The challenge of improving pterostilbene (PTS) solubility for solid and semi-solid dosage forms: The obtention of binary and ternary systems.

Pterostilbene (PTS) is a drug candidate with low water solubility and poor bioavailability. On the other hand, drug:cyclodextrins complexes frequently provide bulk powders with low drug concentrations, which is crucial for obtention solid or semi-solid pharmaceutical dosage forms. In order to determine the optimal conditions for enhancing the solubility of PTS:BCD (ß-cyclodextrin) complex, a Box-Behnken design was performed. Although the optimal conditions have been applied, low complexation efficiency (0.127) and the bulk powder remained. A PTS:BCD:HPMC (HPMC, hydroxypropyl methylcellulose) ternary system was developed to overcome this limitation, comparing two media, water and a mixture of ethanol-water. When ethanol was used as a co-solvent, the PTS:BCD:HPMC ternary system (freeze-dried) contained 116.65 ± 1.40 mg/g of PTS. This value was 3.4-fold higher than the PTS content observed when the same ternary system was obtained in aqueous media (34.8 mg/g) and 2.8-fold higher than the PTS content observed for PTS:BCD complex (freeze-dried) obtained using ethanol as a co-solvent. Dissolution tests revealed that after 120 min, in a buffer with a pH value of 1.2, only 43% of PTS dissolved. In contrast, 80% and 90% of PTS were dissolved from the PTS:BCD complex and PTS:BCD:HPMC ternary system, respectively. Moreover, the dissolution was fast in a buffer with a pH value of 6.8. PTS:BCD complex reached the maximum PTS dissolution at 75 min and PTS:BCD:HPMC at 45 min. In summary, the results of this study demonstrated, for the first time, that low-bulk powders with a high content of PTS can be obtained from PTS:BCD:HPMC ternary systems using ethanol as a co-solvent. This new finding offers a valuable alternative for producing solid or semi-solid formulations containing highly soluble PTS.

The challenge of flavonoid/cyclodextrin complexation in a complex matrix of the quercetin, luteolin, and 3-O-methylquercetin.

The complexation of herbal constituents with cyclodextrin has been a useful tool to improve their aqueous solubility. However, the simultaneous complexation of these compounds still lacks detailed studies. The present study investigated the multicomplexation of quercetin (QCT), luteolin (LUT), and 3-O-methylquercetin (3OMQ) with (2-hydroxypropyl)-ß-cyclodextrin (HPßCD), when they are simultaneously contained in a flavonoid-enriched fraction (FEF) of Achyrocline satureioides. The phase-solubility diagram revealed a linear correlation between the flavonoids solubility and the HPßCD concentration, demonstrating the formation of complexes with a 1:1 stoichiometric ratio, which was confirmed by ESI-MS. Negative ΔG0 values indicated that complexation was spontaneous. Flavonoids/HPßCD interactions were evidenced by FT-IR, DSC, SEM, and 1D and 2D NMR. The last one showed the formation of inclusion complexes by insertion of the B-ring of the flavonoids into the cavity of HPßCD. Unexpectedly, the FEF/HPßCD complex showed a radical scavenger potential lower than the FEF. The HPLC analysis revealed that the complex contained different flavonoid ratio than the fraction. Thus, the antioxidant capacity of the samples was demonstrated to be related to the ratio among the flavonoids, rather than to the total flavonoids. These new findings are very useful for developing herbal cyclodextrin-based products from A. satureioides or other herbal products.

Poloxamer-enhanced solubility of griseofulvin and its related antifungal activity against Trichophyton spp

