Characterization and antimicrobial activity of endophytic fungi from medicinal plant Agave americana.

Endophytic microorganisms associated with medicinal plants are of particular interest as they are a potential source of new bioactive chemicals effective against novel emerging and drug-resistant pathogens. Agave americana is a tropical medicinal plant with antibacterial, antifungal, and anticancer properties. We studied the biodiversity of fungal endophytes of A. americana and their antimicrobial production potential. Isolated endophytic fungi were classified into 32 morphotypes (15 from stem and 17 from leaf) based on their cultural and morphological characteristics. Among the fungal crude extracts tested, 82% of isolates from the leaves and 80% of the isolates from the stem showed antibacterial activity against the bacterial strains (Escherichia coli ATTC 25902, Staphylococcus aureus ATTC 14775, and Bacillus subtilis NRRL 5109) tested. Extracts from four fungal isolates from leaves showed antifungal activity against at least one of the fungal strains (Candida albicans ATTC 10231 and Aspergillus fumigatus NRRL 5109) tested. Crude extracts of seven fungal isolates showed a zone of inhibition of more than 11 mm at 10 mgml-1 against both Gram-positive and Gram-negative bacteria tested. Penicillium, Colletotrichum, Curvularia, Pleosporales, Dothideomycetes, and Pleurotus are the main endophytes responsible for bioactive potential. These results indicate that A. americana harbors endophytes capable of producing antimicrobial metabolites.

The antimicrobial property of JY-1, a complex mixture of Traditional Chinese Medicine, is linked to it abilities to suppress biofilm formation and disrupt membrane permeability.

The substantial increase of infections, caused by novel, sudden, and drug-resistant pathogens, poses a significant threat to human health. While numerous studies have demonstrated the antibacterial and antiviral effects of Traditional Chinese Medicine, the potential of a complex mixture of traditional Chinese Medicine with a broad-spectrum antimicrobial property remains underexplored. This study aimed to develop a complex mixture of Traditional Chinese Medicine (TCM), JY-1, and investigate its antimicrobial properties, along with its potential mechanism of action against pathogenic microorganisms. Antimicrobial activity was assessed using a zone of inhibition assay and the drop plate method. Hyphal induction of Candida albicans was conducted using RPMI1640 medium containing 10% FBS, followed by microscopic visualization. Quantitative real-time PCR (RT-qPCR) was employed to quantify the transcript levels of hyphal-specific genes such as HWP1 and ALS3. The impact of JY-1 on biofilm formation was evaluated using both the XTT reduction assay and scanning electron microscopy (SEM). Furthermore, the cell membrane integrity was assessed by protein and nucleic acid leakage assays. Our results clearly showed that JY-1 significantly inhibits the vegetative growth of Candida spp. and Cryptococcus spp. In addition, this complex mixture is effectively against a wide range of pathogenic bacteria, including Staphylococcus aureus, Vancomycin-resistant enterococci, Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae. More interestingly, JY-1 plays a direct anti-viral role against the mammalian viral pathogen vesicular stomatitis virus (VSV). Further mechanistic studies indicate that JY-1 acts to reduce the expression of hyphal specific genes HWP1 and ALS3, resulting in the suppression of the hyphal formation of C. albicans. The antimicrobial property of JY-1 could be attributed to its ability to reduce biofilm formation and disrupt the cell membrane permeability, a process resulting in microbial cell death and the release of cellular contents. Taken together, our work identified a potent broad-spectrum antimicrobial agent, a complex mixture of TCM which might be developed as a potential antimicrobial drug.

Assessment of antimalarial activity of crude extract of Chan-Ta-Lee-La and Pra-Sa-Chan-Dang formulations and their plant ingredients for new drug candidates of malaria treatment: In vitro and in vivo experiments.

