molecules

Review Research Advances and Detection Methodologies for Microbe-Derived Inhibitors: A Systemic Review

Jingqian Su 1,2,3, Huiying Liu 1,2,3, Kai Guo 1,2,3, Long Chen 4, Minhe Yang 3 and Qi Chen 1,2,3,* 1 Fujian Key Laboratory of Innate Immune Biology, Fujian Normal University, Fuzhou 350117, China; [email protected] (J.S.); [email protected] (H.L.); [email protected] (K.G.) 2 Biomedical Research Center of South China, Fujian Normal University, Fuzhou 350117, China 3 College of Life Science, Fujian Normal University, Fuzhou 350117, China; [email protected] 4 Tumor Invasion Microecological Laboratory, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, China; [email protected] * Correspondence: [email protected]; Tel.: +86-591-2286-8190

Academic Editor: Derek J. McPhee Received: 9 December 2016; Accepted: 16 January 2017; Published: 23 January 2017

Abstract: Acetylcholinesterase inhibitors (AChEIs) are an attractive research subject owing to their potential applications in the treatment of neurodegenerative diseases. Fungi and bacteria are major producers of AChEIs. Their active ingredients of fermentation products include alkaloids, terpenoids, phenylpropanoids, and steroids. A variety of in vitro acetylcholinesterase inhibitor assays have been developed and used to measure the activity of , including modified Ellman’s method, thin layer chromatography bioautography, and the combined liquid chromatography-mass spectrometry/modified Ellman’s method. In this review, we provide an overview of the different detection methodologies, the microbe-derived AChEIs, and their producing strains.

Keywords: Alzheimer’s disease; acetylcholinesterase inhibitors; in vitro assays

Acetylcholinesterase is a secretory carboxylesterase present in the central and peripheral nervous systems. This enzyme rapidly hydrolyze the neurotransmitter into and ultimately terminates the excitatory effects of acetylcholine on the postsynaptic membrane, ensuring the proper transduction of nervous signals and critically affecting the nervous system [1]. In addition to presenting at the neuromuscular junctions, the cholinergic system is also widely expressed in the cortex, basal ganglia, hypothalamus, brainstem nuclei, and cerebellum, and has functions in attention, memory, execution, language, and other cognitive processes [2]. In addition to its role in transducing nervous signals, acetylcholinesterase is also involved in the other cellular processes, such as cell differentiation, apoptosis, and cell adhesion [3]. Acetylcholinesterase inhibitors (AChEIs) extend and enhance the effects of acetylcholine by causing the acetylcholine accumulation in the synapses. AChEIs are usually made by chemical synthesis or produced by plants and microorganisms, and may have applications in treating neurodegenerative disorders such as Alzheimer’s disease. In this review, we illustrate a variety of microorganism-derived AChEIs and summarized recent advances in the detection assays used for testing AChEI activity.

1. Effects of AChEIs AChEIs can be categorized as strong or weak inhibitors. Strong inhibitors include organic phosphates and , which are primarily used as neural toxins and pesticides, while weak inhibitors have been used in the treatments of Alzheimer’s disease (AD), dementia with Lewy bodies,

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Parkinson’s disease, myasthenia gravis, glaucoma, postural tachycardia syndrome, autism, insomnia, and cognitive disorders [4]. Currently, AChEIs are most often studied in the context of their applications in the treatment of AD and myasthenia gravis. AD is a common degenerative disease of the central nervous system in the elderly, with clinical manifestations including memory decline and impaired social recognition [5,6]. To date, several AChEIs have been used in clinical practice for treating AD, including , , galanthamine, and , among which galanthamine and huperzine A are natural compounds [7]. Myasthenia gravis is a chronic autoimmune disease resulting from dysfunction of the acetylcholine receptor at the neuromuscular junction. The symptoms of myasthenia gravis include weakness or abnormalities in the involved skeletal muscles and susceptibility to fatigue, which may increase after intense activities. Symptoms can be partially alleviated after rest or by the use of cholinesterase inhibitors, such as and [8].

2. In Vitro Assays for AChEIs Most original in vitro assays for testing AChEI activity are conducted by using preparations of isolated guinea pig ileum smooth muscles. These assays involve complicated protocols, including a long preparation time, and the use of large amounts of animal tissues and reagents, which constrains the development of large-scale screening and detection of AChEIs. These assays have been replaced by biochemical assays that are more sensitive and less complicated [9], which will be elucidated in the following.

2.1. Ellman’s Method The chemical principle of Ellman’s method involves hydrolysis of acetylcholine by acetylcholinesterase to produce acetic acid and choline, which then reacts with Ellman’s reagent (5,50-bisdithionitrobenzoic acid [DTNB]) to form a compound exhibiting light absorbance at 412 nm. With other factors unchanged, the absorbance value is linearly correlated with the inhibition activity of the enzyme, facilitating calculation of the half-maximal inhibitor concentration (IC50) [10]. Although Ellman’s method does not require the preparation of animal tissues for in vitro analysis of AChEIs, it is restricted to a small pH range (6.5–8.5), in which the disulfide bond breakage in DTNB occurs, thereby limiting the assay for use within this pH range. The –SH group, which detaches from the protein, can also react with DTNB and interfere with the hydrolysis reaction; however, the results are often not ideal and therefore cannot be used in high-throughput screens. To overcome these problems, Gorun et al. [11] made various modifications to the assay as follows. Instead of adding the DTNB reagent and substrates into the reaction system at the same time, DTNB is added into the solution after the incubation of acetylcholinesterase and substrates, followed by termination and color development. In this context, the enzymatic reaction and the chromogenic step which are affected by the pH are separated; these modifications permit the detection of enzyme activity at any pH and thereby increase the assay sensitivity. The use of a microplate/microplate reader also allows for further simplification of Ellman’s method, thereby significantly decreasing the experimental duration [12,13]. Bajda et al. [14] developed the electrophoretically mediated microanalysis (EMMA) technique for rapid screening of AChEIs. The data obtained from the EMMA assay and Ellman’s test had good linear relationships in the range of their respective mass concentration. Ignasik et al. [15] used both methods for testing AChE inhibitory activities of a series of new 2-(diethylaminoalkyl)-isoindoline-1,3-dione derivatives designed using molecular modeling. Both methods gave a satisfactory result, which lends support for EMMA as a reliable tool for rapid screening of novel cholinesterase inhibitors.

2.2. Thin-Layer Chromatography (TLC) Bioautography One of the disadvantages of the modified Ellman’s method in screening for AChEIs is the higher frequency of false-positive results, which stems from the inhibitory activities of acetylcholinesterase by Molecules 2017, 22, 176 3 of 24 residues of organic solvents used in sample preparation and purification. In addition, the production cost increases with the number of isolation and purification steps increases. Therefore, an important tactic is to reduce the number of steps during isolation and purification, as well as to reduce the interference from organic solvents [16]. Rhee et al. combined TLC with the modified Ellman’s method and developed a TLC bioautography method to effectively solve the above problems. In the procedure, test samples are effectively separated by silica gel TLC. After the organic solvents are completely evaporated, the chromogenic reagent DTNB is first sprayed on the chromatography plates, followed by spraying with acetylcholinesterase. After acetylcholinesterase reacts with the sample thoroughly, the acetylcholine substrate is sprayed on the plate. The appearance of white spots in the orange or yellow background indicates the possibility of the existence of compounds with acetylcholinesterase inhibition activity, and further separation and purification can be conducted [17]. To further eliminate the possible errors caused by the naked, Marston et al. used acetylcholinesterase to decompose naphthyl acetate into naphthol, which can react with Fast Blue B to form a lustrous purple diazo compound [18]. The distinction of white spots in a purple background has a better visual effect and increases the sensitivity up to 1 × 10−10 µg/L. Yang et al. [19] optimized the use of acetylcholinesterase, substrate (naphthyl acetate), and chromogenic agent (Fast Blue B), which leads to an 85% reduction in acetylcholinesterase usage while adhering to the same sensitivity of 1 × 10−10 µg/L. Ramallo et al. [20] screened for AChEIs by combining TLC bioautography with high-resolution mass spectrometry (HRMS). In comparison to direct mass spectrometric analysis, the combined system is more affordable and exhibits improved detection specificity.

2.3. Combining Liquid Chromatography-Mass Spectrometry (LC-MS) with the Modified Ellman’s Method Ingkaninan et al. [21] modified the Ellman’s method by synchronously coupling liquid chromatography-mass spectrometry (LC-MS) which allows simultaneous separation, purification, and structural analysis of the sample, as well as detection of AchEI activity. The coupled LC-MS/modified Ellman’s method system requires three reagents: DNTB, AChE, and acetylthiocholine iodide (ATCI). The detection instrument therefore also needs to be equipped with three respective samplers, which causes the inevitable problems in synchronizing the ultraviolet spectrum, mass spectrometry, and biochemical detection in the system. Furthermore, the chromogenic reagent DNTB can result in chromogenic reaction with impurities in the sample, leading to false-positive results. However, despite these problems, this detection system is still advantageous and shows high selectivity and sensitivity. For example, for galanthamine, the detection sensitivity for this method can reach 0.3 nM. To resolve the above problems, Rhee et al. (2003b) [22] have used a synthetic nonfluorescent substrate, 7-acetoxy-1-methyl quinolinium iodide (AMQI), which can be decomposed into a strong fluorescent reagent, 7-hydroxy-1-methyl quinolinium iodide (HMQI), in the improved system, therefore the AChEI activity can be determined by fluorescence intensity. Based on the example of galanthamine, multiple compounds with AChEI activity were isolated and screened from plants of the Amaryllidaceae family. This method only requires one reagent to be added, thus reduces the quantity of reagents used. However, the nonfluorescent substrate AMQI is only stable at low pH and is susceptible to self-decomposition under the optimal pH range, which makes it difficult to detect the actual inhibitory capacity of the compounds being tested. De Jong et al. [23] made further improvements to the experimental system, in which the test samples were separated by high-performance liquid chromatography (HPLC), followed by reacting with acetylcholinesterase and the substrate acetylcholine. Then, mass spectrometry is used to determine the quantity of acetylcholine and choline contents, and the sample’s AchEI activity is then obtained. The studies have shown that this system is simple and stable. Kukula-Koch et al. [24] have used a combined system utilizing countercurrent chromatography, TLC bioautography, liquid chromatography, and time-of-flight (TOF) mass spectrometry to first separate components of root extracts from Argemone mexicana L. by countercurrent chromatography, followed by TLC autography to determine the bioactivity, and finally HPLC-electrospray ionization Molecules 2017, 22, 176 4 of 24

(ESI)-TOF-MS for purification to obtain molecular information of compounds. Using this study, compounds such as berberine, protopine, chelerithrine, sanguinarine, coptisine, palmatine, magnoflorine, and galanthamine have been identified. Additionally, galanthamine was found for the first time in Argemone mexicana L. Different methods of in vitro AChEI detection have their specific advantages and disadvantages (Table1), and current research predominantly employs the modified Ellman’s method and TLC bioautography; further modifications to these procedures should be made according to particular experimental conditions.

Table 1. Comparison of methods used for in vitro AChEI detection.