Abstract Poorly water-soluble drugs, such as the antifungal drug griseofulvin (GF), exhibit limited bioavailability, despite their high membrane permeability. Several technological approaches have been proposed to enhance the water solubility and bioavailability of GF, including micellar solubilization. Poloxamers are amphiphilic block copolymers that increase drug solubility by forming micelles and supra-micellar structures via molecular self-association. In this regard, the aim of this study was to evaluate the water solubility increment of GF by poloxamer 407 (P407) and its effect on the antifungal activity against three Trichophyton mentagrophytes and two T. rubrum isolates. The GF water solubility profile with P407 revealed a non-linear behavior, well-fitted by the sigmoid model of Morgan-Mercer-Flodin. The polymer promoted an 8-fold increase in GF water solubility. Fourier-transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), and 2D nuclear magnetic resonance (NMR Roesy) spectroscopy suggested a GF-P407 interaction, which occurs in the GF cyclohexene ring. These results were supported by an increase in the water solubility of the GF impurities with the same molecular structure. The MIC values recorded for GF ranged from 0.0028 to 0.0172 mM, except for T. Mentagrophytes TME34. Notably, the micellar solubilization of GF did not increase its antifungal activity, which could be related to the high binding constant between GF and P407.

Optimization of Coumarins Extraction from Pterocaulon balansae by Box-Behnken Design and Anti-Trichomonas vaginalis Activity.

Trichomonas vaginalis causes trichomoniasis, a nonviral sexually transmitted infection with a high prevalence worldwide. Oral metronidazole is the drug of choice for the treatment of this disease, although high levels of T. vaginalis resistance to this agent are well documented in the literature. This study describes the anti-T. vaginalis activity of an optimized coumarin-rich extract from Pterocaulon balansae. Optimization was performed to maximize extraction of total coumarins by means of a 3-level Box-Behnken design, evaluating the effect of three factors: extraction time, plant : solvent ratio, and ethanol concentration. Optimum conditions were found to be 5 h extraction time and a plant : solvent ratio of 1% (w/v) and 60% (v/v) ethanol, which resulted in approximately 30 mg of total coumarins/g of dry plant. The coumarin-enriched extract exhibited a minimum inhibitory concentration of 30 µg/mL and an IC50 of 3.2 µg/mL against T. vaginalis, a low cytotoxicity, and a high selectivity index (18 for vaginal epithelial cells and 16 for erythrocytes). The coumarins permeation/retention profile through porcine vaginal mucosa was evaluated in Franz-type diffusion cells. After 8 h of kinetics, coumarins were detected in the tissue (4.93 µg/g) without detecting them in the receptor compartment. A significant increase of coumarins in the mucosa layers (8.18 µg/g) and receptor compartment (0.26 µg/g) was detected when a T. vaginalis suspension (2 × 105 trophozoites/mL) was previously added onto the mucosa. No alterations were visualized in the stratified squamous non-keratinized epithelium of the porcine vaginal mucosa after contact with the extract. Overall, these results suggest that the P. balansae coumarin-rich extract may have potential as a treatment for trichomoniasis.

3-O-Methylquercetin from Achyrocline satureioides-cytotoxic activity against A375-derived human melanoma cell lines and its incorporation into cyclodextrins-hydrogels for topical administration.

3-O-Methylquercetin (3OMQ), a natural 3-O-methylflavonoid, was isolated from Achyrocline satureioides and purified using the high-performance counter current chromatography (HPCCC) on a semi-preparative scale. High-purity 3OMQ (98%) was obtained with excellent recovery (81.8% (w/w)) and good yield (190 mg/100 g of plant). Isolated 3OMQ was evaluated against the A375 human amelanotic melanoma cancer cell line and A375-derived with different degrees of aggressiveness (A375-A7, A375-G10, and A375-PCDNA3). The results showed that 3OMQ reduced the cell viability of all strains, demonstrating time- and dose-dependent responses. 3OMQ was used to obtain hydrogels for the topical treatment of melanoma. Thus, 3OMQ was incorporated into hypromellose hydrogels with/without different cyclodextrins (CDs). The 3OMQ formulations showed permeation/retention in all skin layers, namely stratum corneum, epidermis, and dermis. A significant amount of 3OMQ was found in the replication site of the melanoma cells (epidermis and dermis). Altogether, these results demonstrate that 3OMQ can be isolated from Achyrocline satureioides by HPCCC on a semi-preparative scale and exhibit cytotoxic activity against melanoma cells. Its incorporation into an HPMC hydrogel containing HP-ß-CD yielded a formulation with excellent technological and biopharmaceutical characteristics for evaluating the topical management of melanoma.