The emergence and spread of antimalarial drug resistance have become a significant problem worldwide. The search for natural products to develop novel antimalarial drugs is challenging. Therefore, this study aimed to assess the antimalarial and toxicological effects of Chan-Ta-Lee-La (CTLL) and Pra-Sa-Chan-Dang (PSCD) formulations and their plant ingredients. The crude extracts of CTLL and PSCD formulations and their plant ingredients were evaluated for in vitro antimalarial activity using Plasmodium lactate dehydrogenase enzyme and toxicity to Vero and HepG2 cells using the tetrazolium salt method. An extract from the CTLL and PSCD formulations exhibiting the highest selectivity index value was selected for further investigation using Peter's 4-day suppressive test, curative test, prophylactic test, and acute oral toxicity in mice. The phytochemical constituents were characterized using gas chromatography-mass spectrometry (GC-MS). Results showed that ethanolic extracts of CTLL and PSCD formulations possessed high antimalarial activity (half maximal inhibitory concentration = 4.88, and 4.19 g/mL, respectively) with low cytotoxicity. Ethanolic extracts of the CTLL and PSCD formulations demonstrated a significant dose-dependent decrease in parasitemia in mice. The ethanolic CTLL extract showed the greatest suppressive effect after 4 days of suppressive (89.80%) and curative (35.94%) testing at a dose of 600 mg/kg. Moreover, ethanolic PSCD extract showed the highest suppressive effect in the prophylactic test (65.82%) at a dose of 600 mg/kg. There was no acute toxicity in mice treated with ethanolic CTLL and PSCD extracts at 2,000 mg/kg bodyweight. GC-MS analysis revealed that the most abundant compounds in the ethanolic CTLL extract were linderol, isoborneol, eudesmol, linoleic acid, and oleic acid, whereas ethyl 4-methoxycinnamate was the most commonly found compound in the ethanolic PSCD extract, followed by 3-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-chromen-4-one, flamenol, oleic acid amide, linoleic acid, and oleic acid. In conclusions, ethanolic CTLL and PSCD extracts exhibited high antimalarial efficacy in vitro. The ethanolic CTLL extract at a dose of 600 mg/kg exhibited the highest antimalarial activity in the 4-day suppressive and curative tests, whereas the ethanolic PSCD extract at a dose of 600 mg/kg showed the highest antimalarial activity in the prophylactic test.

Control of pathogenic bacteria using marine actinobacterial extract with antiquorum sensing and antibiofilm activity.

OBJECTIVE: The objectives of this research were to screen the anti-quorum sensing and antibiofilm activity of marine actinobacteria, isolated from several aquatic environments in Indonesia against several pathogenic bacteria, such as Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Vibrio cholerae, Salmonella Typhimurium, and Pseudomonas aeruginosa. RESULTS: Ten out of 40 actinobacteria were found to have anti-quorum sensing activity against wild-type Chromobacterium violaceum (ATCC 12472); however, the validation assay showed that only eight of 10 significantly inhibited the quorum sensing system of Chromobacterium violaceum CV026. The crude actinobacteria extracts inhibited and disrupted biofilm formation produced by pathogens. The highest antibiofilm inhibition was discovered in isolates 11AC (90%), 1AC (90%), CW17 (84%), TB12 (94%), 20PM (85%), CW01 (93%) against Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Vibrio cholerae, Salmonella Typhimurium, and Pseudomonas aeruginosa, respectively. The highest biofilm destruction activity was observed for isolate 1AC (77%), 20PM (85%), 16PM (72%), CW01 (73%), 18PM (82%), 16PM (63%) against Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Vibrio cholerae, Salmonella Typhimurium, and Pseudomonas aeruginosa, respectively. Actinobacteria isolates demonstrated promising anti-quorum and/or antibiofilm activity, interfering with the biofilm formation of tested pathogens. Appropriate formulations of these extracts could be developed as effective disinfectants, eradicating biofilms in many industries.

Immunoregulatory effects of Huaier (Trametes robiniophila Murr) and relevant clinical applications.

Huaier (Trametes robiniophila Murr) is a medicinal fungus of traditional Chinese medicine with more than 1000 years of history of clinical application. Its remarkable anticancer activities has led to its application in treating diverse malignancies. In recent years, the immunomodulatory effects of Huaier have been uncovered and proved to be beneficial in a plethora of immune-related diseases including cancer, nephropathy, asthma, etc. In this review, we comprehensively summarized the active components of Huaier, its regulatory activities on multifaceted aspects of the immune system, its application in various clinical settings as well as toxicologic evidence. Based on currently available literature, Huaier possesses broad-spectrum regulatory activities on various components of the innate and adaptive immune system, including macrophages, dendritic cells, natural killer cells, T and B lymphocytes, etc. Versatile immunologic reactions are under the regulation of Huaier from expression of damage-associated molecular patterns, immune cell activation and maturation to cell proliferation, differentiation, antibody production, expression of cytokines and chemokines and terminal intracellular signal transduction. Moreover, some modulatory activities of Huaier might be context-dependent, typically promoting the restoration toward normal physiological status. With excellent efficacy and minimal side effects, we foresee more extensive application of Huaier for treating immune-related disorders.

In vitro antibacterial activity and synergetic effect of crude extract of the Wohlfahrtia nuba (Diptera: Sarcophagidae) flesh fly larvae.