Method Advantages Disadvantages Large consumption of reagents; difficulty in In vitro testing; no requirement for large-scale preparation of some samples; Ellman’s method animal tissues often nonideal results, inappropriate for high-throughput screens Simplified assay protocol, reduced Complicated protocol for isolation and Modified Ellman’s method experimental time, purification of samples; interference from high-throughput screens organic solvents Reduced steps in sample isolation and purification protocol; TLC bioautography False-positive experimental results elimination of interference from organic solvents Synchronous coupling by the instrument, Combined High specificity and detection requirement of additional samplers; LC-MS/modified sensitivity false-positive experimental results; Ellman’s method expensive materials

3. Types of AChEIs Produced by Microbes The isolation of AChEIs has progressed from initial extraction from plants to the screening of AchEI-producing strains from soil and isolation from cyanobacteria (initially thought to be dead green algae floating on top of stagnant water), lichens, and endophytic fungi within plants. The products mainly include alkaloids, terpenoids, and other compounds. As shown in Table2, as of 2015, a total of 31 strains have shown to contain compounds with AChEI activity in their fermentation products. Except for one strain of lichen (Cladonia macilenta Hoffm), the 36 remaining strains are fungi and bacteria, including 12 Deuteromycotina, 11 Ascomycota, one Cyanophyta, one Basidiomycota, and 11 bacteria, primarily Actinomycetes and Cyanophyta. The homogeneity of these strains could provide insights into future screening of AchEI producers. Molecules 2017, 22, 176 5 of 24

Molecules 2017, 22, 176 5 of 25 MoleculesMolecules 2017, 222017, 176, 22 , 176 Table 2. Microbial producers of AChEIs. 5 of 25 5 of 25 Molecules 2017, 22, 176 5 of 25 Table 2. Microbial producers of AChEIs. Table 2.2. MicrobialMicrobial producers producers of AChEIs.of AChEIs. StrainStrain Classification Classification StructureStructure of of ActiveTable Active Ingredient(s)2. Ingredient(s) Microbial producers of AChEIs.Compound Type Compound TypeIC50 Method Used IC50 Ref. Method Used Ref. 50 Strain Strain ClassificationClassification StructureStructure ofof Active Ingredient(s)Ingredient(s) CompoundCompound Type Type IC IC50 Method UsedMethod UsedRef. Ref. Strain Classification Structure of Active Ingredient(s) Compound Type IC50 Method Used Ref.

Eumycota, DeuteromycotinaEumycotaEumycota, , DeuteromycotinaEumycota,Deuteromycotina, , , HyphomycetesEumycotaDeuteromycotina, Hyphomycetes, , , Aspergillus terreusHyphomycetes , Territrem B 7.6 μM Ellman’s method Ling et al. [25] Aspergillus terreus Aspergillus terreus DeuteromycotinaHyphomycetesMoniliales, , , Territrem B Territrem B7.6 μM Ellman’s method 7.6 µMLing et al. [25] Ellman’s method Ling et al. [25] AspergillusMoniliales terreus , Moniliales, Territrem B 7.6 μM Ellman’s method Ling et al. [25] HyphomycetesMoniliaceae, , Aspergillus terreus MoniliaceaeMoniliales, , Territrem B 7.6 μM Ellman’s method Ling et al. [25] Moniliaceae, MonilialesAspergillus, AspergillusMoniliaceae , Aspergillus MoniliaceaeAspergillus , Aspergillus

OH O OH O OH O OH O Anhydrojavanicin 2.01 μM O AnhydrojavanicinAnhydrojavanicin 2.01 μM 2.01 µM O O O Anhydrojavanicin 2.01 μM O Anhydrojavanicin 2.01 μM Me O O O O O Me O OO Eumycota, Me Deuteromycotina, Me O O Aspergillus terreusEumycota Hyphomycetes, , N Modified Ellman’s Deng et al. O O 8‐O‐Methylbostrycoidin 6.71 μM (No. GX7‐3B)DeuteromycotinaEumycota Moniliales, , , O O method [26] EumycotaDeuteromycotinaMoniliaceae, ,, Aspergillus terreus Hyphomycetes, O N Modified Ellman’s Deng et al. Aspergillus terreus DeuteromycotinaHyphomycetesAspergillus, , N 8‐O‐Methylbostrycoidin 6.71 μM Modified Ellman’s Deng et al. (No. GX7‐3B) Moniliales, 8‐O‐Methylbostrycoidin 6.71 μM method [26] Aspergillus(No. GX7 terreus‐3B) HyphomycetesMoniliales, , OH O N 8-O-MethylbostrycoidinModifiedmethod Ellman’s 6.71 µMDeng[26] et al. Moniliaceae, 8‐O‐Methylbostrycoidin 6.71 μM (No. GX7‐3B) MonilialesMoniliaceae, , O method [26] Aspergillus O MoniliaceaeAspergillus , Eumycota, O OH O DeuteromycotinaAspergillus, OH O OH O NGA0187 Aspergillus terreus Hyphomycetes, 1.89 μM Modified Ellman’s (Terpenoid) Deng et al. [26] (No. GX7-3B) Moniliales, method NGA0187 Moniliaceae, 1.89 μM (Terpenoid)NGA0187 Aspergillus NGA0187 1.89 μM (Terpenoid) 1.89 μM (Terpenoid) NGA0187 (Terpenoid) 1.89 µM

Molecules 2017, 22, 176 6 of 25

Beauvericin Beauvericin3.09 μM 3.09 µM

Eumycota, Deuteromycotina, Aspergillus flavus Hyphomycetes, Modified Ellman’s Wang et al. Huperzine A 0.6 μM LF40 Moniliales, method [27] Moniliaceae, Aspergillus

(8E,12Z)‐10,11‐Dihydroxyoctadeca‐ 8,12‐dienoic acid

Eumycota, Deuteromycotina, Hyphomycetes, Modified Ellman’s Aspergillus flavus No report Qiao et al. [28] Moniliales, method Moniliaceae, 3β,4α‐Dihydroxy‐26‐methoxyergosta‐ Aspergillus 7,24(28)‐dien‐6‐one

Molecules 2017, 22, 176 6 of 25 Molecules 2017, 22, 176 6 of 25 Molecules 2017, 22, 176 6 of 25

Molecules 2017, 22, 176 6 of 24

Beauvericin 3.09 μM Beauvericin 3.09 μM TableBeauvericin 2. Cont. 3.09 μM

Strain Classification Structure of Active Ingredient(s) Compound Type IC50 Method Used Ref. Eumycota, Eumycota, DeuteromycotinaEumycotaDeuteromycotina, , , AspergillusHyphomycetes flavus DeuteromycotinaHyphomycetes, Eumycota,, , Modified Ellman’s Wang et al. Modified Ellman’s Huperzine A 0.6 μM Aspergillus flavus LF40 AspergillusLF40 flavus HyphomycetesMonilialesDeuteromycotina, , , Huperzine AModifiedmethod Ellman’s 0.6 µM Wang[27] et al. Wang et al. [27] MonilialesAspergillus flavus, Hyphomycetes, Huperzine A 0.6 μM Modified Ellman’s Wang et al. method LF40 MonilialesMoniliaceae, , Huperzine A 0.6 μM method [27] MoniliaceaeLF40 , MoniliaceaeAspergillusMoniliales , , method [27] Aspergillus AspergillusMoniliaceae , Aspergillus (8E,12Z)‐10,11‐(8DihydroxyoctadecaE,12Z)-10,11-‐ (88,12E,12‐dienoicZ)‐10,11 acid‐Dihydroxyoctadeca ‐ 8,12(8‐dienoicE,12Z) ‐acid10,11Dihydroxyoctadeca- ‐Dihydroxyoctadeca‐ Eumycota, 8,12‐dienoic8,12-dienoic acid acid Eumycota, EumycotaDeuteromycotina, , DeuteromycotinaDeuteromycotinaHyphomycetesEumycota, ,, , Modified Ellman’s Aspergillus flavus No report Qiao et al. [28] HyphomycetesHyphomycetesMoniliales, Deuteromycotina, , , Modifiedmethod Ellman’s Aspergillus flavus Hyphomycetes, No report Modified Ellman’s Qiao et al. [28] Modified Ellman’s Aspergillus flavus Aspergillus flavus MonilialesMoniliaceae, , 3β,4α‐Dihydroxy‐26‐methoxyergosta‐ No report methodNo report Qiao et al. [28] Qiao et al. [28] Moniliales, Moniliales, method method MoniliaceaeAspergillus , 37,24(28)β,4α‐Dihydroxy‐dien‐6‐oneβ‐26 ‐αmethoxyergosta‐ Moniliaceae, 3 ,4 -Dihydroxy-26- Moniliaceae, Aspergillus 7,24(28)3β,4‐α‐dienDihydroxy‐6‐one ‐26‐methoxyergosta‐ Aspergillus methoxyergosta-7,24(28)- Aspergillus 7,24(28)‐dien‐6‐one dien-6-one

Molecules 2017, 22, 176 7 of 25 Molecules 2017, 22, 176 7 of 25 Molecules 2017, 22, 176 7 of 25

Eumycota, Eumycota, Eumycota, DeuteromycotinaDeuteromycotina, , Aspergillus EumycotaDeuteromycotina, , Aspergillus versicolor AspergillusHyphomycetes DeuteromycotinaHyphomycetes, , , Modified Ellman’s Wang et al. Modified Ellman’s Aspergillusversicolor Hyphomycetes, Avertoxin B Avertoxin B14.9 μM Modified Ellman’s 14.9 µMWang et al. Wang et al. [29] versicolor HyphomycetesMoniliales, , Avertoxin B 14.9 μM Modifiedmethod Ellman’s Wang[29] et al. Y10 MonilialesversicolorY10 , Moniliales, Avertoxin B 14.9 μM method [29] method Y10 MonilialesMoniliaceae, , method [29] MoniliaceaeY10 , Moniliaceae, MoniliaceaeAspergillus , Aspergillus Aspergillus Aspergillus

Quinolactacins A1 280 μM Quinolactacins A1 280 μM µ QuinolactacinsQuinolactacins A1 A1280 μM 280 M

Eumycota, Eumycota, Eumycota, Deuteromycotina, DeuteromycotinaEumycotaDeuteromycotina, , , Penicillium DeuteromycotinaHyphomycetes, , Modified Ellman’s PenicilliumHyphomycetes Hyphomycetes, , Modified Ellman’s Kim et al. [30] Modified Ellman’s Penicillium citrinum Penicilliumcitrinum HyphomycetesMoniliales, , Modifiedmethod Ellman’s Kim et al. [30] Kim et al. [30] citrinum Moniliales, method Kim et al. [30] Monilialescitrinum , MonilialesMoniliaceae, , method method Moniliaceae, Penicillium Moniliaceae, MoniliaceaePenicillium , Penicillium Penicillium Quinolactacins A2 19.8 μM Quinolactacins A2 19.8 μM QuinolactacinsQuinolactacins A2 A219.8 μM 19.8 µM

Eumycota, Eumycota, EumycotaDeuteromycotina, , Deuteromycotina, Penicillium sp. Hyphomycetes, Kakinuma Penicillium sp. DeuteromycotinaHyphomycetes, , Quinolactacins A, B, and C No report Ellman’s method Kakinuma PenicilliumEPF‐6 sp. HyphomycetesMoniliales, , Quinolactacins A, B, and C No report Ellman’s method Kakinumaet al. [31] EPF‐6 Moniliales, Quinolactacins A, B, and C No report Ellman’s method et al. [31] EPF‐6 MonilialesMoniliaceae, , et al. [31] Moniliaceae, Penicillium MoniliaceaePenicillium , Penicillium

Penicillium sp. Eumycota, Modified Ellman’s Penicillium sp. Eumycota, Arigsugacin I 0.64 μM Modified Ellman’s PenicilliumFO‐4259 sp. EumycotaDeuteromycotina, , Arigsugacin I 0.64 μM Modifiedmethod Ellman’s FO‐4259 Deuteromycotina, Arigsugacin I 0.64 μM method FO‐4259 Deuteromycotina, method

Molecules 2017, 22, 176 7 of 25

Eumycota, Deuteromycotina, Aspergillus Hyphomycetes, Modified Ellman’s Wang et al. versicolor Avertoxin B 14.9 μM Moniliales, method [29] Y10 Moniliaceae, Aspergillus

Quinolactacins A1 280 μM

Eumycota, Deuteromycotina, Penicillium Hyphomycetes, Modified Ellman’s Kim et al. [30] Molecules 2017, 22, 176 citrinum Moniliales, method 7 of 24 Moniliaceae, Penicillium

TableQuinolactacins 2. Cont. A2 19.8 μM

Strain Classification Structure of Active Ingredient(s) Compound Type IC50 Method Used Ref.

