Endophytic fungi of Lycium barbarum: isolation, determination, bioactivity and separation of compounds.

Lycium barbarum is widely distributed in China and used as a traditional Chinese medicine herb to treat dizziness, abdominal pain, dry cough, headache and fatigue. Several studies have examined the endophytes of L. barbarum from northwest China; however, few have focused on that from eastern China. The objective of this study was to isolate and determine the endophytic fungi of L. barbarum from Shandong province, as well as to obtain and identify active secondary metabolites from the endophytes. In this study, 17 endophytic fungi were isolated from L. barbarum and denoted as GQ-1 to GQ-17, respectively. These fungi were further classified into ten genera based on the morphological and ITS identification. The crude extracts of these fungi were obtained by using liquid fermentation and EtOAc extraction, and their antibacterial, cytotoxic, and antioxidant activities were evaluated. The results showed that GQ-6 and GQ-16 exhibited high inhibitory activity; GQ-6 and GQ-9 showed high cytotoxic activity and GQ-5 exhibited high scavenging capability for DPPH free radicals. Additionally, Cladosporium sp. GQ-6 was used to investigate the secondary metabolites. The crude extracts were purified by using column chromatography, reverse column, and liquid chromatography, and four monomeric compounds were identified, including two known compounds (α-acetylorcinol (1) and cladosporester B (2)) and two new compounds (cladosporacid F (3) and cladosporester D (4)). The anti-fungal and antibacterial activities of these compounds were confirmed, but no cytotoxic activity was observed. In conclusion, endophytic fungi of L. barbarum from eastern China can serve as a potential source of active natural products with antibacterial and antioxidant properties.

Fusaricide is a Novel Iron Chelator that Induces Apoptosis through Activating Caspase-3.

Nonsmall cell lung cancer (NSCLC) has been a fatal and refractory disease worldwide. Novel therapeutic developments based on fundamental investigations of anticancer mechanisms underlie substantial foundations to win the fight against cancer diseases. In this study, we isolated a natural product fusaricide (FCD) from an endophytic fungus of Lycium barbarum, identified as Epicoccum sp. For the first time, we discovered that FCD potently inhibited proliferation in a variety of human NSCLC cell lines, with relatively less toxicity to normal cells. Our study exhibited that FCD induced apoptosis, caused DNA damage and cell cycle arrest in G0/G1 phase, and activated caspase-3 as well as other apoptosis-related factors in human NSCLC NCI-H460 cells. FCD was proven to be an iron chelator that actively decreased levels of cellular labile iron pool in NCI-H460 cells in our study. FeCl3 supplement reversed FCD-induced apoptosis. The upregulation of transferrin receptor 1 (TfR1) and downregulation of ferritin heavy chain (FTH) expression were observed after FCD treatment. In summary, our study highlighted the potential anticancer effects of FCD against human NSCLCs and demonstrated that the FCD-mediated apoptosis depended on binding to intracellular iron.

[Antimicrobial and anti-tumor activities of endophytes from Lycium barbarum of Ningxia].

The antimicrobial activity and cytotoxicity in vitro of the fermentation broth of 10 endophytic strains isolated from Lycium barbarum were determined to screen high activity endophytic strains. Sequences analysis of ITS and 16S rDNA was used for molecular identification of the strains. The results showed that 5 endophytic fungi had no inhibitory activity against the tested pathogens. Endophytic actinomycete strain AL6 had a certain inhibitory effect on 3 kinds of pathogenic fungi, and strain AL5 only had strong inhibitory activity against Staphylococcus aureus. However, the anti-tumor activity of endophytic fungi was significantly higher than that of actinomycetes. Four endophytic fungi strains exhibited the growth inhibition rate of above 50% against at least one of the tested tumor cells when the concentration of fermentation broth was 0.2 g•L⁻¹. Sequences analysis showed that 5 endophytic fungi strains belonged to genus Aspergillus, Penicillium and Emericella, and the 5 endophytic actinomycetes strains belonged to genus Aspergillus, Penicillium and Emericella. Aspergillus strain FL1 had stronger inhibitory activity against A549 and HeLa cells, and the IC50 values were 0.022，0.028 g•L⁻¹, respectively, which was worthy of further study.

Influence of Rhizoglomus irregulare on nutraceutical quality and regeneration of Lycium barbarum leaves under salt stress.