Multidrug-resistant pathogens have become ubiquitous, and effective treatment alternatives are urgently required. Maggot therapy is a promising agent that is being studied to overcome antibiotic-resistant pathogens. This study evaluated the antibacterial activity of the larvae extract of the Wohlfahrtia nuba (wiedmann) (Diptera: Sarcophagidae) flesh fly on the growth of five pathogenic bacterial species (methicillin-sensitive Staphylococcus aureus [ATCC 29213], methicillin-resistant Staphylococcus aureus [ATCC BAA-1680], Pseudomonas aeruginosa [ATCC 27853], Escherichia coli [ATCC 25922], and Salmonella typhi [ATCC 19430]) in vitro by using different techniques. Resazurin-based turbidimetric assay demonstrated that the W. nuba maggot exosecretion (ES) was potent against all the bacterial species tested, and according to the determined minimum inhibitory concentration (MIC) for each bacterium, gram-negative bacteria were more sensitive than gram-positive bacteria. Additionally, colony-forming unit assay showed that maggot ES was able to inhibit bacterial growth rate for all bacterial species tested, where the highest bacterial reduction was observed with methicillin-sensitive S. aureus (MSSA) followed by S. typhi. Moreover, maggot ES was shown to be concentration-dependent, where 100 µL of ES at 200 mg/mL was bactericidal towards methicillin-resistant S. aureus (MRSA) and P. aeruginosa compared with 100 µL at the MIC of the ES. Moreover, based on the result of agar disc diffusion assay, maggot extract was more efficient against P. aeruginosa and E. coli than the remaining reference strains tested. Furthermore, the combination between regular antibiotics with maggot ES at different concentrations indicated that ES acts synergistically with the tested antibiotics against the five bacterial models.

Restoring Ampicillin Sensitivity in Multidrug-Resistant Escherichia coli Following Treatment in Combination with Coffee Pulp Extracts.

Escherichia coli, particularly multidrug-resistant (MDR) strains, is a serious cause of healthcare-associated infections. Development of novel antimicrobial agents or restoration of drug efficiency is required to treat MDR bacteria, and the use of natural products to solve this problem is promising. We investigated the antimicrobial activity of dried green coffee (DGC) beans, coffee pulp (CP), and arabica leaf (AL) crude extracts against 28 isolated MDR E. coli strains and restoration of ampicillin (AMP) efficiency with a combination test. DGC, CP, and AL extracts were effective against all 28 strains, with a minimum inhibitory concentration (MIC) of 12.5-50 mg/ml and minimum bactericidal concentration of 25-100 mg/ml. The CP-AMP combination was more effective than CP or AMP alone, with a fractional inhibitory concentration index value of 0.01. In the combination, the MIC of CP was 0.2 mg/ml (compared to 25 mg/ml of CP alone) and that of AMP was 0.1 mg/ml (compared to 50 mg/ml of AMP alone), or a 125-fold and 500-fold reduction, respectively, against 13-drug resistant MDR E. coli strains. Time-kill kinetics showed that the bactericidal effect of the CP-AMP combination occurred within 3 h through disruption of membrane permeability and biofilm eradication, as verified by scanning electron microscopy. This is the first report indicating that CP-AMP combination therapy could be employed to treat MDR E. coli by repurposing AMP.

[Effective substances and mechanism of Yishen Guluo Mixture in treatment of chronic glomerulonephritis based on metabolomics and serum pharmacochemistry].