Eumycota, Eumycota, DeuteromycotinaDeuteromycotina, , Penicillium sp. PenicilliumHyphomycetes sp. Hyphomycetes, , Kakinuma QuinolactacinsQuinolactacins A, B, and C A, B,No and report C Ellman’s method No report Ellman’s method Kakinuma et al. [31] EPF-6 MonilialesEPF‐6 , Moniliales, et al. [31] Moniliaceae, Moniliaceae, Penicillium Penicillium

Molecules 2017, 22, 176 8 of 25 Penicillium sp. Eumycota, Modified Ellman’s Arigsugacin I 0.64 μM EumycotaFO‐4259 , Deuteromycotina, method Molecules 2017Hyphomycetes, 22, 176 , 8 of 25 Deuteromycotina, Penicillium sp. HyphomycetesMoniliales,, MoniliaceaeHyphomycetes, , FO-4259 Moniliales, PenicilliumMoniliales , Arigsugacin I 0.64 µM Moniliaceae, Moniliaceae, Penicillium Penicillium Arigsugacins F 0.37 μM Arigsugacins F 0.37 μM Arigsugacins F 0.37 µM

Eumycota, Modified Ellman’s Omura et al.; Huang Deuteromycotina, Omura et al.; Omura et al.; EumycotaEumycota, , RR2 Huang et al. method et al. [32,33] Penicillium sp. HyphomycetesDeuteromycotina, , 2 Huang et al. Deuteromycotina, [32,33] R3R [32,33] sk5GW1LPenicilliumPenicillium sp.Moniliales sp.Hyphomycetes , Hyphomycetes, , 3 sk5GW1L Moniliales, sk5GW1L MoniliaceaeMoniliales, , O O Territrem B 7.03 μM Moniliaceae, O O R4 Territrem B 7.03 μM PenicilliumMoniliaceae, R4 Penicillium R Penicillium 1 Territrem B 7.03 µM R1 CH3 CH3 O CH3 CH3 OH O R1 OH OH H3C R1 CH3 OH H3C CH3 R1 R2 R3 R4 Terreulactone C OCH3 OCH3 OCH3 OCH3 Arisugacin A HR1 OCHR23 OCHR3 3 H R4 Arisugacin B H H OCH H Terreulactone C OCH3 OCH3 OCH3 3 OCH3 Territrem A H -OCH2-OCH3 Arisugacin A H OCH3 OCH3 H Territrem B H OCH3 OCH3 OCH3 Arisugacin B H H OCH3 H Territrem C H OCH3 OH OCH3 Territrem A H -OCH2-OCH3 Eumycota, Territrem B H OCH3 OCH3 OCH3 Deuteromycotina, Penicillium Territrem C H OCH3 OH OCH3 Hyphomycetes, 7.8 μM Modified Ellman’s Huang et al. chermesinumEumycota , Terphenyls EumycotaMoniliales, , 5.2 μM method [34] Deuteromycotina(ZH4‐E2)Deuteromycotina , , Penicillium Penicillium Moniliaceae, HyphomycetesHyphomycetesPenicillium, , 7.8 μM Modified7.8 µM Ellman’s HuangModified et al. Ellman’s chermesinum Terphenyls chermesinum Moniliales, Terphenyls 5.2 μM method [34] Huang et al. [34] (ZH4‐E2) Moniliales, 5.2 µM method (ZH4-E2) MoniliaceaeEumycota, , Moniliaceae, Deuteromycotina, PenicilliumPenicillium Penicillium Hyphomycetes, Modified Ellman’s Wang et al. griseofulvum Huperzine A 0.6 μM Moniliales, method [35] LF146 EumycotaMoniliaceae, , Deuteromycotina, Penicillium Penicillium Hyphomycetes, Modified Ellman’s Wang et al. griseofulvum Huperzine A 0.6 μM Moniliales, method [35] LF146 Moniliaceae, Penicillium

Molecules 2017, 22, 176 8 of 25

Hyphomycetes, Moniliales, Moniliaceae, Penicillium

Arigsugacins F 0.37 μM

Omura et al.; Eumycota, R2 Huang et al. Deuteromycotina, R [32,33] Penicillium sp. Hyphomycetes, 3 sk5GW1L Moniliales, O O Territrem B 7.03 μM Moniliaceae, R4 Penicillium R1

CH3 CH3 O OH

R1 OH H3C CH3

R1 R2 R3 R4 Terreulactone C OCH3 OCH3 OCH3 OCH3 Molecules 2017, 22, 176 Arisugacin A H OCH3 OCH3 H 8 of 24 Arisugacin B H H OCH3 H Territrem A H -OCH2-OCH3 Territrem B H OCH3 OCH3 OCH3 Territrem C H OCH3 OH OCH3 Eumycota, Deuteromycotina, Table 2. Cont. Penicillium Hyphomycetes, 7.8 μM Modified Ellman’s Huang et al. chermesinum Terphenyls Moniliales, 5.2 μM method [34] (ZH4‐E2) Moniliaceae, Strain Classification Structure of Active Ingredient(s) Compound Type IC50 Method Used Ref. Penicillium Eumycota, Eumycota, DeuteromycotinaDeuteromycotina, , Penicillium Penicillium HyphomycetesHyphomycetes, , Modified Ellman’s Wang et al. Modified Ellman’s griseofulvum Huperzine A 0.6 μM griseofulvum Moniliales, Huperzine Amethod 0.6 µM [35] Wang et al. [35] MonilialesLF146 , method LF146 Moniliaceae, Moniliaceae, Penicillium MoleculesPenicillium 2017, 22, 176 9 of 25

Eumycota, DeuteromycotinaEumycota, , Paecilomyces tenuis Paecilomyces tenuis Deuteromycotina, Modified Ellman’s Su and Yang Modified Ellman’s Hyphomycetes, Huperzine A Huperzine A0.6 μM 0.6 µM Su and Yang [36] YS-13 YS‐13 Hyphomycetes, method [36] method PaecilomycesPaecilomyces Paecilomyces Paecilomyces

Eumycota, Eumycota, Deuteromycotina, Acremonium Hyphomycetes, Modified Ellman’s Wang et al. implicatumDeuteromycotina , No clear products Acremonium Moniliales, method [35] HyphomycetesLF30 , Modified Ellman’s implicatum Moniliaceae, No clear products Wang et al. [35] Moniliales, Acremonium method LF30 Moniliaceae, Molecules 2017, 22, 176 9 of 25 Acremonium Eumycota, Deuteromycotina, Acremonium sp. Hyphomycetes, Eumycota, Huperzine A 0.6 μM No report Li et al. [37] 2F09P03B Moniliales, DeuteromycotinaEumycotaMoniliaceae, , , Acremonium sp. PaecilomycesHyphomycetes tenuis DeuteromycotinaAcremonium, , Modified Ellman’s Su and Yang Huperzine A Huperzine A0.6 μM 0.6 µM No report Li et al. [37] 2F09P03B MonilialesYS‐13 , Hyphomycetes, method [36] Paecilomyces Paecilomyces Moniliaceae, Molecules 2017, 22, 176Eumycota , 9 of 25 Acremonium Deuteromycotina, Botrytis sp. EumycotaHyphomycetes, , Deuteromycotina, Huperzine A 0.6 μM No report Ju et al. [38] AcremoniumEumycotaHA23 , Moniliales, HyphomycetesMoniliaceae, , Modified Ellman’s Wang et al. implicatumDeuteromycotina Eumycota, , No clear products MonilialesBotrytis , method [35] Botrytis sp. PaecilomycesHyphomycetesLF30 tenuis Deuteromycotina, , Modified Ellman’s Su and Yang Moniliaceae, Huperzine A Huperzine A0.6 μM 0.6 µM No report Ju et al. [38] HA23 MonilialesYS‐13 , HyphomycetesAcremonium , method [36] PaecilomycesEumycota, Paecilomyces Moniliaceae, Deuteromycotina, EumycotaHyphomycetes, , Modified Ellman’s Dong et al. TrichodermaBotrytis sp. No clear products DeuteromycotinaMoniliales, , method [39] Eumycota, Acremonium sp. HyphomycetesMoniliaceae, , Eumycota, Deuteromycotina, Huperzine A 0.6 μM No report Li et al. [37] Acremonium2F09P03B MonilialesTrichoderma, Hyphomycetes, Modified Ellman’s Wang et al. implicatumDeuteromycotina Moniliaceae, , No clear products Moniliales, method [35] HyphomycetesLF30 Acremonium, Modified Ellman’s Trichoderma sp. EumycotaMoniliaceae, , No clear products Dong et al. [39] ColletotrichumMoniliales , DeuteromycotinaAcremonium , method Modified Ellman’s Zhao et al. gloeosporioidesMoniliaceae , Coelomycetes, Huperzine A 0.6 μM Eumycota, method [40] MoleculesES026 2017 , 22, 176MelanconialesEumycota , , 9 of 25 Trichoderma Deuteromycotina, MelanconiaceaeDeuteromycotina Colletotrichum, Botrytis sp. Hyphomycetes, Acremonium sp. Hyphomycetes, Huperzine A 0.6 μM No report Ju et al. [38] EumycotaHA23 , Moniliales, Huperzine A 0.6 μM No report Li et al. [37] 2F09P03B Moniliales, Moniliaceae, DeuteromycotinaEumycotaMoniliaceae, , , Colletotrichum Botrytis PaecilomycesCoelomycetes tenuis DeuteromycotinaAcremonium, , Modified Ellman’s Su and Yang Modified Ellman’s gloeosporioides Huperzine A Huperzine A0.6 μM 0.6 µM Zhao et al. [40] MelanconialesYS‐13 Hyphomycetes, , method [36] method ES026 PaecilomycesEumycota, Paecilomyces MelanconiaceaeDeuteromycotina, Eumycota, Hyphomycetes, Modified Ellman’s Dong et al. TrichodermaColletotrichum sp. Deuteromycotina, No clear products Moniliales, method [39] Botrytis sp. EumycotaHyphomycetes, , Moniliaceae, Huperzine A 0.6 μM No report Ju et al. [38] HA23 DeuteromycotinaMoniliales, , Acremonium Trichoderma Hyphomycetes, Modified Ellman’s Wang et al. implicatum Moniliaceae, No clear products Moniliales, method [35] LF30 Botrytis EumycotaMoniliaceae, , Colletotrichum DeuteromycotinaAcremonium , Eumycota, Modified Ellman’s Zhao et al. gloeosporioides Coelomycetes, Huperzine A 0.6 μM Deuteromycotina, method [40] ES026 MelanconialesEumycotaHyphomycetes, ,, Modified Ellman’s Dong et al. Trichoderma sp. No clear products MelanconiaceaeDeuteromycotinaMoniliales, Colletotrichum, method [39] Acremonium sp. Hyphomycetes, Moniliaceae, Huperzine A 0.6 μM No report Li et al. [37] 2F09P03B MonilialesTrichoderma, Moniliaceae, Acremonium Eumycota, Colletotrichum Deuteromycotina, Modified Ellman’s Zhao et al. gloeosporioides Coelomycetes, Huperzine A 0.6 μM Eumycota, method [40] ES026 Melanconiales, Deuteromycotina, Melanconiaceae Colletotrichum Botrytis sp. Hyphomycetes, Huperzine A 0.6 μM No report Ju et al. [38] HA23 Moniliales, Moniliaceae, Botrytis

Eumycota, Deuteromycotina, Hyphomycetes, Modified Ellman’s Dong et al. Trichoderma sp. No clear products Moniliales, method [39] Moniliaceae, Trichoderma

Eumycota, Colletotrichum Deuteromycotina, Modified Ellman’s Zhao et al. gloeosporioides Coelomycetes, Huperzine A 0.6 μM method [40] ES026 Melanconiales, Melanconiaceae Colletotrichum

Molecules 2017, 22, 176 9 of 24

Table 2. Cont.