Development and validation of a specific-stability indicating liquid chromatography method for quantitative analysis of pterostilbene: application in food and pharmaceutical products.

Pterostilbene is a natural constituent with numerous preventive and therapeutic properties used for treating a wide range of human diseases. It has been isolated from blueberries in high concentrations. A versatile specific-stability indicating liquid chromatography method for reliable quantitative determination of pterostilbene in blueberry extracts was developed. In the stability-indicating analysis, pterostilbene samples were exposed to stress conditions: temperature, UV light, and oxidative, acid and alkaline media. The robustness of the method was evaluated using a Box-Behnken experimental design. Pterostilbene was stable in acid and alkaline media, but unstable when exposed to oxidation, high temperature, and UV light. Using the proposed method, it was demonstrated that degradation products did not interfere in the analyte analysis. The method revealed to be simple, fast, selective, linear, precise and accurate for the quantitative analysis of pterostilbene in Vaccinium myrtillus extract. Besides being a specific-stability indicating method, it demonstrated to be applicable to quantify pterostilbene in pterostilbene:ß-cyclodextrin complexes and pterostilbene:ß-cyclodextrin:hydrophilic polymer ternary systems. These findings are relevant for the development of food, pharmaceutical or cosmetic products containing pterostilbene or blueberry extracts.

Achyrocline satureioides compounds, achyrobichalcone and 3-O-methylquercetin, induce mitochondrial dysfunction and apoptosis in human breast cancer cell lines.

Natural products are a valuable source of new molecules and are important for drug discovery. Many chemotherapeutics currently in clinical use were originated from natural sources and are effective cytotoxic agents. In this study, we investigated the cytotoxic and pro-apoptotic effects of achyrobichalcone (ACB) and 3-O-methylquercetin (3OMQ), two novel compounds isolated from the Achyrocline satureioides plant. Because extracts from this plant have been shown to have anticancer activity in vitro, we evaluated ACB and 3OMQ using a human breast cancer cell line, MDA-MB-231, and a nontumorigenic human breast epithelial cell line, MCF-12A. We found that ACB demonstrates cytotoxic effects on MDA-MB-231 cells, but not MCF-12A cells. 3OMQ also demonstrated cytotoxic effects on MDA-MB-231 cells, but with lower selectivity compared to treated MCF-12A cells. Cell death by both compounds was associated with caspase-9 and caspase-3/7 activation. Using high-resolution respirometry, we found that ACB and 3OMQ were able to cause acute mitochondrial dysfunction in MDA-MB-231-treated cells. These results suggest that apoptosis in MDA-MB-231 cells is induced through the activation of the mitochondrial-dependent pathway. Collectively, these findings suggest that ACB is a strong candidate for further anticancer in vivo tests.

Box-Behnken Design for Extraction Optimization Followed by High Performance Countercurrent Chromatography: Production of a Flavonoid-enriched Fraction from Achyrocline Satureioides.

The biological properties of Achyrocline satureioides have been mostly ascribed to its major flavonoids quercetin (QCT), luteolin (LUT), and 3-O-methylquercetin (3OMQ). The present study aimed to optimize the extraction by dynamic maceration of the major phenolic compounds in order to obtain in a subsequent step a flavonoid-enriched fraction (FEF) using high performance countercurrent chromatography (HPCCC). A 3-level Box-Behnken design (BBD) was applied to maximize the extraction of the substances, using the plant : solvent ratio (X1 ), extraction time (X2 ), and ethanol concentration (X3 ) as factors. One-step HPCCC semipreparative separation with a solvent system composed of hexane : ethyl acetate : methanol : water (0.9 : 0.9 : 0.8 : 1.0, v/v) was employed to obtain the FEF. The second-order polynomial model was able to fit the experimental data adequately. The linear and quadratic terms of X3 were the most significant factors that affected all the responses. The positive linear term of X3 indicated a substantial increase in extraction yield, while the negative quadratic term showed a nonlinear tendency. Linear terms of X1 suggested a tendency to solvent saturation, except for QCT. The terms of X2 did not affect the responses substantially. The HPCCC method was found to be efficient and rapid for separating the FEF with 71% (w/w) flavonoid content. Overall, the developed extraction procedure coupled with HPCCC proved to be efficient for obtaining an enriched fraction with a very high content of flavonoids from A. satureioides.