This study aimed to investigate the effective substances and mechanism of Yishen Guluo Mixture in the treatment of chronic glomerulonephritis(CGN) based on metabolomics and serum pharmacochemistry. The rat model of CGN was induced by cationic bovine serum albumin(C-BSA). After intragastric administration of Yishen Guluo Mixture, the biochemical indexes related to renal function(24-hour urinary protein, serum urea nitrogen, and creatinine) were determined, and the efficacy evaluations such as histopathological observation were carried out. The serum biomarkers of Yishen Guluo Mixture in the treatment of CGN were screened out by ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) combined with multivariate statistical analysis, and the metabolic pathways were analyzed. According to the mass spectrum ion fragment information and metabolic pathway, the components absorbed into the blood(prototypes and metabolites) from Yishen Guluo Mixture were identified and analyzed by using PeakView 1.2 and MetabolitePilot 2.0.4. By integrating metabolomics and serum pharmacochemistry data, a mathematical model of correlation analysis between serum biomarkers and components absorbed into blood was constructed to screen out the potential effective substances of Yishen Guluo Mixture in the treatment of CGN. Yishen Guluo mixture significantly decreased the levels of 24-hour urinary protein, serum urea nitrogen, and creatinine in rats with CGN, and improved the pathological damage of the kidney tissue. Twenty serum biomarkers of Yishen Guluo Mixture in the treatment of CGN, such as arachidonic acid and lysophosphatidylcholine, were screened out, involving arachidonic acid metabolism, glycerol phosphatide metabolism, and other pathways. Based on the serum pharmacochemistry, 8 prototype components and 20 metabolites in the serum-containing Yishen Guluo Mixture were identified. According to the metabolomics and correlation analysis of serum pharmacochemistry, 12 compounds such as genistein absorbed into the blood from Yishen Guluo Mixture were selected as the potential effective substances for the treatment of CGN. Based on metabolomics and serum pharmacochemistry, the effective substances and mechanism of Yishen Guluo Mixture in the treatment of CGN are analyzed and explained in this study, which provides a new idea for the development of innovative traditional Chinese medicine for the treatment of CGN.

The crude extract from the flowers of Trollius chinensis Bunge exerts anti-influenza virus effects through modulation of the TLR3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The flowers of Trollius chinensis Bunge (Ranunculaceae) is a traditional Chinese medicine used to treat various inflammatory diseases, including upper respiratory infections, chronic tonsillitis, and pharyngitis. Recently, there has been growing research on the antiviral role of the flowers of T. chinensis Bunge. However, little is known about its anti-influenza virus effects and the underlying mechanisms. AIM OF THE STUDY: This study aims to evaluate the therapeutic effects of the crude extract from the flowers of T. chinensis Bunge (CEFTC) on mice infected with influenza virus. We further explored its mechanism by detecting the expression of vital proteins (TLR3, TBK1, TAK1, IKKα, IRF3, and IFN-ß) related to TLR3 signaling pathway. MATERIALS AND METHODS: Mice were infected with influenza A virus (H1N1) through the nasal cavity and were intragastrically administered CEFTC at the dose of 0.2 mg/g once daily. The therapeutic effects of CEFTC were evaluated by blood cell count, lung index, spleen index, alveolar lavage fluid testing, and HE staining. Network pharmacology analysis predicted the potential signaling pathway between the flowers of T. chinensis Bunge and pneumonia. The expression of TLR3, TBK1, TAK1, IKKα, IRF3, and IFN-ß in lung tissues were examined by Western blot assay. In addition, the immunofluorescence assay was applied to assess the effect of CEFTC on the distribution of IRF3 and IFN-ß between nuclei and cytoplasm. RESULTS: Compared with the infected group, the lung index was markedly reduced, and the pathological damage of the lungs was also attenuated in the CEFTC treatment group. The network pharmacology analysis indicated that the NF-κB pathway was a potential signaling pathway in the flowers of T. chinensis Bunge for the treatment of pneumonia, TLR3, IRF3, and TBK1 were crucial targets associated with pneumonia. Western blot assay demonstrated that in the high-dose virus infected group, CEFTC reduced the expression of TLR3, TAK1, TBK1, and IRF3. Furthermore, CEFTC could increase the nuclear distribution of IRF3 in alveolar epithelial cells after virus infection. CONCLUSIONS: These results suggested that different doses of influenza virus could cause varying infection symptoms in mice. Moreover, CEFTC could exert anti-influenza virus effects by regulating the expression of TLR3, IRF3, IFN-ß, TAK1, and TBK1 in the TLR3 signaling pathway.

First Discovery of Beta-Sitosterol as a Novel Antiviral Agent against White Spot Syndrome Virus.