Molecules 2017, 22, 176 9 of 25 StrainMolecules 2017 Classification, 22, 176 Structure of Active Ingredient(s) Compound Type IC50 10 of 25 Method Used Ref. Molecules 2017, 22, 176 10 of 25 MoleculesEumycota 2017, 22, 176 10 of 25 Eumycota, DeuteromycotinaEumycota, , Alternaria sp. Paecilomyces tenuis Deuteromycotina, Modified Ellman’s Su and Yang Deuteromycotina, Huperzine A 0.6 μM AlternariaHyphomycetesYS‐13 sp. EumycotaHyphomycetes, , , Huperzine Amethod 0.6 µM [36] YD-01 Hyphomycetes, Huperzine A 0.6 μM MonilialesYD‐01 , DeuteromycotinaPaecilomyces Paecilomyces, Alternaria sp. Moniliales, EumycotaHyphomycetes, , Huperzine A 0.6 μM MoleculesDematiaceaeYD 2017‐01 , 22, 176 AlternariaDeuteromycotinaDematiaceae Alternaria, 9 of 25 Alternaria sp. Moniliales, Hyphomycetes, Huperzine A 0.6 μM YD‐01 DematiaceaeEumycota, Alternaria TLC Yang et al. Moniliales, TLC bioautography Yang et al. [41] Eumycota, Deuteromycotina, bioautography [41] Acremonium Dematiaceae Alternaria Hyphomycetes, ModifiedTLC Ellman’s WangYang etet al.al. implicatumDeuteromycotina Eumycota, , No clear products Molecules 2017, 22, 176EumycotaMoniliales ,, bioautographymethod [41][35]10 of 25 Cladosporium LF30 Deuteromycotina, TLC Yang et al. PaecilomycesCladosporiumHyphomycetes tenuis EumycotaDeuteromycotinaMoniliaceae, , , , Modified Ellman’s Su and Yang cladosporioides Hyphomycetes, Huperzine A Huperzine A0.6 μM bioautography 0.6 µM [41] cladosporioidesYS‐13 DeuteromycotinaHyphomycetesAcremonium , , Huperzine A 0.6 μM method [36] CladosporiumMoniliales , Moniliales, LF70 LF70 EumycotaHyphomycetesPaecilomyces, Paecilomyces, cladosporioidesDermateaceae DeuteromycotinaDermateaceae, , , Huperzine A 0.6 μM Cladosporium EumycotaMoniliales,, , CladosporiumLF70 HyphomycetesCladosporium , cladosporioides DeuteromycotinaDermateaceae, ,, Huperzine A 0.6 μM Alternaria sp. MonilialesEumycota,, AcremoniumLF70 sp. HyphomycetesCladosporium ,, Huperzine A 0.6 μM YD‐01 DermateaceaeDeuteromycotina, , Huperzine A 0.6 μM No report Li et al. [37] Acremonium2F09P03B Moniliales,, CladosporiumHyphomycetes , Modified Ellman’s Wang et al. implicatumEumycota , DematiaceaeMoniliaceae, Alternaria No clear products EumycotaMoniliales,, method [35] Acremonium DeuteromycotinaLF30 Deuteromycotina, , TLC Yang et al. EumycotaMoniliaceae, , CoelamycetesCoelamycetes, , bioautographyTLC Chapla[41] et al. Phomopsis sp. Phomopsis sp. DeuteromycotinaAcremonium , Cytochalasin HCytochalasin HNo report No report TLC bioautography Chapla et al. [42] SphaeropsidalesEumycotaSphaeropsidales, , , bioautography [42] CoelamycetesEumycota, , TLC Chapla et al. Phomopsis sp. DeuteromycotinaSphaeropsidaceaeEumycota, , Cytochalasin H No report CladosporiumSphaeropsidaceae SphaeropsidalesDeuteromycotina, , , bioautography [42] HyphomycetesCoelamycetesPhomopsisDeuteromycotina , , , TLC Chapla et al. cladosporioidesPhomopsisBotrytis sp. sp. SphaeropsidaceaeHyphomycetes, , HuperzineCytochalasin A H No0.6 report μM AcremoniumPhomopsis sp. MonilialesSphaeropsidalesHyphomycetes, , , Huperzine A 0.6 μM bioautographyNo report Ju et[42] al. [38] HA23LF70 PhomopsisMoniliales, Huperzine A 0.6 μM No report Li et al. [37] 2F09P03B DermateaceaeSphaeropsidaceaeMoniliales, , , Moniliaceae, CladosporiumPhomopsisEumycotaMoniliaceae, , Eumycota, AscomycotinaBotrytis , EumycotaAcremonium, Ascomycetes, 14‐(2′,3′,5′‐ Modified Ellman’s Sekhar Rao et ChrysosporiumAscomycotina sp. Ascomycotina, , 197 μM OnygenalesEumycota, , Trihydroxyphenyl)tetradecan0 0 0 ‐2‐ol method al. [43] Ascomycetes,EumycotaAscomycetes, , 14‐(2′,3′,5′‐ 14-(2 ,3 ,5 - Modified Ellman’s Sekhar Rao et Modified Ellman’s Chrysosporium sp. AscomycotinaOnygenaceae,Deuteromycotina , , 197 μM Chrysosporium sp. EumycotaOnygenales,, , Trihydroxyphenyl)tetradecan‐2‐ol method 197 µM al. [43] Sekhar Rao et al. [43] Onygenales, AscomycetesChrysosporiumHyphomycetes, , 14‐(2′,3′,5′‐ Trihydroxyphenyl)tetradecan-2-olModified Ellman’s SekharDong Raoet al. et method ChrysosporiumTrichoderma sp. sp. DeuteromycotinaOnygenaceae, ,, No clear products 197 μ M OnygenalesMoniliales, , Trihydroxyphenyl)tetradecan‐2‐ol method al.[39] [43] BotrytisOnygenaceae, sp. CoelamycetesChrysosporiumHyphomycetes, , TLC Chapla et al. Phomopsis sp. Onygenaceae,Moniliaceae, CytochalasinHuperzine A H No0.6 report μM No report Ju et al. [38] ChrysosporiumHA23 SphaeropsidalesEumycotaMoniliales,, , bioautography [42] Trichoderma ChrysosporiumSphaeropsidaceaeAscomycotina, , EumycotaMoniliaceae, , Shiraia sp. PhomopsisPyrenomycetes , Modified Ellman’s AscomycotinaBotrytis , Huperzine A 0.6 μM Zhu et al. [44] EumycotaSlf14 , Sphaeriales, method Shiraia sp. EumycotaPyrenomycetes,, , Modified Ellman’s ColletotrichumAscomycotina AscomycotinaHypocreaceaeDeuteromycotina, ,, , Huperzine A 0.6 μM Zhu et al. [44] Slf14 SphaerialesEumycota, , Modifiedmethod Ellman’s Zhao et al. Shiraia sp. gloeosporioidesShiraiaPyrenomycetes sp. PyrenomycetesEumycotaShiraiaCoelomycetes, , , , Huperzine A 0.6 μM Modified Ellman’s Modified Ellman’s HypocreaceaeDeuteromycotina, , Huperzine A Huperzine A0.6 μM method 0.6 µMZhu et[40] al. [44] Zhu et al. [44] Slf14 SphaerialesES026Slf14 , AscomycotinaSphaerialesShiraiaMelanconialesHyphomycetes , ,, , Modifiedmethod Ellman’s Dong et al. method Trichoderma sp. AscomycetesHypocreaceaeMelanconiaceae, , Colletotrichum No clear products 14‐(2′,3′,5′‐ Modified Ellman’s Sekhar Rao et ChrysosporiumHypocreaceae sp. Moniliales, , 197 μM method [39] OnygenalesShiraiaMoniliaceae ,, Trihydroxyphenyl)tetradecan‐2‐ol method al. [43] Shiraia Onygenaceae,Trichoderma Chrysosporium

Eumycota, Eumycota, Eumycota, Colletotrichum Deuteromycotina, Ascomycota, Ascomycotina, Modified Ellman’s Zhao et al. gloeosporioides Coelomycetes, Huperzine A 0.6 μM Xylaria sp. Shiraiapyrenomycetes sp. Pyrenomycetes, , Modifiedmethod Ellman’s [40] Modified Ellman’s ES026 Melanconiales, Huperzine A 0.6 μM Zhu et al. [44] Slf14 Sphaeriales, Huperzine Amethod 0.6 µM Su et al. [45] YS-02 Sphaeriales, Melanconiaceae Colletotrichum method Hypocreaceae, Xylariaceae, Shiraia Xylaria

Molecules 2017, 22, 176Molecules 2017, 22, 176 11 of 25 10 of 24 Molecules 2017, 22, 176 11 of 25 Molecules 2017, 22, 176 11 of 25 Molecules 2017, 22, 176 11 of 25 Eumycota, AscomycotaEumycota, , Xylaria sp. EumycotapyrenomycetesAscomycota, , , Table 2. Cont. Modified Ellman’s Eumycota, Huperzine A 0.6 μM Su et al. [45] XylariaYS‐02 sp. AscomycotaSphaerialespyrenomycetes,, , Modifiedmethod Ellman’s Ascomycota, Huperzine A 0.6 μM Su et al. [45] XylariaYS‐02 sp. pyrenomycetesXylariaceaeSphaeriales,, , Modifiedmethod Ellman’s Xylaria sp. pyrenomycetes, Huperzine A 0.6 μM Modified Ellman’s Su et al. [45] YS‐02 SphaerialesXylariaXylariaceae , , Huperzine A 0.6 μM method Su et al. [45] StrainYS‐02 Classification XylariaceaeSphaerialesXylaria , Structure of Active Ingredient(s) Compound Typemethod IC50 Method Used Ref. XylariaXylariaceae , Xylaria

Eumycota, Eumycota, Ascomycota, AscomycotaEumycota, , pyrenomycetesEumycotapyrenomycetesAscomycota, , , , Xylaria sp. Eumycota, Xyloketal A 29.9 μM Lin et al. [46] Xylaria sp. AscomycotaSphaerialespyrenomycetes,, , Xyloketal A 29.9 µM Lin et al. [46] XylariaSphaeriales sp. , Ascomycota, Xyloketal A 29.9 μM Lin et al. [46] pyrenomycetesXylariaceaeSphaeriales,, , Xylaria sp. pyrenomycetes, Xyloketal A 29.9 μM Lin et al. [46] XylariaXylariaceae sp. , SphaerialesXylariaXylariaceae , , Xyloketal A 29.9 μM Lin et al. [46] Xylaria XylariaceaeSphaerialesXylaria , XylariaXylariaceae , Xylaria

Modified Ellman’s Xyloketal B 109.3 μM Modifiedmethod Ellman’s Xyloketal B 109.3 μM Xyloketal BModifiedmethod Ellman’s 109.3 µM Xyloketal B 109.3 μM Modifiedmethod Ellman’s Xyloketal B 109.3 μM method Modified Ellman’s method

Xyloketal C 109.3 μM Xyloketal C 109.3 μM Xyloketal C Xyloketal C109.3 μM 109.3 µM Xyloketal C 109.3 μM

Xyloketal D 425.6 μM Xyloketal D 425.6 μM Xyloketal D 425.6 μM Xyloketal D Xyloketal D425.6 μM 425.6 µM

Molecules 2017, 22, 176 12 of 25 Molecules 2017, 22, 176 12 of 25

Palmariol B (1) Palmariol B (1)115.31 μg/mL 115.31 µg/mL Eumycota, Palmariol B (1) 115.31 μg/mL Ascomycotina, Hyalodendriella sp. Discomycetes, Modified Ellman’s Meng et al. [47], Ponipodef12 Leotiales, OH method Mao et al. [48] Leotiaceae, OH CH3 Hyalodendriella CH3 4‐Hydroxymellein (2) 116.05 μg/mL O 4‐Hydroxymellein4-Hydroxymellein (2) (116.052) μg/mL 116.05 µg/mL O Eumycota, AscomycotinaEumycota, , Ascomycotina, OH O Meng et al. Hyalodendriella sp. Discomycetes, OH O Modified Ellman’s Meng et al. Hyalodendriella sp. Discomycetes, Modified Ellman’s [47], Ponipodef12 Leotiales, method [47], Ponipodef12 Leotiales, method Mao et al. [48] Leotiaceae, Mao et al. [48] HyalodendriellaLeotiaceae , Hyalodendriella Alternariol 9‐methyl ether (3) 135.52 μg/mL Alternariol 9‐methyl ether (3) 135.52 μg/mL

Botrallin (4) 83.70 μg/mL Botrallin (4) 83.70 μg/mL

Eumycota, AscomycotinaEumycota, , HemiascomycetesAscomycotina, , Blastomyces sp. Hemiascomycetes, Huperzine A 0.6 μM No report Ju et al. [38] Blastomyces sp.Endomycetalcs , Huperzine A 0.6 μM No report Ju et al. [38] EndomycetaccaeEndomycetalcs, , BlastomycesEndomycetaccae , Blastomyces