Whether arbuscular mycorrhizal fungi augment the nutraceutical quality of crops under salt stress is critical as a potential agronomic practice in salinized farmland. To evaluate the effect of Rhizoglomus irregulare on the nutraceutical quality of Lycium barbarum leaves under salt stress, we analyzed growth parameters and the rutin, polysaccharide, acidic polysaccharide, and amino acids contents of 2 harvests. Inoculation of R. irregulare significantly increased the regenerated bud number (partial eta squared (PES) = 0.577, P < 0.0001) and rutin concentration (PES = 0.544, P < 0.001) of L. barbarum leaves, with and without salt stress. The biomass of the 2nd harvest (PES = 0.355, P = 0.0091) and acidic polysaccharide (PES = 0.518, P = 0.001) of L. barbarum leaves were notably enhanced by R. irregulare under 200 mmol/L salt level. Rhizoglomus irregulare had insignificant effect on polysaccharide (PES = 0.092, P = 0.221) and amino acids levels (PES = 0.263, P = 0.130) in the leaves of L. barbarum. However, inoculation by R. irregulare decreased proline level (PES = 0.761, P = 0.001) in the leaves of L. barbarum when subjected to salt stress. Taken together, these results indicate that R. irregulare significantly improved the nutraceutical quality and facilitated the sustainable production of L. barbarum leaves exposed to salt stress.

[Screening and identification of antioxidant endophytes from Lycium barbarum of Ningxia].

In this paper, 29 endophytes were isolated from different organs and tissues of Lycium barbarum of Ningxia by tablet coating method, 18 of them was fungi, and 11 of them was actinomycetes. The endophytes quantity in the different tissues were leaves > flowers > roots >fruits; The hydroxyl radical scavenging activities of 11 endophytes were investigated by Fenton reaction, and total antioxidant capacities of them were examined by a. total antioxidant capacity test kit; culture features and strain-specific sequence analysis were employed to explore the diversity of the 11 endophytes. The result showed that 5 fungi and 6 actinomycetes that having antioxidant activity could be phylogenetically classified into 3 genera, 3 genera and 3 families, respectively. The total antioxidant capacity and hydroxyl radical scavenging activity of the 11 endophytes showed distinct difference. The antioxidant activity of Aspergillus were stronger, among which total antioxidant capacity of fL1 was (188.5 ± 0.549) U · mL⁻¹ and the IC50 was 0.3 mg · L⁻¹; the IC50 of strain fL1 was 0.42 mg · L⁻¹ and the total antioxidant capacity of fL9 was (113.63 ± 1.021) U · mL⁻¹, all of them were stronger than the positive control Vit C. The experimental results indicated that endophytic fungi of L. barbarum of Ningxia have a great developing and application prospect for the development of antioxidant agent.

[Investigation of production status in major wolfberry producing areas of China and some suggestions].

To investigate the production status and the safety influence factors of wolfberry in China. We investigated the detailed factors which affect the quality safe of wolfberry in the periods of July-August 2013 and July-September 2009. The factors include fertilizing patterns, the used pesticide and preliminary process wolfberry. The factors were discussed according to the results of investigation, and suggestions were proposed for the management and production departments of wolfberry.

Endophytic fungi from Lycium chinense Mill and characterization of two new Korean records of Colletotrichum.

Chinese boxthorn or matrimony vine (Lycium chinense Mill) is found primarily in southeastern Europe and Asia, including Korea. The dried ripe fruits are commonly used as oriental medicinal purposes. Endophytic fungi were isolated from surface sterilized tissues and fruits of the medicinal plant in 2013 to identify the new or unreported species in Korea. Among 14 isolates, 10 morphospecies were selected for molecular identification with the internal transcribed spacer (ITS) gene. Phylogenetic analysis revealed that all isolates belonged to Ascomycota including the genera Acremonium, Colletotrichum, Cochliobolus, Fusarium, Hypocrea and Nemania. Two Colletotrichum species were identified at the species level, using three genes including internal transcribed spacer (ITS), glycerol-3-phosphate dehydrogenase (GAPDH) and Actin (ACT) for PCR and molecular data analysis along with morphological observations. The fungal isolates, CNU122031 and CNU122032 were identified as Colletotrichum fructicola and C. brevisporum, respectively. Morphological observations also well supported the molecular identification. C. brevisporum is represented unrecorded species in Korea and C. fructicola is the first record from the host plant.

Effects of a dark-septate endophytic isolate LBF-2 on the medicinal plant Lycium barbarum L.

Dark septate endophytes (DSE) are ubiquitous root associated fungi; however, our understanding of their ecological function remains unclear. Here, we investigated the positive effect of a DSE fungus on its host plant Lycium barbarum L. A DSE isolate, LBF-2, isolated from the roots of L. barbarum, was inoculated onto the roots of plants, which were grown under greenhouse conditions for five weeks. The result of molecular analyses of internal transcribed spacer regions indicated that LBF-2 was 96% similar to Paraphoma chrysanthemicola. Melanized septate hyphae were observed in the root cortical cells of L. barbarum using a light microscope. Inoculation with LBF-2 increased the total biomass by 39.2% and also enhanced chlorophyll fluorescence. Inoculation increased the concentration of total chlorophyll by 22.8% and of chlorophyll a by 21.3%, relative to uninoculated controls. These data indicate that the LBF-2 isolate might be used to facilitate the cultivation of L. barbarum, which has medicinal applications.