The outbreak of white spot syndrome (WSS) is a looming challenge, due to dramatic losses to the crustacean aquaculture industry. However, at present, there are no prophylactic or therapeutic means to control this infectious viral disease. Here, we screened fifteen medicinal plants for their inhibitory activity on the white spot syndrome virus (WSSV), using red swamp crayfish (Procambarus clarkii) as a model species. The results showed that the crude extracts of Pinellia ternata (Thunb.) Breit. had the highest inhibitory effect (91.59%, 100 mg/kg) on WSSV proliferation, and its main component, beta-sitosterol, showed a much higher activity (95.79%, 50 mg/kg). Further, beta-sitosterol potently reduced (p < 0.01) viral loads and viral gene transcription levels in a concentration-dependent fashion, and significantly promoted the survival rate of WSSV-challenged crayfish (57.14%, 50 mg/kg). The co-incubation assay indicated that beta-sitosterol did not influence the infectivity of WSSV particles. Both pre- and post-treatment of beta-sitosterol exerted a significant inhibitory effect (p < 0.01) on the viral load in vivo. Mechanistically, beta-sitosterol not only interfered with the expression of viral genes (immediate early gene 1, ie1; DNA polymerase, DNApol) that are important in initiating WSSV transcription, but it also attenuated the hijacking of innate immune signaling pathways (Toll, IMD, and JAK/STAT pathways) by viral genes to block WSSV replication. Moreover, the expression of several antiviral immune, antioxidant, pro-inflammatory, and apoptosis-related genes changed significantly in beta-sitosterol-treated crayfish. Beta-sitosterol is a potent WSSV inhibitor and has the potential to be developed as an effective anti-WSSV agent against a WSS outbreak in crustacean aquaculture.

Antibacterial Effects of Artemisia afra Leaf Crude Extract Against Some Selected Multi-Antibiotic Resistant Clinical Pathogens.

Background: Due to improper use of antibiotics, some pathogenic bacteria that cause serious and deadly infections have become resistant to commonly used broad spectrum antibiotics. This antibiotic resistance has become major global healthcare problem. Therefore, there is an urgent need to develop novel antibacterial agents; hence, much attention has been made on medicinal plants such as Artemisia afra. Thus, the current study was aimed to evaluate the antibacterial activity of ethanolic, methanolic and n-hexane extracts of this plant leaf against four multi-antibiotic resistant clinical pathogens. Methods: Crude extracts from A.afra leaf were prepared using ethanol, methanol and n-hexane and the antibacterial effect of each extract was tested against Escherichia coli, Streptococcus pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. In addition, minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were evaluated. Results: Among the crude extracts, the highest zone of inhibition (25.33±0.58mm) was recorded against E. coli when methanolic extract was applied. On the other hand, the lowest inhibition was exhibited when n-hexane extract was applied against S.aureus (5.67±1.56 mm). Concerning MIC values of the different extracts, varied results were obtained. MIC value of 6.25mg/mL was recorded when methanolic extract was applied against all clinical pathogens. Moreover, both methanolic and ethanolic extracts showed MBC value of 12.5mg/mL against the four clinical pathogens. However, the methanolic extract gave MBC value of 6.25mg/mL against E. coli. Conclusion: From this study, it can be concluded that it is possible to develop and formulate of new, efficacious, less toxic and inexpensive herbal medicine from A.afra leaf extract that act against multi-antibiotic resistant clinical pathogens.

Antibacterial effect of Asphodelus fistulosus aqueous and ethanolic crude extracts on gram positive and gram negative bacteria.

Asphodelus fistulosus (A. fistulosus) is a wild plant grows in Jordan. Traditionally, it is used to treat different medical conditions and diseases such as respiratory ailments, against burns and dermatomucosal infections.This study aims to find out the effects of A. fistulosus aqueous and ethanolic crude extracts on Staphylococcus aureus(S. aureus) as gram positive bacteria and Escherichia coli (E. coli) as gram negative bacteria and to investigate which one will be affected either by aqueous and/or ethanolic crude extracts of A. fistulosus shooting parts that were collected from Jerash in the north of Jordan. Agar well diffusion method was used to evaluate the antibacterial activity of the crude extracts. In addition, MIC (minimum inhibitory concentration) as well as MBC (minimum bactericidal concentration) were determined against both types of bacteria. The results showed that flower aqueous extract of A. fistulosus was very effective against E. coli (20.0 ± 0.50) mm and caused a (14.0 ± 0.50) mm inhibition to S. aureus. The ethanolic extract of stem was very effective cauesed a (19.0 ± 0.50) mm inhibition in both bacterial species. Respectively, both S. aureus and E. coli were inhibited by ethanolic and aqueous extracts (mixture1 and mixture2) (15.0 ± 0.00 mm and 10.5 ± 0.50 mm). The highest antimbacterial activity was observed for the leaves aqueous extract against E.coli (0.06120 mg/mL). The obtained MIC values from A. fistulosus parts extracts demonstrated antibacterial activity ranged between 7.606 and 0.06120 mg/mL. The highest antimicrobial activity was recorded in the leaves aqueous extract against E. coli.The MBC value of stem aqueous extract was 5.00 mg/mL against both S. aureus and E. coli. On the other hand, ethanolic and aqueous extracts of the leaves gave MBC values 5.00 mg/mL, and 0.156 mg/mL, respectively, against E. coli.Based on the results of this study, it can be concluded that there is good inhibitory effect of aqueous and ethanolic of A. fistulosus shooting parts extracts on growth of E. coli and S. aureus. Adding to that, stem ethanolic extract has the most effective against S. aureus while aqueous extract of flower has the most effective against E. coli.So, it is recommended to have further future studies on the A. fistulosus shooting parts crude extract bioactive components and the mechanism of how these constituents affect these types of bacteria.

