Preparation and Physicochemical Properties of Vinblastine Microparticles by Supercritical Antisolvent Process

The objective of the study was to prepare vinblastine microparticles by supercritical antisolvent process using N-methyl-2-pyrrolidone as solvent and carbon dioxide as antisolvent and evaluate its physicochemical properties. The effects of four process variables, pressure, temperature, drug concentration and drug solution flow rate, on drug particle formation during the supercritical antisolvent process, were investigated. Particles with a mean particle size of 121 ± 5.3 nm were obtained under the optimized process conditions (precipitation temperature 60 °C, precipitation pressure 25 MPa, vinblastine concentration 2.50 mg/mL and vinblastine solution flow rate 6.7 mL/min). The vinblastine was characterized by scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, mass spectrometry and dissolution test. It was concluded that physicochemical properties of crystalline vinblastine could be improved by physical modification, such as particle size reduction and generation of amorphous state using the supercritical antisolvent process. Furthermore, the supercritical antisolvent process was a powerful methodology for improving the physicochemical properties of vinblastine.     

Micronization of taxifolin by supercritical antisolvent process and evaluation of radical scavenging activity

The aim of this study was to prepare micronized taxifolin powder using the supercritical antisolvent precipitation process to improve the dissolution rate of taxifolin. Ethanol was used as solvent and carbon dioxide was used as an antisolvent. The effects of process parameters, such as temperature (35-65 °C), pressure (10-25 MPa), solution flow rate (3-6 mL/min) and concentration of the liquid solution (5-20 mg/mL) on the precipitate crystals were investigated. With a lower temperature, a stronger pressure and a lower concentration of the liquid solution, the size of crystals decreased. The precipitation temperature, pressure and concentration of taxifolin solution had a significant effect. However, the solution flow rate had a negligible effect. It was concluded that the physicochemical properties and dissolution rate of crystalline taxifolin could be improved by physical modification such as particle size reduction using the supercritical antisolvent (SAS) process. Further, the SAS process was a powerful methodology for improving the physicochemical properties and radical scavenging activity of taxifolin.     

Preparation and Physicochemical Properties of 10-Hydroxycamptothecin (HCPT) Nanoparticles by Supercritical Antisolvent (SAS) Process

The goal of the present work was to study the feasibility of 10-hydroxycamptothecin (HCPT) nanoparticle preparation using supercritical antisolvent (SAS) precipitation. The influences of various experimental factors on the mean particle size (MPS) of HCPT nanoparticles were investigated. The optimum micronization conditions are determined as follows: HCPT solution concentration 0.5 mg/mL, the flow rate ratio of CO(2) and HCPT solution 19.55, precipitation temperature 35 °C and precipitation pressure 20 MPa. Under the optimum conditions, HCPT nanoparticles with a MPS of 180 ± 20.3 nm were obtained. Moreover, the HCPT nanoparticles obtained were characterized by Scanning electron microscopy, Dynamic light scattering, Fourier-transform infrared spectroscopy, High performance liquid chromatography-mass spectrometry, X-ray diffraction and Differential scanning calorimetry analyses. The physicochemical characterization results showed that the SAS process had not induced degradation of HCPT. Finally, the dissolution rates of HCPT nanoparticles were investigated and the results proved that there is a significant increase in dissolution rate compared to unprocessed HCPT.     

Supercritical antisolvent processing of γ-Indomethacin: Effects of solvent, concentration, pressure and temperature on SAS processed Indomethacin

γ-Indomethacin (IMC) is successfully processed with the supercritical antisolvent (SAS) technique. Pure, acicular (needle-like) particles of the α-polymorph are consistently obtained with SAS as the solvent, concentration, temperature and pressures are varied. Controlled changes in process parameters yield significant changes in particle size. Enhanced dissolution profiles are observed with IMC processed with SAS as opposed to the unprocessed IMC. The reduced particle size, as well as the α-polymorphic form of IMC, contributes to the enhanced dissolution rate.     

Screening design of experiment applied to supercritical antisolvent precipitation of amoxicillin: Exploring new miscible conditions

Microparticles of amoxicillin (AMC) have been precipitated by supercritical antisolvent process (SAS) using carbon dioxide and N-methylpyrrolidone (NMP) as antisolvent and solvent, respectively. A fractional factorial design of experiment (DOE) with 2⁷⁻⁴ experiments has been used. Mean particle size (PS) and particle size distribution (PSD) of the processed amoxicillin have been chosen as responses to evaluate the process performance. In a previous work, a DOE was applied too, but now, the range of operating conditions investigated has been changed to let the process take place in a single supercritical phase. Within this range, concentration is again the key factor having most effect on both PS and PSD and thus, the most important factor for controlling the formation of sub-microparticles of amoxicillin by the SAS technique. Moreover, all the experiments included in the new design matrix led to a successful precipitation of amoxicillin.     