Molecules 2017, 22, 176 12 of 25 Molecules 2017, 22, 176 12 of 25 Molecules 2017, 22, 176 12 of 25

Palmariol B (1) 115.31 μg/mL Palmariol B (1) 115.31 μg/mL Palmariol B (1) 115.31 μg/mL Molecules 2017, 22, 176 11 of 24

OH OH CH OH CH3 CH3 3 Table4‐Hydroxymellein 2. Cont. (2) 116.05 μg/mL O 4‐Hydroxymellein (2) 116.05 μg/mL O 4‐Hydroxymellein (2) 116.05 μg/mL Eumycota, O Strain ClassificationEumycotaAscomycotina, , Structure of Active Ingredient(s) Compound Type IC50 Method Used Ref. Eumycota, OH O Meng et al. Hyalodendriella sp. AscomycotinaDiscomycetes,, OH O Modified Ellman’s Ascomycotina, Meng[47], et al. HyalodendriellaPonipodef12 sp. DiscomycetesLeotiales, , OH O Modifiedmethod Ellman’s Meng et al. Hyalodendriella sp. Discomycetes, Modified Ellman’s Mao [47],et al. [48] Ponipodef12 LeotialesLeotiaceae, , method [47], Ponipodef12 Leotiales, method Mao et al. [48] LeotiaceaeHyalodendriella, Mao et al. [48] HyalodendriellaLeotiaceae, Hyalodendriella Alternariol 9‐methyl ether (3) 135.52 μg/mL3 µ Alternariol 9‐methylAlternariol ether (3) 9-methyl135.52 ether μg/mL ( ) 135.52 g/mL Alternariol 9‐methyl ether (3) 135.52 μg/mL

Botrallin (4) 83.70 μg/mL Botrallin (4) Botrallin (4)83.70 μg/mL 83.70 µg/mL Botrallin (4) 83.70 μg/mL

Eumycota, Eumycota, EumycotaAscomycotina, , Eumycota, AscomycotinaHemiascomycetes, , BlastomycesAscomycotina sp. Ascomycotina, , Huperzine A 0.6 μM No report Ju et al. [38] HemiascomycetesEndomycetalcs, , BlastomycesHemiascomycetes sp. Hemiascomycetes, , Huperzine A 0.6 μM No report Ju et al. [38] Blastomyces sp. EndomycetalcsEndomycetaccae, , Huperzine A 0.6 μM No report Ju et al. [38] Blastomyces sp. Endomycetalcs, Huperzine A 0.6 µM No report Ju et al. [38] EndomycetalcsEndomycetaccaeBlastomyces, , BlastomycesEndomycetaccae , EndomycetaccaeBlastomyces, Molecules 2017, 22, 176 13 of 25 MoleculesBlastomyces 2017, 22, 176 13 of 25

11-Oxo-ganoderiol D (1) Lanosta-8-en-7,11-dioxo-3b-

11‐Oxo‐ganoderiolacetyloxy-24,25,26- D (1) Lanosta11‐Oxo‐‐ganoderiol8‐en‐7,11trihydroxy‐dioxo D (1)‐ 3b‐ (2) Lanosta‐8‐en‐7,11‐dioxo‐3b‐ acetyloxy‐24,25,26Lanosta-8-en-7-oxo-3b-‐trihydroxy (2) Lanostaacetyloxy‐8‐24,25,26en‐7‐oxo‐trihydroxy‐3b‐acetyloxy (2)‐ 11b,24,25,26Lanosta‐8‐en‐tetrahydroxy‐7acetyloxy-11b,24,25,26-‐oxo‐3b‐acetyloxy (3) ‐

Ganoderiol11b,24,25,26 D‐tetrahydroxy (tetrahydroxy10) (3) (3) LanostaGanoderiol‐8‐en D‐7 (Ganoderiol‐10one) ‐3b‐acetyloxy D‐ (10) Eumycota, 24,25,26Lanosta‐‐trihydroxy8‐en‐7‐one‐ (3b11‐)acetyloxy ‐ 24,25,26‐trihydroxyLanosta-8-en-7-one-3b- (11) Basidiomycotina, acetyloxy-24,25,26- Hymenomycetes, Modified Ellman’s Haddowia longipes trihydroxy (11) No report Zhang et al. [49] Aphyllophorales, method Ganodermataceae, Eumycota, Eumycota, OH Haddowia Basidiomycotina, OH Basidiomycotina, R2 Hymenomycetes, R Modified Ellman’s Zhang et al. Haddowia longipes Hymenomycetes, 2 No report Modified Ellman’s Zhang et al. Haddowia longipes Aphyllophorales, No report method [49] Aphyllophorales, R3 method [49] Ganodermataceae, R3 HaddowiaGanodermataceae , Haddowia Lanosta‐7,9(11)Lanosta-7,9(11)-dien-3b-‐dien‐3b‐acetyloxy‐ 24,25,26Lanosta‐‐trihydroxy7,9(11)acetyloxy-24,25,26-‐dien (‐43b) ‐acetyloxy‐ Ganodermanondiol24,25,26‐trihydroxy (412) ) R1 Ganodermanondioltrihydroxy (12) (4) R Lucidumol B (13) 1 Lucidumol B (13Ganodermanondiol) (12)

Lucidumol B (13)

11β‐Hydroxy‐lucidadiol (7) Lucidadiol11β‐Hydroxy (15‐lucidadiol) (7) Lucidadiol (15)

Molecules 2017, 22, 176 13 of 25

11‐Oxo‐ganoderiol D (1) Lanosta‐8‐en‐7,11‐dioxo‐3b‐ acetyloxy‐24,25,26‐trihydroxy (2) Lanosta‐8‐en‐7‐oxo‐3b‐acetyloxy‐ 11b,24,25,26‐tetrahydroxy (3) Ganoderiol D (10) Lanosta‐8‐en‐7‐one‐3b‐acetyloxy‐ 24,25,26‐trihydroxy (11)

Eumycota, OH Basidiomycotina, R Hymenomycetes, 2 Modified Ellman’s Zhang et al. Haddowia longipes No report Aphyllophorales, method [49] R3 Molecules 22 Ganodermataceae, 2017, , 176 Haddowia 12 of 24 Lanosta‐7,9(11)‐dien‐3b‐acetyloxy‐ 24,25,26‐trihydroxy (4) Ganodermanondiol (12) R 1 Lucidumol B (13) Table 2. Cont.

Strain Classification Structure of Active Ingredient(s) Compound Type IC50 Method Used Ref.

11β‐Hydroxy‐lucidadiol11β-Hydroxy-lucidadiol (7) (7) Lucidadiol (15Lucidadiol) (15)

MoleculesEumycota 2017, 22, 176 14 of 25 Basidiomycotina, Molecules 2017, 22, 176 14 of 25 MoleculesHymenomycetes 2017, 22, 176 , 14 of 25 Modified Ellman’s Haddowia longipes Molecules 2017, 22, 176 No report 14 of 25 Zhang et al. [49] Aphyllophorales, method Ganodermataceae, Haddowia Lucidone H (8)Lucidone H (8)

lucidadone A (lucidadone17) A (17) Lucidone H (8)

Lucidonelucidadone H A(8 )( 17) Lucidone H (8) lucidadone A (17) lucidadone A (17)

Bacteria, Bacteria, Firmicutes, TLC Pandey et al.; BacilliBacteria,, bioautography and BacillusFirmicutes subtilis , No clear product Wang et al. TLC bioautography Bacteria,Bacillales,Firmicutes , ModifiedTLC Ellman’s Pandey et al.; Bacteria, Pandey[50,51] et al.; Bacillus subtilis Bacilli, FirmicutesBacillaceaeBacilli, ,, No clear product bioautographymethodTLC and and Modified Bacillus subtilis Firmicutes, No clear product TLC PandeyWang et al.;al. Wang et al. [50,51] Bacillales, BacillaceaeBacilliBacillusBacillales,, , bioautographyModified Ellman’s and Pandey et al.; Ellman’s method Bacillus subtilis Bacilli, No clear product bioautography and Wang[50,51] et al. Bacillus subtilis Bacillales,Bacillaceae , No clear product Modifiedmethod Ellman’s Wang et al. Bacillus Bacillales, Modified Ellman’s [50,51] BacillaceaeBacillus , method [50,51] BacillusPhylumBacillaceae Actinobacteria, , method Streptomyces sp. ActinomycetalesBacillus , Modified Ellman’s Murao and 41 μM PhylumAH‐14 ActinobacteriaStreptomycetaceaePhylum Actinobacteria, , , method Hayashi [52] Streptomyces sp. StreptomycesActinomycetales sp. PhylumStreptomycesActinomycetales, Actinobacteria , , Modified Ellman’s Murao and Modified Ellman’s Murao and Phylum Actinobacteria, Physostigmine 41 μM StreptomycesAH‐14 sp. ActinomycetalesStreptomycetaceae, , PhysostigmineModifiedmethod Ellman’s 41 µMHayashiMurao and [52] AH-14 StreptomycesStreptomycetaceae sp. Actinomycetales, , Physostigmine 41 μM Modified Ellman’s Murao and method Hayashi [52] AH‐14 StreptomycetaceaeStreptomyces , Physostigmine 41 μM method Hayashi [52] StreptomycesAH‐14 StreptomycesStreptomycetaceae , method Hayashi [52] Streptomyces

Phylum Actinobacteria, Streptomyces sp. Actinomycetales, Modified Ellman’s Ohlendorf Geranylphenazinediol 2.62 μM PhylumLB173 ActinobacteriaStreptomycetaceaePhylum Actinobacteria, , , method et al. [53] Streptomyces sp. PhylumStreptomycesActinomycetales Actinobacteria , , Modified Ellman’s Ohlendorf Phylum Actinobacteria, Geranylphenazinediol 2.62 μM Streptomyces sp. StreptomycesActinomycetalesLB173 sp. ActinomycetalesStreptomycetaceae, , , Modifiedmethod Ellman’s Ohlendorfet al. [53] Modified Ellman’s Streptomyces sp. Actinomycetales, GeranylphenazinediolGeranylphenazinediol 2.62 μM Modified Ellman’s 2.62 µMOhlendorf Ohlendorf et al. [53] LB173 StreptomycetaceaeLB173 StreptomycetaceaeStreptomyces, , Geranylphenazinediol 2.62 μM method et al. [53] method LB173 StreptomycesStreptomycetaceae , method et al. [53] StreptomycesStreptomyces

Phylum Actinobacteria, Streptomyces Actinomycetales, Modified Ellman’s Kurokawa et Oxygen heterocyclic compound 7.6 μM lavendulae StreptomycetaceaePhylum Actinobacteria, , method al. [54] Streptomyces PhylumStreptomycesActinomycetales Actinobacteria , , Modified Ellman’s Kurokawa et Phylum ActinobacteriaPhylum Actinobacteria, , Oxygen heterocyclic compound 7.6 μM Streptomyceslavendulae ActinomycetalesStreptomycetaceae, , Modifiedmethod Ellman’s Kurokawaal. [54] et Streptomyces StreptomycesActinomycetales Actinomycetales, , Oxygen heterocyclicOxygen compound heterocyclic 7.6 μM Modified Ellman’s Kurokawa et Modified Ellman’s lavendulae StreptomycetaceaeStreptomyces , Oxygen heterocyclic compound 7.6 μM method 7.6 µM al. [54] Kurokawa et al. [54] lavendulae lavendulaeStreptomycetaceae StreptomycesStreptomycetaceae, , compound method al. [54] method Streptomyces Streptomyces

Molecules 2017, 22, 176 13 of 24

Table 2. Cont.