Dried Plum's Polyphenolic Compounds and Carbohydrates Contribute to Its Osteoprotective Effects and Exhibit Prebiotic Activity in Estrogen Deficient C57BL/6 Mice.

Evidence of dried plum's benefits on bone continues to emerge. This study investigated the contribution of the fruit's polyphenol (PP) and carbohydrate (CHO) components on a bone model of postmenopausal osteoporosis to explore their prebiotic activity. Osteopenic ovariectomized mice were fed diets supplemented with dried plum, a crude extract of dried plum's polyphenolic compounds, or the PP or CHO fraction of the crude extract. The effects of treatments on the bone phenotype were assessed at 5 and 10 weeks as well as the prebiotic activity of the different components of dried plum. Both the CHO and PP fractions of the extract contributed to the effects on bone with the CHO suppressing bone formation and resorption, and the PP temporally down-regulating formation. The PP and CHO components also altered the gut microbiota and cecal short chain fatty acids. These findings demonstrate that the CHO as well as the PP components of dried plum have potential prebiotic activity, but they have differential roles in mediating the alterations in bone formation and resorption that protect bone in estrogen deficiency.

Evaluation of the antimicrobial and cytotoxic potential of endophytic fungi extracts from mangrove plants Rhizophora stylosa and R. mucronata.

Mangrove endophytic fungi are tolerant to numerous stresses and are inevitably capable of exhibiting excellent biological activity by producing impressive numbers of metabolites with special biological functions, based on previous work on the biological potential of mangrove-derived endophytic fungi. To obtain marked antimicrobial and cytotoxic fermentation products of culturable endophytic fungi from mangrove forests, our research evaluated the antimicrobial and cytotoxic activities of crude extracts of endophytic fungi from Rhizophora stylosa and Rhizophora mucronata. Forty-six fungal isolates were cultured on four different media, namely, dextrose agar (PDA), Czapek's agar (CZA), rice medium (RM) and grain medium (GM) and harvested by ethyl acetate solvent at 40 days. The extracts were tested for antimicrobial activity by the microdilution method against the gram-negative bacteria Pseudomonas adaceae (PA), gram-positive bacteria Enterococcus faecalis (EF), methicillin-resistant Staphylococcus aureus (MRSA) and pathogenic fungus Monilia albicans (MA). The cytotoxic activity of the extracts was evaluated by MTT assay using A549 human lung cancer cells, HeLa human cervical carcinoma cells, and HepG2 human hepatocellular cells. The results showed that rice medium could promote the secretion of antimicrobial and antitumour secondary metabolites of endophytic fungi in comparison with other cultivation media. Seventeen strains (68%) from R. stylosa exhibited inhibitory effects on indicators, especially N. protearum HHL46, which could inhibit the growth of four microbes with MIC values reaching 0.0625 mg/mL. Fifteen strains (71.4%) from R. mucronata displayed activities against human pathogenic microbes; in particular, Pestalotiopsis sp. HQD6 and N. protearum HQD5 could resist the growth of four microbes with MIC values ranging from 0.015 to 1 mg/mL. In the cytotoxicity assay, the extracts of 10 strains (40%), 9 strains (40%) and 13 strains (52%) of R. stylosa and 13 strains (61.9%), 10 strains (47.6%) and 10 strains (47.6%) of R. mucronata displayed cytotoxicity against A549, HeLa and HepG2 cancer cells with cell viability values ≤ 50%. Neopestalotiopsis protearum HHL46, Phomopsis longicolla HHL50, Botryosphaeria fusispora HQD83, Fusarium verticillioides HQD48 and Pestalotiopsis sp. HQD6 displayed significant antitumour activity with IC50 values below 20 µg/mL. These results highlighted the antimicrobial and antitumour potential of endophytic fungi from R. stylosa and R. mucronata and the possibility of exploiting their antimicrobial and cytotoxic agents.

Purification and characterization of a novel thermostable anticoagulant protein from medicinal leech Whitmania pigra Whitman.

