Effects of Salts and Other Contaminants on Ciprofloxacin Removal Efficiency of Green Synthesized Copper Nanoparticles.

Ciprofloxacin (CIP), a broad-spectrum fluoroquinolone antibiotic, is commonly used in aquaculture to prevent and treat bacterial infections in aquatic animals. For this reason, aquatic environments contain CIP and its derivatives, which lead to the development of drug-resistant bacteria. In the present study, copper nanoparticles were prepared using Garcinia mangostana extract (GME-CuNPs) as a reducing agent and evaluated for their CIP removal efficiency (CRE). The results demonstrate that within 20 min, GME-CuNPs at 25 mM possess a CRE of 92.02 ± 0.09% from CIP-containing aqueous media with pH 6-7. The CRE is influenced by both monovalent and divalent salts. A high salt concentration significantly reduces the CRE. Contaminants in fish wastewater can reduce the CRE, but phenolics, flavonoids, tannins, and ammonia do not affect the CRE. Our results reveal that the CRE is controlled by electrostatic attraction between the negatively charged GME-CuNPs and the cationic species of CIP. The CRE is reduced by wastewater with a pH higher than 8.0, in which the CIP molecules have a negative charge, resulting in a repulsive force due to the negative charge of GME-CuNPs. In fish wastewater with a pH lower than 7.0, GME-CuNPs show the potential to achieve a CRE above 80%. Therefore, pH adjustment to a range of 6-7 in fish wastewater before treatment is deemed imperative. It is concluded that the newly developed GME-CuNPs possess excellent activity in CIP elimination from actual fish wastewater samples. Our findings suggest that GME-CuNPs can be a promising tool to effectively eliminate antibiotics from the environment.

Modification of Polyethylene Glycol-Hydroxypropyl Methacrylate Polymeric Micelles Loaded with Curcumin for Cellular Internalization and Cytotoxicity to Wilms Tumor 1-Expressing Myeloblastic Leukemia K562 Cells.

Curcumin loaded in micelles of block copolymers of ω-methoxypoly(ethylene glycol) and N-(2-hydroxypropyl) methacrylamide modified with aliphatic dilactate (CD) or aromatic benzoyl group (CN) were previously reported to inhibit human ovarian carcinoma (OVCAR-3), human colorectal adenocarcinoma (Caco-2), and human lymphoblastic leukemia (Molt-4) cells. Myeloblastic leukemia cells (K562) are prone to drug resistance and differ in both cancer genotype and phenotype from the three mentioned cancer cells. In the present study, CD and CN micelles were prepared and their effects on K562 and normal cells were explored. The obtained CD and CN showed a narrow size distribution with diameters of 63 ± 3 and 50 ± 1 nm, respectively. The curcumin entrapment efficiency of CD and CN was similarly high, above 80% (84 ± 8% and 91 ± 3%). Both CD and CN showed suppression on WT1-expressing K562 and high cell-cycle arrest at the G2/M phase. However, CD showed significantly higher cytotoxicity to K562, with faster cellular uptake and internalization than CN. In addition, CD showed better compatibility with normal red blood cells and peripheral blood mononuclear cells than CN. The promising CD will be further investigated in rodents and possibly in clinical studies for leukemia treatment.

Solubility and Physical Stability Enhancement of Loratadine by Preparation of Co-Amorphous Solid Dispersion with Chlorpheniramine and Polyvinylpyrrolidone.