Compatibility study of rosmarinic acid with excipients used in pharmaceutical solid dosage forms using thermal and non-thermal techniques.

Rosmarinic acid (RA) is a phenolic compound that presents well-documented anti-inflammatory, antioxidant and antitumor activities, and based on its pharmacological potential and poor bioavailability, several solid dosage forms have been developed to RA delivery. Therefore, in literature, there are no reports about RA compatibility with excipients. In this regard, the aim of the present study was to evaluate, for the first time, the compatibility of RA with excipients commonly used in solid dosage forms at a 1:1 (RA:excipient) ratio using differential scanning calorimetry (DSC), thermogravimetry (TG), Fourier-transform infrared (FTIR), solid-state nuclear magnetic resonance (ssNMR), and isothermal stress testing (IST) coupled with liquid chromatography (LC). The excipients selected were hydroxypropyl methylcellulose (HPMC), microcrystalline cellulose (MCC), lactose monohydrate (LAC), polyvinylpyrrolidone (PVP), talc (TALC), croscarmellose sodium (CCS), and magnesium stearate (MgSTE). According to DSC results, physical interactions were found between RA and HPMC, LAC, CCS, and MgSTE. The TG analyses confirmed the physical interactions and suggested chemical incompatibility. FTIR revealed physical interaction of RA with TALC and MgSTE and the ssNMR confirmed the physical interaction showed by FTIR and excluded the presence of chemical incompatibility. By IST, the greatest loss of RA content was found to CCS and MgSTE (>15%), demonstrating chemical incompatibilities with RA. High temperatures used in DSC and TG analyses could be responsible for incompatibilities in binary mixtures (BMs) with HPMC and LAC, while temperature above 25 °C and presence of water were factors that promote incompatibilities in BMs with CCS and MgSTE. Overall results demonstrate that RA was compatible with MCC and PVP.

Semi-preparative isolation and purification of phenolic compounds from Achyrocline satureioides (Lam) D.C. by high-performance counter-current chromatography.

INTRODUCTION: Phenolic compounds present in Achyrocline satureioides are known to have therapeutic benefits like antioxidant, anti-inflammatory, and antitumour properties. The main polyphenols present in the plant are quercetin (QCT), luteolin (LUT), 3-O-methylquercetin (3OMQ), and achyrobichalcone (ACB). However, the effective isolation and purification of these compounds from A. satureioides inflorescences are not an easy task. OBJECTIVE: To develop an efficient high-performance counter-current chromatography (HPCCC) method for quick separation and purification of naturally occurring phenolic compounds from the extract of A. satureioides. METHODOLOGY: A two-step HPCCC semi-preparative isolation method was developed using a solvent system composed of n-hexane/ethyl acetate/methanol/water (0.8:1.0:0.8:1.0) and dichloromethane/methanol/water (3.5:3.5:2.5). RESULTS: The HPCCC method was used to obtain two fractions. The first fraction (F1 ) contained high levels of ACB, among other constituents, while the second fraction (F2 ) contained mostly QCT, LUT, and 3OMQ. Besides the high ACB content, F1 contained three other flavonoid-aglycones (kaempferol, 97.3%; isokaempferide, 92.4%; and 3,3'-di-O-methylquercetin, 95.2%) identified by an ultra-performance liquid chromatography system coupled to a quadrupole time-of-flight with high-definition mass spectrometry (UPLC-QTOF/HDMS) and nuclear magnetic resonance (NMR) analysis. Purity levels of ACB, 3OMQ, QCT, and LUT were 98.0, 97.0, 97.5, and 90.2%, respectively. CONCLUSION: This is the first time that high purity ACB and six other flavonoids were obtained from A. satureioides inflorescences by HPCCC. These excellent results reveal the potential and versatility of HPCCC as a technique to produce different types of products from this plant species on a semi-preparative scale: enriched fractions, new metabolites, or high purity compounds.