ETHNOPHARMACOLOGICAL RELEVANCE: The prevalence of cardiovascular disease (CVD) is increasing worldwide. Despite significant improvements in novel targeted treatment agents, natural products purified from medicinal animals with minimal side effects have attracted much attention. Several native proteins explored from suck-blood leeches, such as non-thermostable hirudin and its variants, revealed potent anticoagulant activity. Traditional Chinese medicine clinics have proved that non-suck-blood leech Whitmania pigra Whitman (W. pigra) also played notable roles in CVD treatments even after decoction. However, only a few natural proteins and peptides have been identified from the fresh material of this medicinal species. AIM OF THE STUDY: We aimed to purify and characterize thermostable anticoagulant proteins from W. pigra for further development of a therapeutic agent for thrombosis. MATERIALS AND METHODS: W. pigra crude extract was prepared by decoction in water. Anticoagulant proteins were purified by DEAE cellulose DE-52, Sephadex G-75, and reversed-phase liquid chromatography sequentially and analyzed by SDS-PAGE and LC-MS/MS for structural information. In addition, we conducted in vitro anticoagulant experiments, including plasma recalcification time (PRT) assay, fibrinolytic assay, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fib) assay, and cell viability assays. Furthermore, a carrageenan-induced chronic thromboembolism model was employed in ICR mice, and four coagulation factors (APTT, PT, TT, and Fib) activities were determined after intragastric administration. RESULTS: The anticoagulant protein WP-77 has a relative molecular weight of ca. 20.8 kDa. It was effective over a broad temperature range from 20 °C to 100 °C and a pH 2-8 condition. The anticoagulant activity of WP-77 was retained after incubation with pepsin but was greatly inhibited by trypsin (P < 0.01). It significantly prolonged APTT and TT (P < 0.05) but had little effect on PT and Fib in vitro. Furthermore, WP-77 of a low concentration resulted in the recovery of injured EA.hy926 by thrombin. The protein also significantly prolonged APTT and TT (P < 0.01) and inhibited thrombus formation in carrageenan-induced thrombosis mice, demonstrating its antithrombotic effect in vivo. CONCLUSION: Our results suggest that WP-77 from W. pigra plays a distinct role in treating thrombotic diseases, and it is an essential substance of anticoagulant activity of non-suck-blood medicinal leeches. This thermostable anticoagulant protein could be a promising candidate for the development of clinical antithrombosis medicines.

Bioactivity and Mycochemical Profile of Extracts from Mycelial Cultures of Ganoderma spp.

Fungal mycelium cultures are an alternative to natural sources in order to obtain valuable research materials. They also enable constant control and adaptation of the process, thereby leading to increased biomass growth and accumulation of bioactive metabolites. The present study aims to assess the biosynthetic potential of mycelial cultures of six Ganoderma species: G. adspersum, G. applanatum, G. carnosum, G. lucidum, G. pfeifferi, and G. resinaceum. The presence of phenolic acids, amino acids, indole compounds, sterols, and kojic acid in biomass extracts was determined by HPLC. The antioxidant and cytotoxic activities of the extracts and their effects on the inhibition of selected enzymes (tyrosinase and acetylcholinesterase) were also evaluated. The total content of phenolic acids in the extracts ranged from 5.8 (G. carnosum) to 114.07 mg/100 g dry weight (d.w.) (G. pfeifferi). The total content of indole compounds in the extracts ranged from 3.03 (G. carnosum) to 11.56 mg/100 g d.w. (G. lucidum) and that of ergosterol ranged from 28.15 (G. applanatum) to 74.78 mg/100 g d.w. (G. adspersum). Kojic acid was found in the extracts of G. applanatum and G. lucidum. The tested extracts showed significant antioxidant activity. The results suggest that the analyzed mycelial cultures are promising candidates for the development of new dietary supplements or pharmaceutical preparations.

Antiviral Activity and Underlying Action Mechanism of Euglena Extract against Influenza Virus.

It is difficult to match annual vaccines against the exact influenza strain that is spreading in any given flu season. Owing to the emergence of drug-resistant viral strains, new approaches for treating influenza are needed. Euglena gracilis (hereinafter Euglena), microalga, used as functional foods and supplements, have been shown to alleviate symptoms of influenza virus infection in mice. However, the mechanism underlying the inhibitory action of microalgae against the influenza virus is unknown. Here, we aimed to study the antiviral activity of Euglena extract against the influenza virus and the underlying action mechanism using Madin-Darby canine kidney (MDCK) cells. Euglena extract strongly inhibited infection by all influenza virus strains examined, including those resistant to the anti-influenza drugs oseltamivir and amantadine. A time-of-addition assay revealed that Euglena extract did not affect the cycle of virus replication, and cell pretreatment or prolonged treatment of infected cells reduced the virus titer. Thus, Euglena extract may activate the host cell defense mechanisms, rather than directly acting on the influenza virus. Moreover, various minerals, mainly zinc, in Euglena extract were found to be involved in the antiviral activity of the extract. In conclusion, Euglena extract could be a potent agent for preventing and treating influenza.