Supercritical antisolvent precipitation of andrographolide from Andrographis paniculata extracts: Effect of pressure, temperature and CO2 flow rate

Andrographis paniculata extracts were precipitated using the so-called supercritical antisolvent (SAS) technique. Ethanol was used as the solvent and compressed CO₂ as the antisolvent. The effects of process operating conditions (pressure: 5-24 MPa, temperature: 308-328 K and CO₂ flow rate: 0.5-1.5 g/min) on particle size and morphology of precipitated andrographolide were evaluated. X-ray diffraction (XRD) patterns showed significant changes in andrographolide morphology depending on process operating conditions; both column-like and slice-like crystals were observed depending on operating conditions. Crystals with mean diameters of 3.30-228.35 μm were produced, smaller crystals were obtained at high pressure, low temperature and high CO₂ flow rate and vice versa for large crystals. In addition, SAS process also produced high precipitation yields, since solubility of andrographolide is small in the supercritical CO₂ plus ethanol. When operating under subcritical conditions, amorphous particles were produced.     

Triterpenoids from the Roots of Rhaphiolepis indica var. tashiroi and Their Anti-Inflammatory Activity

Two new triterpenoids, 2α,3β-dihydroxyolean-11,13(18)-dien-19β,28-olide (1) and 3β,5β-dihydroxyglutinol (2), together with eight known compounds (3–10) were isolated from the roots of Rhaphiolepis indica var. tashiroi (Rosaceae). The structures of 1–10 were determined by spectroscopic techniques. Among these isolates, 2α,3β-dihydroxyolean-13(18)-en-28-oic acid (9) exhibited inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide production, with an IC₅₀ value of 16.50 μM.     

Bioactive Chemical Constituents from the Brown Alga Homoeostrichus formosana

A new chromene derivative, 2-(4'',8''-dimethylnona-3''E,7''-dienyl)-8-hydroxy-2,6-dimethyl-2H-chromene (1) together with four known natural products, methylfarnesylquinone (2), isololiolide (3), pheophytin a (4), and β-carotene (5) were isolated from the brown alga Homoeostrichus formosana. The structure of 1 was determined by extensive 1D and 2D spectroscopic analyses. Acetylation of 1 yielded the monoacetylated derivative 2-(4'',8''-dimethylnona-3''E,7''-dienyl)-8-acetyl-2,6-dimethyl-2H-chromene (6). Compounds 1–6 exhibited various levels of cytotoxic, antibacterial, and anti-inflammatory activities. Compound 2 was found to display potent in vitro anti-inflammatory activity by inhibiting the generation of superoxide anion (IC₅₀ 0.22 ± 0.03 μg/mL) and elastase release (IC₅₀ 0.48 ± 0.11 μg/mL) in FMLP/CB-induced human neutrophils.     

Recrystallization of sulfathiazole and chlorpropamide using the supercritical fluid antisolvent process

The supercritical antisolvent (SAS) process was used to modify the solid–state properties of sulfathiazole and chlorpropamide. Acetone, methanol, and ethyl acetate were employed as solvents for the pharmaceutical compounds, and carbon dioxide was used as an antisolvent. The effects of process parameters on the precipitate crystals such as carbon dioxide injection rate, type of solvent, and temperature were investigated. The SAS processed crystals show more ordered appearances with clean surfaces and sharp angles compared with the unprocessed particles. The crystal habit changed from tabular to acicular when the carbon dioxide injection rate increased. The X-ray diffraction patterns of the two compounds revealed variations of crystallinity and crystal orientations depending upon the injection rate, where the degree of crystallinity was found to be inversely proportional to the rate of injection. The analysis of differential scanning calorimetry indicated that both the injection rate and temperature influence the crystal's thermal stability which is related to the solid–solid transition and fusion. The crystal size significantly increased when the nucleation and crystal growth took place at a slow rate.     

