Preparation and evaluation of a niosomal delivery system containing G. mangostana extract and study of its anti-Acanthamoeba activity.

Garcinia mangostana extract (GME) has severe pharmacokinetic deficiencies and is made up of a variety of bioactive components. GME has proven its anti-Acanthamoeba effectiveness. In this investigation, a GME-loaded niosome was developed to increase its potential therapeutic efficacy. A GME-loaded niosome was prepared by encapsulation in a mixture of span60, cholesterol, and chloroform by the thin film hydration method. The vesicle size, zeta potential, percentage of entrapment efficiency, and stability of GME-loaded niosomes were investigated. The values for GME-loaded niosome size and zeta potential were 404.23 ± 4.59 and -32.03 ± 0.95, respectively. The delivery system enhanced the anti-Acanthamoeba activity, which possessed MIC values of 0.25-4 mg mL-1. In addition, the niosomal formulation decreased the toxicity of GME by 16 times. GME-loaded niosome must be stored at 4 °C, as the quantity of remaining GME encapsulated is greater at this temperature than at room temperature. SEM revealed the damage to the cell membrane caused by trophozoites and cysts, which led to dead cells. In light of the above, it was found that GME-loaded niosomes had better anti-Acanthamoeba activity. The study suggested that GME-loaded niosomes could be used as an alternative to Acanthamoeba's therapeutic effects.

Evaluation of Antibacterial, Antibiofilm, Antioxidant, and Anti-Inflammatory Activities of Kratom Leaves (Mitragyna speciosa) Fermentation Supernatant Containing Lactobacillus rhamnosus GG.

Kratom (Mitragyna speciosa) leaves are commonly used to enhance endurance and treat various diseases. This study evaluated the effect of kratom leaf fermentation with Lactobacillus rhamnosus. Antibacterial activity was investigated against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Escherichia coli, and E. coli O157:H7. Biofilm inhibition and eradication assays were also performed. Antioxidant properties were determined by measuring the total phenolic and flavonoid content and DPPH and ABTS scavenging activities. Nitric oxide and TNF-α, IL-1ß, and IL-6 expressions in LPS-stimulated RAW 264.7 macrophage cells were also measured. Aqueous kratom extract exhibited promising effects against free radicals and pro-inflammatory cytokines. Notably, all fermented kratoms showed significant antibacterial activity against the tested pathogens and antibiofilm formation by S. aureus and MRSA. Furthermore, the eradication of established biofilms of fermented kratoms was observed in S. aureus (day 2, 50 mg/mL) and E. coli (day 2, 100 mg/mL and day 4, 50 mg/mL). To the best of our knowledge, this study is the first to report that fermented and non-fermented kratoms could be nutraceutical sources of antibacterial, antibiofilm, antioxidant, and anti-inflammatory substances against related diseases and can be applied further in dietary or cosmetic products with health-promoting effects.

Characterization of probiotic properties and development of banana powder enriched with freeze-dried Lacticaseibacillus paracasei probiotics.

Lacticaseibacillus paracasei is one of the probiotic bacteria widely identified from fermented foods. The application of L. paracasei is commonly used in dairy and non-dairy products. To investigate the probiotic properties of L. paracasei cells including their acid, pepsin, pancreatin, and bile salt tolerances; adhesion ability; antipathogen activity; and antibiotic susceptibility, L. paracasei cells were incorporated into skim milk and lyophilized by freeze drying. Freeze-dried probiotic cells were add to green banana powder and low moisture additive food matrices and a storage analysis of the product was performed. The result showed that L. paracasei cells possessed potentially beneficial probiotic properties to survive stress in the gastrointestinal tract (GIT) and functional abilities as an anti-enteropathogenic agent; they were also safe to use and displayed antibiotic properties. Furthermore, the probiotic freeze-drying technique preserved high probiotic cell survivability (1011 CFU/g). In term of prolonged storage (60 days), the powder product was stable and maintained probiotic survival (107 CFU/g) while excluding non-probiotic growth. In conclusion, L. paracasei displayed probiotic properties in the GIT and was judged to be a highly acceptable product as a probiotics-banana rehydrated beverage.

Anticancer mechanism of 7-α-hydroxyfrullanolide on microtubules and computational prediction of its target binding in triple-negative breast cancer cells.