Grifola frondosa (Maitake) Extract Reduces Fat Accumulation and Improves Health Span in C. elegans through the DAF-16/FOXO and SKN-1/NRF2 Signalling Pathways.

In recent years, food ingredients rich in bioactive compounds have emerged as candidates to prevent excess adiposity and other metabolic complications characteristic of obesity, such as low-grade inflammation and oxidative status. Among them, fungi have gained popularity for their high polysaccharide content and other bioactive components with beneficial activities. Here, we use the C. elegans model to investigate the potential activities of a Grifola frondosa extract (GE), together with the underlying mechanisms of action. Our study revealed that GE represents an important source of polysaccharides and phenolic compounds with in vitro antioxidant activity. Treatment with our GE extract, which was found to be nongenotoxic through a SOS/umu test, significantly reduced the fat content of C. elegans, decreased the production of intracellular ROS and aging-lipofuscin pigment, and increased the lifespan of nematodes. Gene expression and mutant analyses demonstrated that the in vivo anti-obesity and antioxidant activities of GE were mediated through the daf-2/daf-16 and skn-1/nrf-2 signalling pathways, respectively. Taken together, our results suggest that our GE extract could be considered a potential functional ingredient for the prevention of obesity-related disturbances.

Modulation of circadian clock by crude drug extracts used in Japanese Kampo medicine.

Circadian rhythm is an approximately 24 h endogenous biological rhythm. Chronic disruption of the circadian clock leads to an increased risk of diabetes, cardiovascular disease, and cancer. Hence, it is important to develop circadian clock modulators. Natural organisms are a good source of several medicines currently in use. Crude drugs used in Japanese traditional Kampo medicine or folk medicines are an excellent source for drug discovery. Furthermore, identifying new functions for existing drugs, known as the drug repositioning approach, is a popular and powerful tool. In this study, we screened 137 crude drug extracts to act as circadian clock modulators in human U2OS cells stably expressing the clock reporter Bmal1-dLuc, and approximately 12% of these modulated the circadian rhythm. We further examined the effects of several crude drugs in Rat-1 fibroblasts stably expressing Per2-luc, explant culture of lung from Per2::Luciferase knockin mice, and zebrafish larvae in vivo. Notably, more than half of the major ingredients of these crude drugs were reported to target AKT and its relevant signaling pathways. As expected, analysis of the major ingredients targeting AKT signaling confirmed the circadian clock-modulating effects. Furthermore, activator and inhibitor of AKT, and triple knockdown of AKT isoforms by siRNA also modulated the circadian rhythm. This study, by employing the drug repositioning approach, shows that Kampo medicines are a useful source for the identification of underlying mechanisms of circadian clock modulators and could potentially be used in the treatment of circadian clock disruption.

Amber Extract Reduces Lipid Content in Mature 3T3-L1 Adipocytes by Activating the Lipolysis Pathway.

Amber-the fossilized resin of trees-is rich in terpenoids and rosin acids. The physiological effects, such as antipyretic, sedative, and anti-inflammatory, were used in traditional medicine. This study aims to clarify the physiological effects of amber extract on lipid metabolism in mouse 3T3-L1 cells. Mature adipocytes are used to evaluate the effect of amber extract on lipolysis by measuring the triglyceride content, glucose uptake, glycerol release, and lipolysis-related gene expression. Our results show that the amount of triacylglycerol, which is stored in lipid droplets in mature adipocytes, decreases following 96 h of treatment with different concentrations of amber extract. Amber extract treatment also decreases glucose uptake and increases the release of glycerol from the cells. Moreover, amber extract increases the expression of lipolysis-related genes encoding perilipin and hormone-sensitive lipase (HSL) and promotes the activity of HSL (by increasing HSL phosphorylation). Amber extract treatment also regulates the expression of other adipocytokines in mature adipocytes, such as adiponectin and leptin. Overall, our results indicate that amber extract increases the expression of lipolysis-related genes to induce lipolysis in 3T3-L1 cells, highlighting its potential for treating various obesity-related diseases.