Anti-Inflammatory Components from the Root of Solanum erianthum

Two new norsesquiterpenoids, solanerianones A and B (1–2), together with nine known compounds, including four sesquiterpenoids, (−)-solavetivone (3), (+)-anhydro-β-rotunol (4), solafuranone (5), lycifuranone A (6); one alkaloid, N-trans-feruloyltyramine (7); one fatty acid, palmitic acid (8); one phenylalkanoid, acetovanillone (9), and two steroids, β-sitosterol (10) and stigmasterol (11) were isolated from the n-hexane-soluble part of the roots of Solanum erianthum. Their structures were elucidated on the basis of physical and spectroscopic data analyses. The anti-inflammatory activity of these isolates was monitored by nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells. The cytotoxicity towards human lung squamous carcinoma (CH27), human hepatocellular carcinoma (Hep 3B), human oral squamous carcinoma (HSC-3) and human melanoma (M21) cell lines was also screened by using an MTT assay. Of the compounds tested, 3 exhibited the strongest NO inhibition with the average maximum inhibition (E_max) at 100 μM and median inhibitory concentration (IC₅₀) values of 98.23% ± 0.08% and 65.54 ± 0.18 μM, respectively. None of compounds (1–9) was found to possess cytotoxic activity against human cancer cell lines at concentrations up to 30 μM.     

In situ optical monitoring of the solution concentration influence on supercritical particle precipitation

We report a novel approach for the measurement of the location of particle formation in the supercritical antisolvent process (SAS). The measurement strategy is based on in situ Raman and elastic light scattering. In the SAS process, paracetamol was used as the solute, ethanol as the solvent and carbon dioxide as the antisolvent. Experiments were performed under miscible conditions for the binary system ethanol and carbon dioxide at 313 K and pressures between 10 MPa and 17.5 MPa. For high paracetamol concentrations in the injected ethanol solution, particles were found to start precipitating after jet breakup in a multi-phase flow. For low paracetamol concentrations, precipitation starts later in a one-phase flow, when the transient interface (phase boundary) between the injected solution and the supercritical carbon dioxide has diminished.     

Micronization of cilostazol using supercritical antisolvent (SAS) process: Effect of process parameters

The aim of this study was to improve dissolution rate of poorly water-soluble drug, cilostazol, using supercritical antisolvent (SAS) process. The effect of process variables, such as pressure, temperature, drug concentration, type of solvents, feed rate ratio of CO₂/drug solution, on drug particle formation during SAS process was investigated. Particles with mean particle size ranging between 0.90 and 4.52 μm were obtained by varying process parameters such as precipitation vessel pressure and temperature, drug solution concentration, solvent type, feed rate ratio of CO₂/drug solution. In particular, mean particle size and distribution were markedly influenced by drug solution concentration during SAS process. Moreover, the drug did not change its crystal form and the operating parameters might control the ‘crystal texture’ due to the change in crystallinity and preferred orientation during SAS process, as confirmed by differential scanning calorimetry and powder X-ray diffraction study. In addition, the dissolution rate of drug precipitated using SAS process was highly increased in comparison with unprocessed drug. Therefore, it is concluded that the dissolution rate of drug is significantly increased by micronization of cilostazol, leading to the reduction in particle size and increased specific surface area after SAS process.     

Dissolution rate enhancement of the anti-inflammatory drug diflunisal by coprecipitation with a biocompatible polymer using carbon dioxide as a supercritical fluid antisolvent

Dissolution rate enhancement of the anti-inflammatory drug diflunisal was achieved using for the first time a supercritical fluid technology. The supercritical fluid antisolvent (SAS) method was applied to precipitate diflunisal alone and to coprecipitate the drug together with the biocompatible polymer polyvinylpyrrolidone (PVP K-30 and K-10). The untreated and SAS processed diflunisal, and the coprecipitates were characterized in terms of size, morphology, crystallinity, compositions, drug-polymer interactions, and drug release. SAS processed diflunisal exhibited a polymorphic form different from that of the untreated drug. Diflunisal crystallinity disappeared in the coprecipitates. Three different drug: polymer mass ratios were studied: 75:25, 50:50, and 25:75. Microparticle size decreased and aggregation disappeared as the relative amount of polymer increased. The 25:75 coprecipitate consisted of loose spherical particles exhibiting mean particle size of 410 nm while the 75:25 coprecipitate consisted of bigger aggregated particles. The SAS method was shown to be a suitable technology to form solid dispersions of a poorly soluble drug.     