Loratadine (LRD), a non-sedating and slow-acting antihistamine, is often given in combination with short-onset chlorpheniramine maleate (CPM) to increase efficacy. However, LRD has poor water solubility resulting in low bioavailability. The aim of this study was to improve LRD solubility by preparing co-amorphous LRD-CPM. However, the obtained co-amorphous LRD-CPM recrystallized rapidly, and the solubility of LRD returned to a poor state again. Therefore, co-amorphous LRD-CPM solid dispersions using polyvinylpyrrolidone (PVP) as a carrier were prepared. The obtained solid dispersions were characterized using X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FT-IR). The solubility, dissolution, and mechanism of drug release from the LRD-CPM/PVP co-amorphous solid dispersions were studied and compared with those of intact LRD, LRD/PVP solid dispersions, and co-amorphous LRD-CPM mixtures. The results from XRPD and DSC confirmed the amorphous form of LRD in the co-amorphous solid dispersions. The FTIR results indicated that there was no intermolecular interaction between LRD, CPM, and PVP. In conclusion, the obtained LRD-CPM/PVP co-amorphous solid dispersions can successfully increase the water solubility and dissolution of LRD and extend the amorphous state of LRD without recrystallization.

Effects of plant-based copper nanoparticles on the elimination of ciprofloxacin.

Ciprofloxacin (CIP) is frequently detected in the environment and causes the emergence of drug-resistant bacteria. High levels of CIP in the environment are also harmful to humans and animals. Therefore, effective elimination of CIP is required. In this study, plant-based copper nanoparticles (CuNPs) have been fabricated for the purpose of eliminating CIP. Aqueous extracts of 6 plants were compared for their phytochemicals and reducing activity. Among all the extracts, Garcinia mangostana extract (GM) was the most potent with the highest total phenolic compounds, flavonoids, tannins, terpenoids, and reducing activity. CuNPs synthesized using GM (GM-CuNPs) were characterized using UV-VIS spectroscopy and dynamic light scattering. The results showed that the maximum absorption of GM-CuNPs was at 340 nm. The average size of GM-CuNPs is in the nanoscale range of 159.2 ± 61 nm, with a narrow size distribution and a negative zeta potential of - 4.13 ± 6.97 mV. The stability of GM-CuNPs is not solely due to their zeta potential but also phytochemicals in the extract. GM-CuNPs at 25 mM showed the highest efficiency of 95% in removing CIP from aqueous medium pH 6-7 at 25-35°C within 20 min. The results indicated that the electrostatic attraction between the negative charge of GM-CuNPs and the positive charge of CIP controlled the drug adsorption on the nanoparticles. In conclusion, the developed GM-CuNPs have excellent CIP removal efficiency. These synthesized GM-CuNPs are expected to be environmentally friendly for the removal of antibiotics and other drugs.

The Binding of Alpinia galanga Oil and Its Nanoemulsion to Mammal GABAA Receptors Using Rat Cortical Membranes and an In Silico Modeling Platform.

The anesthetic effect of Alpinia galanga oil (AGO) has been reported. However, knowledge of its pathway in mammals is limited. In the present study, the binding of AGO and its key compounds, methyl eugenol, 1,8-cineole, and 4-allylphenyl acetate, to gamma-aminobutyric acid type A (GABAA) receptors in rat cortical membranes, was investigated using a [3H]muscimol binding assay and an in silico modeling platform. The results showed that only AGO and methyl eugenol displayed a positive modulation at the highest concentrations, whereas 1,8-cineole and 4-allylphenyl acetate were inactive. The result of AGO correlated well to the amount of methyl eugenol in AGO. Computational docking and dynamics simulations into the GABAA receptor complex model (PDB: 6X3T) showed the stable structure of the GABAA receptor-methyl eugenol complex with the lowest binding energy of -22.16 kcal/mol. This result shows that the anesthetic activity of AGO and methyl eugenol in mammals is associated with GABAA receptor modulation. An oil-in-water nanoemulsion containing 20% w/w AGO (NE-AGO) was formulated. NE-AGO showed a significant increase in specific [3H]muscimol binding, to 179% of the control, with an EC50 of 391 µg/mL. Intracellular studies show that normal human cells are highly tolerant to AGO and the nanoemulsion, indicating that NE-AGO may be useful for human anesthesia.

Masculinizing Effects of Chrysin-Loaded Poloxamer Micelles on Siamese Fighting Fish.

