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.

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.

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.

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.