Molecules 2017, 22, 176 15 of 25 StrainMolecules 2017 Classification, 22, 176 Structure of Active Ingredient(s) Compound Type IC50 15 of 25 Method Used Ref. Molecules 2017, 22, 176 15 of 25 Molecules 2017, 22, 176 15 of 25 Molecules 2017, 22, 176 15 of 25

2‐(2‐(3‐Hydroxy2-(2-(3-Hydroxy-1-(1‐1‐(1H‐indol‐3‐yl)‐2‐ H-indol-3-yl)-2- methoxypropyl)2‐(2‐(3‐Hydroxymethoxypropyl)-1‐1H‐(1‐indolH‐indol‐3‐yl)acetic‐3‐yl)‐2‐ H-indol-3-yl)acetic11.8 μM 11.8 µM acid2methoxypropyl)‐(2‐ (3‐Hydroxy‐1‐H(1‐indolH‐indol‐3‐‐yl)acetic3‐yl)‐2‐ 11.8 μM 2‐(2‐(3‐Hydroxy‐1‐(1H‐indol‐3‐yl)‐2‐ acidmethoxypropyl) acid‐1H‐indol‐3‐yl)acetic 11.8 μM 2‐(2‐(3‐Hydroxy‐1‐(1H‐indol‐3‐yl)‐2‐ Phylum Actinobacteria, methoxypropyl)acid ‐1H‐indol‐3‐yl)acetic 11.8 μM methoxypropyl)acid ‐1H‐indol‐3‐yl)acetic 11.8 μM RubrobacteridaePhylum, Actinobacteria, acid Rubrobacter RubrobacteridaePhylum Actinobacteria, , Modified Ellman’s Rubrobacter Modified Ellman’s RubrobacteralesRubrobacteralesPhylumRubrobacteridae, Actinobacteria,, , Li et al. [55] Li et al. [55] radiotolerans radiotoleransRubrobacter Phylum Actinobacteria, Modifiedmethod Ellman’s method Rubrobacteraceae,Rubrobacteraceae,RubrobacteridaeRubrobacterales,, Li et al. [55] radiotoleransRubrobacter PhylumRubrobacteridae Actinobacteria, , Modifiedmethod Ellman’s Rubrobacter RubrobacterRubrobacteralesRubrobacteraceae, , Modified Ellman’s Li et al. [55] radiotoleransRubrobacter RubrobacteridaeRubrobacterales,, method Li et al. [55] Rubrobacter Rubrobacteraceae,Rubrobacter Modified Ellman’s radiotolerans Rubrobacterales, method Li et al. [55] radiotolerans Rubrobacteraceae,Rubrobacter method Rubrobacteraceae,Rubrobacter 3‐(3‐(2‐Hydroxyethyl)‐1H‐indol‐2‐yl)‐ Rubrobacter 13.5 μM 3‐(1Hindol(3‐(2‐Hydroxyethyl)‐3‐yl)propane3-(3-(2-Hydroxyethyl)-1‐1H‐1,2‐indol‐diol‐2 ‐yl)‐ H-indol-2-yl)- 3‐(3‐(2‐Hydroxyethyl)‐1H‐indol‐2‐yl)‐ 13.5 μM 13.5 µM 3‐(1Hindol‐3‐yl)propane3-(1Hindol-3-yl)propane-1,2-diol‐1,2‐diol 13.5 μM 3‐(3(1Hindol‐(2‐Hydroxyethyl)‐3‐yl)propane‐1H‐1,2‐indol‐diol‐2 ‐yl)‐ 3‐(3‐(2‐Hydroxyethyl)‐1H‐indol‐2‐yl)‐ 13.5 μM 3‐(1Hindol‐3‐yl)propane‐1,2‐diol 13.5 μM 3‐(1Hindol‐3‐yl)propane‐1,2‐diol

Phylum Actinobacteria, Modified Ellman’s Dong et al. N98‐1021 Phylum Actinobacteria, Same structure as terferol 20 μM Modified Ellman’s Dong et al. N98‐1021 Actinomycetales Same structure as terferol 20 μM method [56] Phylum ActinobacteriaPhylum Actinobacteria, , Modified Ellman’s Dong et al. Modified Ellman’s N98‐1021 Actinomycetales Same structure as terferol 20 μM method [56] N98-1021 PhylumActinomycetales Actinobacteria , Same structure as terferolModifiedmethod Ellman’s 20 µ M Dong[56] et al. Dong et al. [56] ActinomycetalesN98‐1021 Phylum Actinobacteria, Same structure as terferol 20 μM Modified Ellman’s Dong et al. method N98‐1021 Actinomycetales Same structure as terferol 20 μM method [56] Actinomycetales method [56]

YXJ‐E1 NovelYXJ‐E1 compound extracted from No report secondaryYXJNovel‐E1 compound metabolites extracted of from YXJ‐E1 YXJ-E1 No report Phylum Actinobacteria, ActinobacteriaNovelsecondary compound metabolites for the extracted first of time from YXJNovel‐E1 compound extracted from No report Streptosporangium ActinomycetalesPhylum Actinobacteria, , secondaryActinobacteria metabolites forNovel the first of compound time extractedNo report Modified Ellman’s Yang et al. Novelsecondary compound metabolites extracted of from No report Streptosporangiumsp. StreptomycetaceaePhylumActinomycetales Actinobacteria, , , Actinobacteria forfrom the first secondary time metabolitesNo report of Modifiedmethod Ellman’s Yang[57] et al. Phylum Actinobacteria, secondaryActinobacteria metabolites for the first of time StreptosporangiumPhylumsp. Actinobacteria StreptosporangiumActinomycetalesStreptomycetaceae, , , Modifiedmethod Ellman’s Yang[57] et al. Streptosporangium PhylumActinomycetales Actinobacteria, , Actinobacteria forActinobacteria the first time for the first time Modified Ellman’s Yang et al. sp. StreptomycetaceaeStreptosporangium, method [57] StreptosporangiumActinomycetales Actinomycetales, , Modified Ellman’s Yang et al. Modified Ellman’s Streptosporangium sp. sp. StreptomycetaceaeStreptosporangium, method [57] Yang et al. [57] Streptomycetaceaesp. Streptomycetaceae, , 7,4′‐Dihydroxy flavone No report method [57] Streptosporangium 7,4′‐Dihydroxy flavone No report method StreptosporangiumStreptosporangium 7,4′‐Dihydroxy flavone No report 7,4′‐Dihydroxy flavone No report 7,4′‐Dihydroxy7,4 flavone0-Dihydroxy flavoneNo report No report

Molecules 2017, 22, 176 14 of 24

Table 2. Cont.

Molecules 2017, 22, 176 16 of 25 StrainMolecules 2017 Classification, 22, 176 Structure of Active Ingredient(s) Compound Type IC50 16 of 25 Method Used Ref. Molecules 2017, 22, 176 16 of 25

Phylum Actinobacteria, Phylum Actinobacteria, TalaromycesActinomycetales sp. , Modified Ellman’s Modified Ellman’s PhylumActinomycetales Actinobacteria, , Talaromycesone A 7.49 μM µ Wu et al. [58] Talaromyces sp. LF458 TalaromycesLF458 sp. Phylum Actinobacteria, Talaromycesone AModifiedmethod Ellman’s 7.49 M Wu et al. [58] TalaromycesArthrobacter sp. ActinomycetalesArthrobacter Talaromyces, Talaromycesone A 7.49 μM Modified Ellman’s Wu et al. [58] method LF458 Actinomycetales, Talaromycesone A 7.49 μM method Wu et al. [58] LF458 Arthrobacter Talaromyces method Talaromyces Arthrobacter Talaromyces

Lichenes, BiruloquinoneBiruloquinone Cladonia macilenta Lichenes,Ascolichens , Modified Ellman’s Lichenes, AscolichensLichenes, , Biruloquinone9,10‐Phenanthrene quinone, a rare 27.1 μg/mL Luo et al. [59] Cladonia macilenta CladoniaHoffm. macilenta AscolichensCladoniaceae, , Biruloquinone9,10-Phenanthrene quinone, Modifiedmethod Ellman’s Modified Ellman’s CladoniaCladoniaceae macilenta ,Ascolichens, 9,10natural‐Phenanthrene quinone compound quinone, a rare 27.1 μg/mL Modified Ellman’s27.1 µ g/mLLuo et al. [59] Luo et al. [59] Hoffm. CladoniaceaeCladonia , 9,10‐Phenanthrene quinone, a rare 27.1 μg/mL method Luo et al. [59] Hoffm. Hoffm. Cladoniaceae, natural quinonea compound rare natural quinone method method Cladonia Cladonia natural quinone compound Cladonia compound

Cyanophyta, Cyanophyta,Nostocales, Modified Ellman’s Becher et al. NostocCyanophyta, 78‐12A Cyanophyta, Nostocarboline 5.3 μM NostocalesNostocaceae, , Modifiedmethod Ellman’s Becher[60] et al. NostocNostocales 78‐12A , Nostocales, Nostocarboline 5.3 μM Modified Ellman’s Becher et al. Modified Ellman’s Nostoc 78-12A Nostoc 78‐12A NostocaceaeNostoc , NostocarbolineNostocarboline 5.3 μM method 5.3 µM [60] Becher et al. [60] Nostocaceae, NostocaceaeNostoc , method [60] method Nostoc Nostoc

Molecules 2017, 22, 176 15 of 24

The active ingredients of fermentation products mainly include alkaloids (50% of all active ingredients), phenylpropanoids, terpenoids, and steroids. The active ingredients of two strains have not been successfully isolated. Out of the 12 Deuteromycotina strains, seven produce alkaloids (six of which produce huperzine A), four produce terpenoids or steroids, one produces multiple types of active ingredients, and one produces unknown active ingredients. Out of the 11 Ascomycotina strains, five produce alkaloids (three of which produce huperzine A), four produce phenylpropanoid derivatives, and two produce terpenoid derivatives. Out of 11 strains of bacteria, six produce alkaloids (two of which produce huperzine A), two produce phenylpropanoid derivatives, two produce other types of compounds, and one produces unknown active ingredients.

3.1. AChEIs from Fungi As shown in Table2, 20 AchEI producing fungi include Aspergillus terreus, Aspergillus terreus (No. GX7-3B), Acremonium implicatum LF30, Acremonium sp. 2F09P03B, Aspergillus flavus LF40, Botrytis sp. HA23, Cladosporium cladosporioides LF70, Paecilomyces tenuis YS-13, Alternaria sp. YD-01, Aspergillus flavus, Phomopsis sp., Aspergillus versicolor Y10, Trichoderma sp., Chrysosporium sp., Penicillium citrinum, Penicillium sp. EPF-6, Penicillium sp. FO-4259, Penicillium sp. sk5GW1L, Penicillium chermesinum ZH4-E2, Xylaria sp., Hyalodendriella sp. Ponipodef12, Penicillium griseofulvum LF146, Shiraia sp. Slf14, and Xylaria sp. YS-02. These fungi belong to the Deuteromycotina and Ascomycota phyla subdivisions, among which five stains belong to Penicillium, accounting for 25% of the studied strains (Table2).