Siamese fighting fish (Betta splendens) are freshwater fish that are commonly found in Thailand and other Southeast Asian countries. In the present study, chrysin-loaded polymeric micelles (CPs) were developed and investigated for the masculinizing effects, survival rate, growth indices, and toxicity on Siamese fighting fish. CPs were prepared using a poloxamer. The micelle system of CPs that were formed at a chrysin-to-polymer ratio of 1:2 was found to be the most suitable monodispersed system and exhibited a nanosized diameter (74.2 ± 1.6 nm) with a narrow size distribution (0.288 ± 0.012). In vivo studies were performed using Siamese fighting fish larvae as animal models. In the in vivo toxicity study, the fish larvae were immersed in aqueous systems containing CPs that had five different chrysin concentrations of 1, 10, 100, 1000, and 10,000 ng/mL for 24, 48, and 72 h. Blank polymeric micelles and water were used as controls. The in vivo masculinization effect of CPs with different chrysin concentrations on the fish larvae was evaluated after 5 weeks of exposure. The results demonstrated that CPs with a chrysin concentration of 1000 ng/mL showed a masculinization effect of 94.59 ± 2.76% with a high fish larvae survival rate of 72.45 ± 5.09% and low toxicity. It was concluded that the developed CPs had a significant effect on the sex reversal of Siamese fighting fish larvae with a high survival rate.

Formulation optimization, anesthetic activity, skin permeation, and transportation pathway of Alpinia galanga oil SNEDDS in zebrafish (Danio rerio).

Alpinia galanga oil (AGO) has an anesthetic activity but its water insoluble property limits its clinical applications. The aim of the present study was to develop a self-nanoemulsifying drug delivery system of AGO (SNEDDS-AGO) to avoid the use of organic solvent and investigate AGO transportation pathway and anesthetic activity. Three optimized formulations from a contour plots of droplet size; SNEDDS-AGO-1, SNEDDS-AGO-2, and SNEDDS-AGO-3, composed of AGO, Miglyol 812, Cremophor RH 40, Capmul MCM EP, and ethanol at the ratios of 40:10:35:10:5, 40:20:15:20:5, and 60:10:15:10:5, respectively were selected as they possessed different droplet size of 62 ± 0.5, 107 ± 2.8, and 207 ± 4.3 nm, respectively. It was found that the droplet size played an important role in fish anesthesia. SNEDDS-AGO-3 showed the longest anesthetic induction time (270 sec) (p < 0.03). Transportation pathway and skin permeation of SNEDDS-AGO-2 were investigated using nile red labelled AGO and detected by fluorescence microscope. AGO was found mostly in brain, gills, and skin suggesting that the transportation pathway of AGO in zebrafish is passing through the gills and skin to the brain. SNEDDS-AGO formulations showed significantly higher permeation through the skin than AGO ethanolic solution. In conclusion, SNEDDS is a promising delivery system of AGO.

Development of Microemulsion Containing Alpinia galanga Oil and Its Major Compounds: Enhancement of Antimicrobial Activities.

The aim of the present study was to develop a microemulsion (ME) containing Alpinia galanga oil (AGO), 1,8-cineole (C), or methyl eugenol (M) as an active pharmaceutical ingredient (API) for enhancing their antimicrobial activities. Agar diffusion, broth microdilution, and killing kinetics were used for antimicrobial evaluations. The ME composed of 30% API, 33.4% Tween 80, 16.6% ethanol, and 20% water appeared as translucent systems with droplet size and polydispersity index of 101.1 ± 1.3 nm and 0.3 ± 0.1, 80.9 ± 1.1 nm and 0.4 ± 0.1, and 96.6 ± 2.0 nm and 0.2 ± 0.1 for ME-AGO, ME-C, and ME-M, respectively. These ME formulations showed minimum bacterial concentrations of 3.91-31.25 µg/mL and 50% fungal inhibition concentrations of 1.83 ± 0.27-0.46 ± 0.13 µg/mL, 2-4 times stronger, and faster kinetic killing rate than their respective API alone. Keeping the ME formulations at 4 °C, 25 °C, and 40 °C for 12 weeks did not affect their activities against fungi and Gram-negative bacteria, but the high temperature of 40 °C decreased their activities against Gram-positive bacteria. It is concluded that ME is a promising delivery system for AGO and its major compounds to enhance their water miscibility and antimicrobial activities.

