chloro-Eremophilane Sesquiterpenes from an Antarctic Deep-Sea Derived Fungus,Penicillium sp. PR19N-1

A new chloro-trinoreremophilane sesquiterpene 1, three new chlorinated eremophilane sesquiterpenes 2–4, together with a known compound, eremofortine C (5), were isolated from an Antarctic deep-sea derived fungus, Penicillium sp. PR19N-1. Structures were established using IR, HRMS, 1D and 2D NMR techniques. In addition, the plausible metabolic network of these isolated products is proposed. Compound 1 showed moderate cytotoxic activity against HL-60 and A549 cancer cell lines.     

Dichotocejpins A–C: New Diketopiperazines from a Deep-Sea-Derived Fungus Dichotomomyces cejpii FS110

Three new diketopiperazines, dichotocejpins A–C (1–3), together with eight known analogues (4–11), were isolated from the culture of the deep-sea sediment derived fungus Dichotomomyces cejpii FS110. Their structures, including absolute configurations, were elucidated by a combination of HRESIMS, NMR, X-ray crystallography, and ECD calculations. Compounds 4–6, 10–11 showed significant cytotoxic activities against MCF-7, NCI-H460, HepG-2, and SF-268 tumor cell lines. Compound 1 exhibited excellent inhibitory activity against α-glucosidase with an IC₅₀ of 138 μM.     

Fungal endophytes reduce regrowth and affect competitiveness of meadow fescue in early succession of pastures

Systemic‐ and seed‐transmitted fungal endophytes are suggested to enhance competitive dominance of agronomic grasses by increasing plant growth and defence against herbivores. We studied whether Neotyphodium uncinatum endophyte infection frequencies of meadow fescue (Schedonorus pratensis) and botanical composition of pastures are affected by 4, 5, 7 and 21 years of grazing by dairy cattle. We then examined with one greenhouse and two field experiments, whether endophyte infection and clipping affect regrowth of young or mature plants relative to nutrient availability in soil. The frequencies of infected plants and the number of plant species were less in grazed parts of the pastures. Endophytes significantly reduced relative regrowth and dry biomass of regrowth of the grass irrespective of nutrient levels in a 1‐year‐old field (on an average 18% in 2 months) and under high nutrient conditions in the greenhouse experiment (on an average 3% in 3 months) respectively. However, effects of endophytes were not detected in 5‐year‐old fields and under low nutrient conditions in the greenhouse. In contrast to past studies, our results demonstrate that grazing may negatively affect endophyte–grass symbiosis and number of plant species of successional pastures, and suggest that the effects of endophytes may be linked to the ontogeny of the host.     

Assessing biodiversity of a freshwater benthic macroinvertebrate community through non-destructive environmental barcoding of DNA from preservative ethanol

Background

Previous work has shown that leaf-cutting ants prefer to cut leaf material with relatively low fungal endophyte content. This preference suggests that fungal endophytes exact a cost on the ants or on the development of their colonies. We hypothesized that endophytes may play a role in their host plants’ defense against leaf-cutting ants. To measure the long-term cost to the ant colony of fungal endophytes in their forage material, we conducted a 20-week laboratory experiment to measure fungal garden development for colonies that foraged on leaves with low or high endophyte content.

Results

Colony mass and the fungal garden dry mass did not differ significantly between the low and high endophyte feeding treatments. There was, however, a marginally significant trend toward greater mass of fungal garden per ant worker in the low relative to the high endophyte treatment. This trend was driven by differences in the fungal garden mass per worker from the earliest samples, when leaf-cutting ants had been foraging on low or high endophyte leaf material for only 2 weeks. At two weeks of foraging, the mean fungal garden mass per worker was 77% greater for colonies foraging on leaves with low relative to high endophyte loads.

Conclusions

Our data suggest that the cost of endophyte presence in ant forage material may be greatest to fungal colony development in its earliest stages, when there are few workers available to forage and to clean leaf material. This coincides with a period of high mortality for incipient colonies in the field. We discuss how the endophyte-leaf-cutter ant interaction may parallel constitutive defenses in plants, whereby endophytes reduce the rate of colony development when its risk of mortality is greatest.

     

Anti-Leukemic Properties of Aplysinopsin Derivative EE-84 Alone and Combined to BH3 Mimetic A-1210477

Aplysinopsins are a class of marine indole alkaloids that exhibit a wide range of biological activities. Although both the indole and N-benzyl moieties of aplysinopsins are known to possess antiproliferative activity against cancer cells, their mechanism of action remains unclear. Through in vitro and in vivo proliferation and viability screening of newly synthesized aplysinopsin analogs on myelogenous leukemia cell lines and zebrafish toxicity tests, as well as analysis of differential toxicity in noncancerous RPMI 1788 cells and PBMCs, we identified EE-84 as a promising novel drug candidate against chronic myeloid leukemia. This indole derivative demonstrated drug-likeness in agreement with Lipinski’s rule of five. Furthermore, EE-84 induced a senescent-like phenotype in K562 cells in line with its cytostatic effect. EE-84-treated K562 cells underwent morphological changes in line with mitochondrial dysfunction concomitant with autophagy and ER stress induction. Finally, we demonstrated the synergistic cytotoxic effect of EE-84 with a BH3 mimetic, the Mcl-1 inhibitor A-1210477, against imatinib-sensitive and resistant K562 cells, highlighting the inhibition of antiapoptotic Bcl-2 proteins as a promising novel senolytic approach against chronic myeloid leukemia.     

Tetrahydrofuran cembranoids from the cultured soft coral Lobophytum crassum

Three new cembranoids, culobophylins A-C (1-3), along with two known compounds (4 and 5) were isolated from the cultured soft coral Lobophytum crassum. The structures of these compounds were elucidated on the basis of their spectroscopic data and comparison of the NMR data with those of known analogues. Among these metabolites, 2 is rarely found in cembranoids possessing an isopropyl moiety with an epoxide group. Compound 1 exhibited significant cytotoxic activity against HL60 and DLD-1 cancer cell lines.