3.1.1. Alkaloid AChEIs and the Producing Fungi Huperzine A is the most studied alkaloid AchEI originating from fungi. The nine strains shown to produce huperzine A by fermentation account for 39% of all strains studied (Table2). Huperzine A possesses a multitude of pharmacological effects. In addition to being a highly selective and efficient AchEI in the central nervous system, huperzine A can also provide protection for cells by inhibiting excitotoxicity induced by glutamate and by blocking oxidative stress and apoptosis. Additionally, huperzine A may exert therapeutic effects in a variety of degenerative diseases, including myasthenia gravis, memory disorders, vascular dementia, mental retardation in children due to iodine deficiency, and chronic insomnia. In recent years, several research groups in China have isolated and screened several huperzine A-producing endophytic fungal strains from Huperziaceae and determined the product quantities of each strain [27,35–38,40,44,45,61–63]. The productivity of most strains is less than 60 µg/L, with Shiraia sp. Slf14 being the best producer (327.8 µg/L of the fermentation product) [44]. The optimal culturing conditions and molecular properties have been set for this strain [27,44,64–66]. Li et al. [37] optimized the fermentation conditions for huperzine A production in endophytic fungi through both one-factor and orthogonal tests. Additionally, based on a model system two huperzine A-producing endophytic fungal strains, Xylaria sp. YS-02 and Paecilonmyces tennis YS-13, with production levels of 26.4 and 21.0 µg/L, were identified, respectively, through screening 127 isolated endophytic fungus strains in three Huperziaceae (Phlegmariurus austrosinicus, Phlegmariurus petiolatus, and Huperzia serrata). The two strains producing huperzine A by fermentation were reported for the first time. Kakinuma et al. [31] isolated the strain Penicillium sp. EPF-6, which produces quinolactacins A, B, and C, from the larvae of Diaphania pyloalis (Lepidoptera: Pyralidae) for the first time. These three types of quinolactacins share a unique quinolone backbone and a common chromophore (C12H8N2O2). The quinolactacins A showed inhibitory activity on TNF production by murine macrophages and macrophage-like J774.1 cells stimulated with LPS. The mixture of quinolactacins A and B, as well as racemate quinolactacin C, inhibited the yeast α-glucosidase in a concentration-dependent fashion with IC50 values of 273.3 µM and 57.3 µM[67]. Molecules 2017, 22, 176 16 of 24

Kim et al. [30] isolated quinolactacins A1 and A2 from the fermentation solid of Penicillium citrinum. Structural analysis revealed that the chemical structure of quinolactacin A1 is identical to that of quinolactacin A and that the structure of quinolactacin A2 is identical to that of quinolactacin B. MoleculesQuinolactacin 2017, 22, x A1 FOR is PEER a diastereomer REVIEW of quinolactacin A2. Kim et al. [30] used Ellman’s method18 of 27 to detectrmine the AchEI activity of quinolactacins A1 and A2 and showed that quinolactacin exhibitedA2 exhibited stronger stronger AchEI AchEI activity activity (IC (IC50 =50 19.8= 19.8 μM).µM). Based Based on on the the “OSMAC” “OSMAC” (one (one strain-many compounds)compounds) technique, technique, Teles Teles et al. [68] [68] isolated and purified purified paecilomide, a a pyridone pyridone alkaloid, alkaloid, from from Paecilomyces lilacinus lilacinus usingusing a a combination combination of of the the modified modified E Ellman’sllman’s method method and and TLC TLC bioautography. bioautography. The acetylcholinesterase inhibition efficiency efficiency of paecilomide is 57.5% ±± 5.50%5.50% at at the the dose dose of of 10 10 mg/mL.

3.1.2.3.1.2. Terpenoid Terpenoid AChEIs AChEIs and and the the Producing Producing Fungi Fungi TerpenoidTerpenoid AChEIs extracted from from fungi are are mostly diterpenoids diterpenoids and and meroterpenoids, meroterpenoids, respectively. respectively. LingLing et et al. al. [25] [25 ]isolated isolated a astrain strain of ofAspergillusAspergillus terreus terreus fromfrom unhusked unhusked rice (paddy) rice (paddy) and obtained and obtained three diterpenoidthree diterpenoid compounds, compounds, i.e., territrem i.e., territrem A, territrem A, territrem B, and territrem B, and territrem C, from the C, fromfermentation the fermentation products (Figureproducts 1). (Figure Peng et1 ).al. Peng[69] confirmed et al. [ 69 that] confirmed five derivatives that five of derivativesterritrem B possess of territrem the AchEI B possess activity. the UsingAchEI AchEI activity. assays, Using Peng AchEI et al. assays, [69] confirmed Peng et al.that [ 69territrem] confirmed B is able that to territrem inhibit acetylcholinesterase B is able to inhibit (ICacetylcholinesterase50 = 7.6 μM). (IC50 = 7.6 µM). AsAs early early as as 1995, 1995, researchers researchers at at Taiwan Taiwan University University initiated initiated screens screens for for mi microbe-originatedcrobe-originated AchEI, AchEI, andand Omura et al. [32], [32], in Japan, reported a series of Arisugacins compounds (F (Figureigure 11)) thatthat effectivelyeffectively inhibitinhibit the the acetylcholinesterase acetylcholinesterase activity. activity. Arisugacins Arisugacins are shown are shown to be produced to be produced by Penicillium by Penicillium sp. FO-4259sp. isolatedFO-4259 from isolated soil fromin the soil Minato-ku in the Minato-ku region of region Tokyo, of Tokyo,Japan. Arisugacins Japan. Arisugacins A and AB andshare B the share same the backbonesame backbone with the with territrems the territrems reported reported by Ling by et Lingal. (1984) et al. [70]. (1984) This [ 70implies]. This that implies the same that backbone the same ofbackbone these compounds of these compoundshas much effect has in much mediating effect their in mediating anti-AChE their activity. anti-AChE Moreover, activity. Huang Moreover, et al. [33] extractedHuang et a al. strain [33] extractedof Penicillium a strain sp., of sk5GW1L,Penicillium fromsp., Kandelia sk5GW1L, candel, from Kandeliafrom which candel, three from terpenoid which compounds,three terpenoid i.e., compounds, arigsugacin i.e.,I, arigsugacin arigsugacin F, I, and arigsugacin territrem F, B and (Figure territrem 1), were B (Figure isolated1), wereand purified; isolated theseand purified; compounds these exhibited compounds prominent exhibited AchEI prominent activity, AchEI with activity, IC50 values with ICof50 0.64,values 0.37, of 0.64,and 7.03 0.37, μ andM, respectively.7.03 µM, respectively.

FigureFigure 1. 1. StructuresStructures of of the the arisugacins arisugacins A–H, A–H, te territremsrritrems A–C, A–C, and and terreulactones C and D.

FromFrom fermentation products products of a a AspergillusAspergillus terreus strain isolated isolated from from soil, soil, Cho Cho et et al. al. [71] [71] were were ableable to isolate and purifypurify fourfour polyterpenoidpolyterpenoid compoundscompounds with with polyketide polyketide structures structures (terreulactones (terreulactones A Aand and B B FigureFigure 22)) that that show show the the AChEI AChEI activity. activity. Terreulactone Terreulactone A is a meroterpenoid A is a meroterpenoid with a sesquiterpene with a lactone structure consisting of a sesquiterpene portion and a lactone backbone; the IC50 of terreulactone A is 0.23 μM. In 2005, Yoo et al. [72] also reported a new meroterpenoid (isoterreulactone A; Figure 3) isolated from the fermentation products of Aspergillus terreus, with an IC50 of 2.5 μM. Terreulactone C shares the same backbone with arisugacins C–H and has an IC50 of 0.23 μM. Terreulactone D shares the same backbone as arisugacins A and B, and territrems A–C, and its IC50 is 0.42 μM. In 2014, Kim et al. [73] obtained a strain of Streptomyces sp. from screens, and anithiactins A–C were isolated and purified from the metabolites. All three compounds were shown to have moderate AChEI effects,

Molecules 2017, 22, 176 17 of 24 sesquiterpene lactone structure consisting of a sesquiterpene portion and a lactone backbone; the IC50 of terreulactone A is 0.23 µM. In 2005, Yoo et al. [72] also reported a new meroterpenoid (isoterreulactone A; Figure3) isolated from the fermentation products of Aspergillus terreus, with an IC50 of 2.5 µM. Terreulactone C shares the same backbone with arisugacins C–H and has an IC50 of 0.23 µM. Terreulactone D shares the same backbone as arisugacins A and B, and territrems A–C, and its IC50 is 0.42 µM. In 2014, Kim et al. [73] obtained a strain of Streptomyces sp. from screens, Molecules 2017, 22, x FOR PEER REVIEW 19 of 27 and anithiactins A–C were isolated and purified from the metabolites. All three compounds were andshown the to IC have50 for moderatethese three AChEI compounds effects, are and 63, the 53, IC andfor 68 these μM. threeThe anithiactins compounds A–C are 63,did 53,not and show 68 anyµM. and the IC50 for these three compounds are 63, 53, and50 68 μM. The anithiactins A–C did not show any significantThe anithiactins cytotoxicity A–C did against not show A-498 any and significant ACHN human cytotoxicity cancer against cell, at A-498concentrations and ACHN of lines human up cancerto 200 µ μcell,M with at concentrations a modified MTT of lines assay. up The to 200analysisM withof correla a modifiedtion of acetylcholinesterase MTT assay. The analysis activity of correlationand chemical of α structureacetylcholinesterase indicated that activity α-Pyrone and merosesquiterpenoids chemical structure indicated possessing that an angular-Pyrone tetracyclic merosesquiterpenoids carbon skeleton arepossessing frequently an angularisolated tetracyclic from the carbon genera skeleton Penicillium are frequentlyand Aspergillus isolated as from anti-cholinesterase the genera Penicillium active constituentsand Aspergillus [74].as anti-cholinesterase active constituents [74].

Figure 2. StructuresStructures of the terreulactones A and B.

Figure 3. Structure of isoterreulactone A. Figure 3. Structure of isoterreulactone A. 3.1.3. Other Types of AChEIs from Fungi 3.1.3. Other Types Types of of AChEIs AChEIs from from Fungi Fungi Marine microorganisms which can be produced in a large quantity have also become a nature Marine microorganisms which can be produced in a large quantity have also become a nature source used for isolating AChEIs. Lin et al. [46] and Luo et al. [75] isolated a strain of Xylaria sp. from source used used for for isolating isolating AChEIs. AChEIs. Lin Lin et etal. al.[46] [46 and] and Luo Luo et al. et [75] al. [isolated75] isolated a strain a strain of Xylaria of Xylaria sp. fromsp. mangrove; five structurally similar oxygen heterocyclic compounds xyloketals A–E were further mangrove;from mangrove; five structurally five structurally similar similar oxygen oxygen hete heterocyclicrocyclic compounds compounds xyloketals xyloketals A–E A–E were were further isolated from the fermentation broth. The xyloketals A–E have a highly substituted core aromatic isolated fromfrom thethe fermentation fermentation broth. broth. The The xyloketals xyloketals A–E A–E have have a highly a highly substituted substituted core core aromatic aromatic ring ring and one or more ketals. Using the modified Ellman’s method, xyloketals A–D are shown the ringand oneand orone more or more ketals. ketals. Using Us theing modified the modified Ellman’s Ellm method,an’s method, xyloketals xyloketals A–D are A–D shown are shown the AChEI the AChEI activity in vitro, with IC50 values of 29.9, 137.4, 109.3, and 425.6 μM. Analysis of enzyme kinetic AChEI activity in vitro, with IC50 values of 29.9, 137.4, 109.3, and 425.6 μM. Analysis of enzyme kinetic activity in vitro, with IC50 values of 29.9, 137.4, 109.3, and 425.6 µM. Analysis of enzyme kinetic curves curves has indicated that xyloketals A–D are a type of highly selective, noncompetitive, reversible has indicated that xyloketals A–D−/− are a type of highly selective, noncompetitive, reversible AChEIs. AChEIs. In atherosclerotic apoE−/− mice, xyloketal B attenuated the atherosclerotic lesion area, which showed neuroprotective and scavenged DPPH free radical activities [76]. Liu et al. [77] isolated 43 strains of marine fungi from seabed sediments in Lianyungang, China. From them, the ethyl acetate extracts were made with Ellman’s method. Of these 43 strains, 15 extracts display 50% AChEI activity and 3 extracts (L1705, S1101 and SH0701) with more than 80% AChEI activity at the concentration of 500 mg/mL, with IC50 values of 11.3 ± 1.2, 72.1 ± 2.3 and 7.8 ± 2.8 mg/mL, respectively. Particularly, the 500 mg/mL, with IC50 values of 11.3 ± 1.2, 72.1 ± 2.3 and 7.8 ± 2.8 mg/mL, respectively. Particularly, the fungus SH0701 was identified as Aspergillus ochraceus SH0701. After polarity gradient fractionation, the results showed that the ethyl acetate fraction of Aspergillus ochraceus SH0701 possessed the highest