Background: Triple-negative breast cancer (TNBC) responds poorly to the available drugs; thus, the mortality rate associated with TNBC remains high. 7-α-Hydroxyfrullanolide (7HF) possesses anticancer properties and arrests cells in the G2/M-phase via modulation of several proteins involved in the G2/M-phase transition, as well as the mitotic checkpoint in MDA-MB-468 (TNBC) cells. Microtubules (MTs) dynamically regulate cell division in the G2/M phase and are related to cancer cell stress response. However, antimitotic drug cytotoxicity to multiple cancer resistance developed in response to drugs are obstacles faced to date. Here, the activity and mechanism via which 7HF controls MTs dynamics was investigated in MDA-MB-468 cells. Methods: 7HF uptake by MDA-MB-468 cells was assessed using spectrophotometry. The drug-like properties of 7HF were predicted using the Swiss-absorption, distribution, metabolism, and excretion (ADME) webtool. Then, the effect of 7HF treatment (6, 12, and 24 µM) on the dynamic arrangement of MTs was assessed for 1, 12, and 24 h using indirect immunofluorescence. Polymerization of α- and ß-tubulin was assessed using different 7HF concentrations in a cell-free system for 1 h. Cell proliferation assay with bromodeoxyuridine plus propidium iodide staining and flow cytometry was performed at different 7HF concentrations and time points. The mechanism of action was assessed by detecting the expression of proteins, including Bub3, cyclin B1, p-Cdk1 (Tyr15), Rb, p-Rb (Ser780), Chk1, p-Chk1 (Ser345), Chk2, p-Chk2 (Ser516), and p-H2AX (Ser139), using western blotting. Molecular docking was used to predict the molecular interactions between 7HF and tubulins in MTs. Results: We observed that 7HF was able to enter the MDA-MB-468 cells. The ADME webtool analysis predicted that it possesses the high passive permeation and gastrointestinal absorption properties of drugs. Various concentrations of 7HF disrupted the dynamic arrangement of spindle MTs by causing radial spindle array shrinkage and expansion of fibrous spindle density and radial array lengths in a time-dependent manner. 7HF reduced polymerization of α-, ß-tubulin in dose-dependent manner. 7HF also triggered DNA damage response by inducing G2/M and G1 phase arrests in a concentration and time-dependent manner, which occurred due to the upregulation of Bub3, Chk1, p-Chk1 (Ser345), p-Cdk1 (Tyr15), and cyclin B1. According to molecular docking analysis, 7HF preferred to bind to ß-tubulin over α-tubulin. The lactone, ketone, and hydroxyl groups of 7HF supported the 7HF-tubulin interactions. Hydrogen bonding with a hydrocarbon ring and salt bridge attractive forces were responsible for the binding versatility of 7HF. Conclusions: This is the first study to investigate the molecular mechanism, MTs interacting sites, and the internalization and drug-like properties of 7HF in TNBC cells. The findings will be useful for developing 7HF-based treatment for patients with TNBC.

Anticancer Effects and Molecular Action of 7-α-Hydroxyfrullanolide in G2/M-Phase Arrest and Apoptosis in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is a breast cancer subtype characterized by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 expression. TNBC cells respond poorly to targeted chemotherapies currently in use and the mortality rate of TNBC remains high. Therefore, it is necessary to identify new chemotherapeutic agents for TNBC. In this study, the anti-cancer effects of 7-α-hydroxyfrullanolide (7HF), derived from Grangea maderaspatana, on MCF-7, MDA-MB-231 and MDA-MB-468 breast cancer cells were assessed using MTT assay. The mode of action of 7HF in TNBC cells treated with 6, 12 and 24 µM of 7HF was determined by flow cytometry and propidium iodide (PI) staining for cell cycle analysis and annexin V/fluorescein isothiocyanate + PI staining for detecting apoptosis. The molecular mechanism of action of 7HF in TNBC cells was investigated by evaluating protein expression using proteomic techniques and western blotting. Subsequently, 7HF exhibited the strongest anti-TNBC activity toward MDA-MB-468 cells and a concomitantly weak toxicity toward normal breast cells. The molecular mechanism of action of low-dose 7HF in TNBC cells primarily involved G2/M-phase arrest through upregulation of the expression of Bub3, cyclin B1, phosphorylated Cdk1 (Tyr 15) and p53-independent p21. Contrastingly, the upregulation of PP2A-A subunit expression may have modulated the suppression of various cell survival proteins such as p-Akt (Ser 473), FoxO3a and ß-catenin. The concurrent apoptotic effect of 7HF on the treated cells was mediated via both intrinsic and extrinsic modes through the upregulation of Bax and active cleaved caspase-7-9 expression and downregulation of Bcl-2 and full-length caspase-7-9 expression. Notably, the proteomic approach revealed the upregulation of the expression of pivotal protein clusters associated with G1/S-phase arrest, G2/M-phase transition and apoptosis. Thus, 7HF exhibits promising anti-TNBC activity and at a low dose, it modulates signal transduction associated with G2/M-phase arrest and apoptosis.

Occurrence, Antimicrobial Resistance Profile, and Characterization of Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolates from Minced Meat at Local Markets in Thailand.

Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli exhibits strong multidrug resistance (MDR) to ampicillin and third-generation cephalosporins. This study examined the occurrence, antimicrobial susceptibility, and molecular genetic features of ESBL-producing E. coli isolates from three commonly consumed minced meat varieties, namely pork, chicken, and beef. In total, 150 samples were collected from 10 local markets in Thailand. ESBL-producing E. coli was identified in 78 samples (52%), and minced chicken meat was most contaminated (79.17%). The isolates exhibited potential susceptibility to amikacin (96.16%) and carbapenems (91-95%). However, ESBL-producing E. coli displayed strong resistance to ampicillin and cefpodoxime (100%) and high MDR to 3-5 antibiotic classes (94.87%). Most presumptive ESBL producers harbored ESBL resistance genes (97.44%), most commonly blaTEM (78.21%). Indeed, our results demonstrated that raw minced meat has a high occurrence of ESBL-producing E. coli harboring ESBL resistance genes, highlighting the importance of implementation of sanitary handling practices to reduce microbial contamination in commercial meat as well as the need for consumer education on safe handling and cooking of meat products.

Prevalence and characterization of extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolated from raw vegetables retailed in Southern Thailand.

BACKGROUND: The increasing prevalence of broad-spectrum ampicillin-resistant and third-generation cephalosporin-resistant Enterobacteriaceae, particularly Escherichia coli and Klebsiella pneumoniae, has become a global concern, with its clinical impacts on both human and veterinary medicine. This study examined the prevalence, antimicrobial susceptibility, and molecular genetic features of extended-spectrum ß-lactamase (ESBL)-producing E. coli and K. pneumoniae isolates from 10 types of raw vegetables. METHODS: In total, 305 samples were collected from 9 markets in Nakhon Si Thammarat, Thailand, in 2020. RESULTS: ESBL-producing E. coli and K. pneumoniae isolates were found in 14 of the 305 samples obtained from 7 out of 10 types of vegetables (4.6% of the total). Further, 14 ESBL-producing E. coli (n = 5/14) and K. pneumoniae isolates (n = 9/14) (1.6% and 3.0%, respectively) were highly sensitive to ß-lactam/carbapenem antibiotics (imipenem, 100%). ESBL-producing E. coli (n = 4) and K. pneumoniae isolates (n = 8) were also sensitive to non-ß-lactam aminoglycosides (amikacin, 80.00% and 88.89%, respectively). ESBL producers were most resistant to ß-lactam antibiotics, including ampicillin (85.71%) and the cephalosporins cefotaxime and ceftazidime (64.29%). The most frequently detected gene in ESBL-producing E. coli and K. pneumoniae was blaSHV . However, two ESBL-producing E. coli isolates also carried three other ESBL-encoding variants, blaTEM , blaCTX-M1 , blaGES and blaTEM, blaSHV, blaCTX-M9 , which may be due to their association with food chains and humans. DISCUSSION: Indeed, our results suggest that raw vegetables are an important source of ESBL-resistant E. coli and K. pneumoniae, which are potentially transmittable to humans via raw vegetable intake.

Anti-breast cancer potential of frullanolide from Grangea maderaspatana plant by inducing apoptosis.

Breast cancer is the leading cause of female mortality worldwide. Although there are several modern treatments for breast cancer, there is a high rate of recurrence for the majority of treatments; therefore, the search for effective anticancer agents continues. The present study aimed to investigate the anti-breast cancer potential of frullanolide, a compound which is isolated and purified from the Grangea maderaspatana plant, for selected human breast cancer cell lines (MCF-7, MDA-MB-468 and MDA-MB-231). The MTT assay was used to assess cytotoxic activity in breast cancer cell lines of treatment with frullanolide at 1.25, 2.5, 5.0, 10.0 and 20.0 µg/ml. Additionally, the apoptotic induction ability of frullanolide at various concentrations [0.5×, 1× and 2× half maximal inhibitory concentration (IC50)] was investigated by flow cytometry and western blot analysis. Frullanolide exhibited strong anti-breast cancer activity against MDA-MB-468 (IC50, 8.04±2.69 µg/ml) and weak cytotoxicity against the MCF-7 (IC50, 10.74±0.86 µg/ml) and MDA-MB-231 (IC50, 12.36±0.31 µg/ml) cell lines. The IC50 of frullanolide was high in the human normal epithelial breast cell line (MCF-12A) and mouse fibroblast cell line (L-929). Density plot diagrams revealed that frullanolide induced apoptosis in MCF-7, MDA-MB-468 and MDA-MB-231 cells. Notably, a plausible anticancer mechanism was elucidated via cellular apoptosis by p53-independence in the treated MCF-7 cell line and p53-dependence in the treated MDA-MB-468 and MDA-MB-231 cell lines. In conclusion, the present study demonstrated that frullanolide may exert anticancer activity on breast cancer cell lines by inducing apoptosis. Frullanolide offers a possible novel approach to breast cancer therapy.