Anesthetic activity of plant essential oils on Cyprinus carpio (koi carp).

The aims of this study were to investigate the anesthetic and cytotoxic effects of essential oils (EOs) of Ocimum basilicum (OBO), O. canum (OCO), and O. sanctum (OSO) on Cyprinus carpio (koi carp). For anesthetic effect, induction time to surgical anesthesia and recovery time were determined. For cytotoxicity effect, viability of fish peripheral blood nuclear cells (PBMCs) was investigated. Results indicated that increasing oil concentration caused significant (p < 0.01) decrease of induction time. OSO at 100, 200, and 300 mg/L gave the induction time of 169.5 ± 10.2, 62.8 ± 2.3, 45.3 ± 2.2 sec, respectively, significantly shorter than OCO, and OBO. The recovery time of anesthetized fish was dose dependent (p <0.01). Among them, OCO showed the longest recovery time of 313.0 ± 8.1, 420.7 ± 12.6, 616.6 ± 12.1 sec for concentrations of 100, 200, and 300 mg/L, respectively, followed by OSO and OBO, respectively. Within 10 min contact time of the EOs and fish PBMCs, the fish PBMC viability was higher than 80%. Increase contact time and EO concentration caused an increase in cytotoxicity to fish PBMC. OBO showed less toxic than OSO and OCO. Based on the desired induction and recovery times for anesthetizing koi carp, OBO, OCO, and OSO at 300, 200, and 100 mg/L, respectively were suggested to be the most suitable. It was concluded that OBO, OCO, and OSO can be used as natural anesthetics for fish.

Cytotoxicity and inhibition of leukemic cell proliferation by sesquiterpenes from rhizomes of Mah-Lueang (Curcuma cf. viridiflora Roxb.).

Curcuma cf. viridiflora Roxb., also known as Mah-Lueang in Thai, belongs to the Zingiberaceae family and is grown from rhizomes. The rhizome of the plant has been used for medicinal purposes, in particular, to treat paralysis in Thai traditional medicine. However, no biologically active compounds have been reported from Mah-Lueang yet. In this study, natural compounds were isolated from Mah-Lueang and structurally determined by spectroscopic methods, including electrospray ionization mass spectrometry and nuclear magnetic resonance. The four isolated compounds were identified as furanodiene (1), dehydrocurdione (2), germacrone-4,5-epoxide (3), and zedoarondiol (4). These sesquiterpenes were investigated for antileukemic activities against KG1a and Molt4 cells. Leukemic cell proliferation is regulated by the Wilms' tumor 1 (WT1) transcription factor. Compound 1 showed the strongest cytotoxicity against both KG1a and Molt4 cells. Noncytotoxic concentrations (20% inhibitory concentration values) of all compounds were able to decrease the WT1 protein expression and total cell numbers in both cell lines. The four compounds showed good inhibitory activities for WT1 protein expression. Compounds 3 and 4 showed excellent antileukemic activities for both cell lines. In summary, four sesquiterpene compounds with antileukemic activities against the KG1a and Molt4 cell lines were identified in Mah-Lueang extracts.

Self-microemulsifying drug delivery system and nanoemulsion for enhancing aqueous miscibility of Alpinia galanga oil.