Potential Protection Effect of ER Homeostasis of N6-(2-Hydroxyethyl)adenosine Isolated from Cordyceps cicadae in Nonsteroidal Anti-Inflammatory Drug-Stimulated Human Proximal Tubular Cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) belong to a class of universally and commonly used anti-inflammatory analgesics worldwide. A diversity of drawbacks of NSAIDs have been reported including cellular oxidative stress, which in turn triggers the accumulation of unfolded proteins, enhancing endoplasmic reticulum stress, and finally resulting in renal cell damage. Cordyceps cicadae (CC) has been used as a traditional medicine for improving renal function via its anti-inflammatory effects. N⁶-(2-hydroxyethyl)adenosine (HEA), a physiologically active compound, has been reported from CC mycelia (CCM) with anti-inflammatory effects. We hypothesize that HEA could protect human proximal tubular cells (HK–2) from NSAID-mediated effects on differential gene expression at the mRNA and protein levels. To verify this, we first isolated HEA from CCM using Sephadex^® LH–20 column chromatography. The MTT assay revealed HEA to be nontoxic up to 100 µM toward HK–2 cells. The HK–2 cells were pretreated with HEA (10–20 µM) and then insulted with the NSAIDs diclofenac (DCF, 200 µM) and meloxicam (MXC, 400 µM) for 24 h. HEA (20 µM) effectively prevented ER stress by attenuating ROS production (p < 0.001) and gene expression of ATF–6, PERK, IRE1α, CDCFHOP, IL1β, and NFκB within 24 h. Moreover, HEA reversed the increase of GRP78 and CHOP protein expression levels induced by DCF and MXC, and restored the ER homeostasis. These results demonstrated that HEA treatments effectively protect against DCF- and MXC-induced ER stress damage in human proximal tubular cells through regulation of the GRP78/ATF6/PERK/IRE1α/CHOP pathway.     

Deletion of Specific Conserved Motifs from the N-Terminal Domain of αB-Crystallin Results in the Activation of Chaperone Functions

Smaller oligomeric chaperones of α-crystallins (αA- and αB-) have received increasing attention due to their improved therapeutic potential in preventing protein aggregating diseases. Our previous study suggested that deleting 54–61 residues from the N-terminal domain (NTD) of αB-crystallin (αBΔ54–61) decreases the oligomer size and increases the chaperone function. Several studies have also suggested that NTD plays a significant role in protein oligomerization and chaperone function. The current study was undertaken to assess the effect of deleting conserved 21–28 residues from the activated αBΔ54–61 (to get αBΔ21–28, Δ54–61) on the structure–function of recombinant αBΔ21–28, Δ54–61. The αBΔ21–28, Δ54–61 mutant shows an 80% reduction in oligomer size and 3- to 25-fold increases in chaperone activity against model substrates when compared to αB-WT. Additionally, the αB∆21–28, ∆54–61 was found to prevent β-amyloid (Aβ_1–42) fibril formation in vitro and suppressed Aβ_1–42-induced cytotoxicity in ARPE-19 cells in a more effective manner than seen with αB-WT or αB∆54–61. Cytotoxicity and reactive oxygen species (ROS) detection studies with sodium iodate (SI) showed that the double mutant protein has higher anti-apoptotic and anti-oxidative activities than the wild-type or αB∆54–61 in oxidatively stressed cells. Our study shows that the residues 21–28 and 54–61 in αB-crystallin contribute to the oligomerization and modulate chaperone function. The deletion of conserved 21–28 residues further potentiates the activated αBΔ54–61. We propose that increased substrate affinity, altered subunit structure, and assembly leading to smaller oligomers could be the causative factors for the increased chaperone activity of αBΔ21–28, Δ54–61.     

The Effect of the Aerial Part of Lindera akoensis on Lipopolysaccharides (LPS)-Induced Nitric Oxide Production in RAW264.7 Cells

Four new secondary metabolites, 3α-((E)-Dodec-1-enyl)-4β-hydroxy-5β-methyldihydrofuran-2-one (1), linderinol (6), 4′-O-methylkaempferol 3-O-α-l-(4″-E-p-coumaroyl)rhamnoside (11) and kaempferol 3-O-α-l-(4″-Z-p-coumaroyl) rhamnoside (12) with eleven known compounds—3-epilistenolide D₁ (2), 3-epilistenolide D₂ (3), (3Z,4α,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (4), (3E,4β,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (5), matairesinol (7), syringaresinol (8), (+)-pinoresinol (9), salicifoliol (10), 4″-p-coumaroylafzelin (13), catechin (14) and epicatechin (15)—were first isolated from the aerial part of Lindera akoensis. Their structures were determined by detailed analysis of 1D- and 2D-NMR spectroscopic data. All of the compounds isolated from Lindera akoensis showed that in vitro anti-inflammatory activity decreases the LPS-stimulated production of nitric oxide (NO) in RAW 264.7 cell, with IC₅₀ values of 4.1–413.8 μM.     