Molecules 2017, 22, 176 18 of 24

In atherosclerotic apoE−/− mice, xyloketal B attenuated the atherosclerotic lesion area, which showed neuroprotective and scavenged DPPH free radical activities [76]. Liu et al. [77] isolated 43 strains of marine fungi from seabed sediments in Lianyungang, China. From them, the ethyl acetate extracts were made with Ellman’s method. Of these 43 strains, 15 extracts display 50% AChEI activity and 3 extracts (L1705, S1101 and SH0701) with more than 80% AChEI activity at the concentration of 500 mg/mL, with IC50 values of 11.3 ± 1.2, 72.1 ± 2.3 and 7.8 ± 2.8 mg/mL, respectively. Particularly, Moleculesthe fungus 2017 SH0701, 22, x FOR was PEER identified REVIEW as Aspergillus ochraceus SH0701. After polarity gradient fractionation,20 of 27 the results showed that the ethyl acetate fraction of Aspergillus ochraceus SH0701 possessed the highest AChEI activity with an inhibitioninhibition rate ofof 71.55%71.55% atat aa concentrationconcentration ofof 1010 mg/mL.mg/mL. Thus, the above results revealed that marine microorganisms couldcould bebe anotheranother importantimportant sourcesource ofof novelnovel AChEIs.AChEIs. Additionally, researchers researchers have have isolated isolated fungi fungi from from soil soil and and plants, plants, and and obtained obtained a multitude a multitude of compoundsof compounds with with the AChEI the AChEI activity activity from fromfermentation fermentation products. products. For example, For example, Rao et al. Rao [43] et reported al. [43] thereported isolation the of isolation a strain of of Chrysosporium a strain of Chrysosporium sp. from the fermentationsp. from the brot fermentationh, from which broth, an AchEI from (14-(2 which′,3′,5 an′- AchEItrihydroxyphenyl)tetradecan-2-ol) (14-(20,30,50-trihydroxyphenyl)tetradecan-2-ol) was extracted. was extracted. Notably, asas aa aromaticaromatic alkylalkyl compound,compound, 14-(214-(20′,3,30′,5,50′-trihydroxyphenyl)tetradecan-2-ol was shown to have an IC 50 ofof 231 231 μµMM as as measured measured by by a a modified modified method method of of Ellman’s. Ellman’s. Moreover, Moreover, Zheng et al. [78] [78] isolated a fungal strain from the soil of of Yunnan, Yunnan, China. The fermentation products of this strain were shown to contain an AchEI (F99-909A; Figure 44)) withwith anan ICIC5050 of 20 μµM using the modifiedmodified Ellman’s method. Chemical structure analysis revealed that this compound has a spatial structure identical to that of radclonic acid, an active compound with stimulatorystimulatory effectseffects onon plantplant growth.growth.

Figure 4. Structure of the compound F99-909A.

Meng et al. (2012) [47] and Mao et al. [48] isolated an endophytic fungal strain Hyalodendriella sp. Meng et al. (2012) [47] and Mao et al. [48] isolated an endophytic fungal strain Hyalodendriella sp. Ponipodef12 from Populus cathayana and screened its metabolites to obtain four benzopyran ketone Ponipodef12 from Populus cathayana and screened its metabolites to obtain four benzopyran ketone compounds: palmariol B, 4-hydroxymellein, alternariol 9-methyl ether, and botrallin. Using the modified compounds: palmariol B, 4-hydroxymellein, alternariol 9-methyl ether, and botrallin. Using the Ellman’s method, these compounds were shown to have IC50 values of 115.31, 116.05, 135.52, and modified Ellman’s method, these compounds were shown to have IC values of 115.31, 116.05, 135.52, 83.70 μg/mL. This was the first report on the antinematodal and AChEI50 activities of palmariol B, and 83.70 µg/mL. This was the first report on the antinematodal and AChEI activities of palmariol B, 4-hydroxymellein and alternariol 9-methyl ether. The experimental results showed that palmariol B 4-hydroxymellein and alternariol 9-methyl ether. The experimental results showed that palmariol B had stronger antimicrobial, antinematodal and AChEI activities than alternariol 9-methyl ether, which had stronger antimicrobial, antinematodal and AChEI activities than alternariol 9-methyl ether, which indicated that the chlorine substitution at position 2 may contribute to its bioactivity. As the same indicated that the chlorine substitution at position 2 may contribute to its bioactivity. As the same endophytic fungus Hyalodendriella sp. Ponipodef12, Mao et al. [48] described the isolation and applied endophytic fungus Hyalodendriella sp. Ponipodef12, Mao et al. [48] described the isolation and applied high-speed counter-current chromatography (HSCCC) and semi-preparative HPLC technology for high-speed counter-current chromatography (HSCCC) and semi-preparative HPLC technology for preparative separation of dibenzo-a-pyrones from the fungus for the first time. preparative separation of dibenzo-a-pyrones from the fungus for the first time. Chapla et al. [42,79] screened and isolated two endophytic fungal strains Phomopsis sp. and Chapla et al. [42,79] screened and isolated two endophytic fungal strains Phomopsis sp. and Colletotrichum gloeosporioides from Senna spectabilis and Michelia champaca, respectively. Seven compounds, Colletotrichum gloeosporioides from Senna spectabilis and Michelia champaca, respectively. Seven including cytochalasin H, were isolated and purified from the fermentation broth of Phomopsis. compounds, including cytochalasin H, were isolated and purified from the fermentation broth of Cytochalasin H has strong inhibitory activity against acetylcholinesterase in vitro, as shown by TLC Phomopsis. Cytochalasin H has strong inhibitory activity against acetylcholinesterase in vitro, as shown bioautography. The results suggested that C. gloeosporioides plays an important ecological role in by TLC bioautography. The results suggested that C. gloeosporioides plays an important ecological role protecting M. champaca against phytopathogens. The potent antifungal activity of the novel natural in protecting M. champaca against phytopathogens. The potent antifungal activity of the novel natural product cytochalasin H, which demonstrated activity against pathogenic fungi, suggested that the product cytochalasin H, which demonstrated activity against pathogenic fungi, suggested that the endophyte and the host plant can produce substances that show antifungal activity against possible endophyte and the host plant can produce substances that show antifungal activity against possible phytopathogenic fungi or that are harmful to predators of the plant. phytopathogenic fungi or that are harmful to predators of the plant. Wang et al. [29] isolated Aspergillus versicolor Y10 from Huperzia serrata and purified five compounds Wang et al. [29] isolated Aspergillus versicolor Y10 from Huperzia serrata and purified five from its fermentation products. Using huperzine A as the positive reference (IC50 = 0.6 μM), the AchEI compounds from its fermentation products. Using huperzine A as the positive reference IC50 of the purified compound avertoxin B was shown to be 14.9 μM with the modified Ellman’s method. (IC50 = 0.6 µM), the AchEI IC50 of the purified compound avertoxin B was shown to be 14.9 µM with 3.2.the modifiedAChEIs from Ellman’s Bacteria method. From the 1980s to the 1990s, while the world was focusing on the development of plant and chemical synthetic AChEIs, Murao et al. [52] isolated a physostigmine-producing strain of Streptomyces sp. AH-14 from Sakai’s soil in Japan for the first time. Additionally, Ohlendorf et al. [53] obtained a new phenazine-derived natural product, geranylphenazinediol, from the fermentation products of Streptomyces sp. isolated from the seabed sediment. This compound shows strong AchEI activity with an IC50 value of 2.62 μM when analyzed using the modified Ellman’s method. Kurokawa et al. [54] reported the isolation of an oxygen heterocycle compound, cyclophostin, from Streptomyces lavendulae. In vitro activity assays demonstrated that cyclophostin possesses high AchEI activity, with an IC50 of 7.6 μM as measured by the modified Ellman’s method. Additionally,

Molecules 2017, 22, 176 19 of 24

3.2. AChEIs from Bacteria From the 1980s to the 1990s, while the world was focusing on the development of plant and chemical synthetic AChEIs, Murao et al. [52] isolated a physostigmine-producing strain of Streptomyces sp. AH-14 from Sakai’s soil in Japan for the first time. Additionally, Ohlendorf et al. [53] obtained a new phenazine-derived natural product, geranylphenazinediol, from the fermentation products of Streptomyces sp. isolated from the seabed sediment. This compound shows strong AchEI activity with an IC50 value of 2.62 µM when analyzed using the modified Ellman’s method. Kurokawa et al. [54] reported the isolation of an oxygen heterocycle compound, cyclophostin, from Streptomyces lavendulae. In vitro activity assays demonstrated that cyclophostin possesses high AchEI activity, with an IC50 of 7.6 µM as measured by the modified Ellman’s method. Additionally, from the metabolites of Actinobacillus N98-1021, Dong et al. [56] isolated the compound N98-1021A, which exhibits the AchEI activity using the modified Ellman’s method and shows structural similarity to terferol. Moreover, Pandey et al. [50] extracted a Bacillus subtilis strain-M18SP4P from Fasciospongia cavernosa and showed that this strain blocks 54% of acetylcholinesterase activity. Using galanthamine as a positive reference, TLC bioautography identified two components in the metabolites of M18SP4P with the AchEI activity. This was the first report on the AChE inhibition activity in the microbial associates of the marine invertebrates and sediment. Li et al. [55] isolated and purified two compounds from the marine actinomycete Rubrobacter radiotolerans. Both of these compounds show moderate AchEI activity as measured by the modified Ellman’s method, with IC50 values of 11.8 and 13.5 µM, respectively. Wu et al. [58] obtained nine compounds from the fermentation broth and mycelia of a Talaromyces sp. strain LF458. Of these nine compounds, talaromycesone A, talaroxanthenone, and AS-186c showed moderate inhibitory activities using the modified Ellman’s method, with IC50 values of 7.49, 1.61, and 2.60 µM, respectively. This study first demonstrated the AchEI activity of oxaphenalenone.

3.3. AChEIs from Other Species Additional AchEI-producing species include Cladonia macilenta and Haddowia longipes. Luo et al. [59] isolated and purified a rare natural phenanthrenequinone compound (biruloquinone) from the fermentation broth of Cladonia macilenta. Biruloquinone has strong AchEI activity as measured by the modified Ellman’s method, with an IC50 of 27.1 µg/mL. From the ethyl acetate extracts of Haddowia longipes, Zhang et al. [49] isolated nine lanosteroids and nine known compounds. Using as a positive reference, 100 µM test samples were prepared from each compound. Of the 18 compounds, 13 exhibited the AchEI activity, with inhibition efficiencies between 10.3% and 42.1%, which was assayed by the spectrophotometric method developed by Ellman. The inhibition efficiency of tacrine is 65.8%. In coincidence with the structure-activity analyses, the compound 4 among these lanostanoids displayed stronger AChEI activities than compound 5, which suggest an important role of the methylation of hydroxyl group substituted in C-25 of the lanostanoid backbone. At the same time, compound 8 had significantly higher activity than compounds 16 and 17, which indicating that the hydroxyl group at C-15 was required and seemed more important for activity comparing the hexa-nor lanostanoids with the same skeleton.

4. Conclusions As the clinical demand for AChEIs continues to increase, in vitro assays for AChEIs are also undergoing rapid development, particularly with the development of Ellman’s method, which allows for in vitro high-throughput screening. The development of the modified Ellman’s method, TLC bioautography, combined LC-MS and modified Ellman’s method, and other methods has significantly improved isolation and screening techniques for AchEI-producing microorganisms, which predominantly consist of fungi and bacteria. The AchEI-producing fungi are mainly Ascomycotina and Deuteromycotina, whereas the primary AchEI-producing bacteria include Actinobacteria and Cyanophyta. Molecules 2017, 22, 176 20 of 24

AchEI produced by fungi include alkaloids, terpenoids, phenylpropanoids, and steroids. Current research continues to focus on the isolation and screening of producing strains, and few reports have described strain modification, product yield increases, metabolic pathways, and genetic engineering. Thus, these areas still need to be further explored. Additionally, further improvements of in vitro AchEI detection methods may be achieved through construction of molecular models, a combination of bioinformatics simulation and biological analysis to predict the amino acids within the active site of acetylcholinesterase, and analysis of the functional mechanisms of the tested compounds.

Acknowledgments: This work was funded by the Key Project of the Department of Science and Technology of Fujian Province, China (grant number 2012N0013) and the Middle and young teachers education and scientific research projects in Fujian Province, China (grant number JAT160107). Author Contributions: Q.C., M.Y. and J.S. conceived and designed the experiments; J.S. analyzed the data; H.L., K.G. and L.C. contributed analysis tools; J.S. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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