Alpinia galanga oil (AGO) possesses various activities but low aqueous solubility limits its application particularly in aquatic animals. AGO has powerful activity on fish anesthesia. Ethanol used for enhancing water miscible of AGO always shows severe side effects on fish. The present study explores the development of self-microemulsifying drug delivery systems (SMEDDS) and nanoemulsions (NE) to deliver AGO for fish anesthesia with less or no alcohol. Pseudoternary phase diagrams were constructed to identify the best SMEDDS-AGO formulation, whereas NE-AGO were developed by means of high-energy emulsification. The mean droplet size of the best SMEDDS-AGO was 82 ± 0.5 nm whereas that of NE-AGO was 48 ± 1.6 nm. The anesthetic effect of the developed SMEDDS-AGO and NE-AGO in koi (Cyprinus carpio) was evaluated and compared with AGO ethanolic solution (EtOH-AGO). It was found that the time of induction the fish to reach the surgical stage of anesthesia was dose dependent. NE-AGO showed significantly higher activity than SMEDDS-AGO and EtOH-AGO, respectively. EtOH-AGO caused unwanted hyperactivity in the fish. This side effect did not occur in the fish anesthetized with SMEDDS-AGO and NE-AGO. In conclusion, SMEDDS and NE are promising delivery systems for AGO.

Effects of Alpinia galanga oil on anesthesia and stress reduction in Oreochromis niloticus.

Oreochromis niloticus (Nile tilapia) is one widely cultured fish in Thailand. Handling processes and transportation causes high stress in Nile tilapia. This study explores anesthetic effect and stress reduction of Alpinia galanga oil (AGO) on Nile tilapia. The anesthetic activity was evaluated by the time for fish induction to anesthesia and full recovery. It was found that the suitable dose of AGO that caused desirable anesthesia of Nile tilapia was 700 mg/L. This dose gave induction and recovery times of approximately 257 and 438 sec, respectively. Blood glucose and plasma cortisol of the fish anesthetized with AGO showed nearly normal levels indicating that the fish stress during handling was not increased. Study on loading densities of fish mimicked general fish transportation and showed that loading density of fish was a crucial factor on fish stress. The highest water quality was found in the lowest loading density of fish. Water containing AGO at a concentration of 150 mg/L showed significantly higher potential for reducing fish activity and water improvement than without AGO. Therefore, AGO is a promising natural edible plant oil for anesthesia in Nile tilapia.

Eulophia macrobulbon - an orchid with significant anti-inflammatory and antioxidant effect and anticancerogenic potential exerted by its root extract.

BACKGROUND: The Orchidaceae family is one of the largest families of flowering plants. Orchids are widely used for the traditional herbal medicine, acting as aphrodisiac, antisepic, antimicrobial, anti-cancer agent, etc. PURPOSE: This study was designed to elucidate the anti-inflammatory, antioxidant and cytotoxic potential of a 50% ethanolic extract of Eulophia macrobulbon roots (EME) in vitro, an orchid growing in Southern Asia. Furthermore, the main active compounds were isolated, and the bioactivity of the single constituents was determined. METHODS: The anti-inflammatory activity of EME and its compounds was evaluated by the secretion of pro- and anti-inflammatory cytokines and by the expression of inducible nitric oxide synthase (iNOS) in a lipopolysaccharide (LPS)-stimulated macrophage model, as determined by an enzyme linked immunosorbent assay (ELISA) and Western blot. Antioxidant activity was assessed using a DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) photometric assay. Cytotoxic effects were determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay. RESULTS: EME and its compounds significantly reduced the production of the proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α), the expression of iNOS and subsequently increased the production of the anti-inflammatory cytokine interleukin 10 (IL-10) in LPS-stimulated macrophages. Additionally it could be demonstrated that EME is rich in radical scavengers. Furthermore, EME and its components showed notable cytotoxic effects on the human cervical adenocarcinoma cell line HeLa, the human colorectal adenocarcinoma cell line CaCo-2 and the human breast adenocarcinoma cell line MCF-7. The most active constituents were identified as 4-methoxy-9,10-dihydro-2,7-phenanthrenediol (8), 4-methoxy-2,7-phenanthrenediol (9), 1,5-dimethoxy-2,7-phenanthrenediol (10), 1,5,7-trimethoxy-2,6-phenanthrenediol (11), 1-(4-hydroxybenzyl)-4,8-dimethoxy-2,7-phenanthrenediol (15). CONCLUSION: Based on this data, EME provides various beneficial anti-inflammatory, antioxidant and cytotoxic attributes and may be used as herbal remedy in the pharmaceutical or food industries.