Terpenoids from the Octocoral Sinularia gaweli

Two eudesmane sesquiterpenoids, verticillatol (1) and 5α-acetoxy-4(14)-eudesmene-1β-ol (2) and two cembrane diterpenoids, (–)-leptodiol acetate (3) and sinulacembranolide A (4) were isolated from the octocoral Sinularia gaweli and compounds 2–4 are new isolates. The structures of new terpenoids 2–4 were elucidated by spectroscopic methods and by comparison the spectral data with those of known analogues. Terpenoid 4 was found to inhibit the accumulation of the pro-inflammatory inducible nitric oxide synthase (iNOS) protein of the lipopolysaccharide (LPS)-stimulated RAW264.7 marcophage cells.     

Influence of pressure, temperature and concentration on the mechanisms of particle precipitation in supercritical antisolvent micronization

In this work, supercritical antisolvent micronization (SAS) is used to produce nanoparticles, microparticles and expanded microparticles of a model compound, gadolinium acetate (GdAc), using dimethylsulfoxide (DMSO) as the liquid solvent with the aim of studying the dependence of particles’ diameter and morphology on some process parameters like pressure, temperature and concentration of the starting solution. Experiments are performed varying the precipitation pressure between 90 and 200 bar, the precipitation temperature between 35 and 60 °C and the concentration of GdAc in the liquid solution in the range from 20 to 300 mg/mL. The experimental evidences show that the formation of particles with specific sizes in the micrometric and nanometric range depends on specific values of each one of these parameters. An explanation of the results is proposed in terms of the competition between two characteristic times of the SAS process that can control the precipitation process. The time of jet break-up of the liquid solution that produces liquid droplet formation, and the dynamic surface tension vanishing time, that induces gas mixing with the precipitation of nanoparticles from the gaseous phase. Indeed, GdAc sub-microparticle, or microparticle (diameter in the range 0.23-1.6 μm with mean diameters in the range 0.28-0.52 μm) formation can be attributed to micro-droplet drying, whereas nanoparticles (mean diameter in the range 90-210 nm) are consistently produced when gas mixing is the possible governing process. In conclusion, the precipitation mechanisms can be modulated varying one SAS parameter a time, thus selecting the range of particle diameters required for the specific application.     

Novel Epoxides of Soloxolone Methyl: An Effect of the Formation of Oxirane Ring and Stereoisomerism on Cytotoxic Profile,Anti-Metastatic and Anti-Inflammatory Activities In Vitro and In Vivo

It is known that epoxide-bearing compounds display pronounced pharmacological activities, and the epoxidation of natural metabolites can be a promising strategy to improve their bioactivity. Here, we report the design, synthesis and evaluation of biological properties of αO-SM and βO-SM, novel epoxides of soloxolone methyl (SM), a cyanoenone-bearing derivative of 18βH-glycyrrhetinic acid. We demonstrated that the replacement of a double-bound within the cyanoenone pharmacophore group of SM with α- and β-epoxide moieties did not abrogate the high antitumor and anti-inflammatory potentials of the triterpenoid. It was found that novel SM epoxides induced the death of tumor cells at low micromolar concentrations (IC₅₀^(24h) = 0.7–4.1 µM) via the induction of mitochondrial-mediated apoptosis, reinforced intracellular accumulation of doxorubicin in B16 melanoma cells, probably by direct interaction with key drug efflux pumps (P-glycoprotein, MRP1, MXR1), and the suppressed pro-metastatic phenotype of B16 cells, effectively inhibiting their metastasis in a murine model. Moreover, αO-SM and βO-SM hampered macrophage functionality in vitro (motility, NO production) and significantly suppressed carrageenan-induced peritonitis in vivo. Furthermore, the effect of the stereoisomerism of SM epoxides on the mentioned bioactivities and toxic profiles of these compounds in vivo were evaluated. Considering the comparable antitumor and anti-inflammatory effects of SM epoxides with SM and reference drugs (dacarbazine, dexamethasone), αO-SM and βO-SM can be considered novel promising antitumor and anti-inflammatory drug candidates.