Several beneficial effects on the skin have been reported for coumestrol (COU), such as protection against photoaging and improvement of skin elasticity and thickness in postmenopausal women. However ...no reports on the effect of COU on wound healing were found. Nevertheless, COU has low aqueous solubility, which is a crucial limitation for biological tests. The present study was designed as a two-step experiment to evaluate the wound healing effect of COU. First, we used fibroblasts and the experimental in vitro artificial wound model, scratch assay, to compare the effects of COU free, dissolved in dimethyl sulfoxide (DMSO) or Dulbecco's modified Eagle's medium (DMEM), or associated with hydroxypropyl-β-cyclodextrin (HPβCD). The 50 μM (66.1%) and 10 μM (56.3%) COU/HPβCD association induced cell proliferation and migration in inflicted wounds. Subsequently, the in vivo wound healing experimental model (Wistar rats) revealed that COU/HPβCD incorporated into hypromellose (HPMC) hydrogel had similar efficacy in wound healing in comparison to the positive control (Dersani®), with the advantage that 50% wound healing was achieved within a shorter period. In summary, the results successfully demonstrated, for the first time, the wound healing effect of COU/HPβCD incorporated into HPMC hydrogel and describe the feasibility of the biological tests with the use of HPβCD instead DMSO.
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Lactobionic acid and its salts are substances that have several applications in the pharmaceutical area. These products were obtained by bioconversion through the enzymatic complex ...glucose-fructose-oxidoreductase (GFOR)/gluconolactonase (GL) present in calcium alginate immobilized cells of Zymomonas mobilis. In the reactions catalyzed by this enzyme system, the medium pH must be controlled at slightly acid values. For this purpose, NaOH, KOH, or Ca(OH)2 were used, and as a result, the respective salts were formed. The kinetic study on the formation of sodium, potassium and calcium lactobionates was followed by the steps of purification and characterization aiming the potential use of these compounds in the pharmaceutical area. In the assays for the bioproduction of sodium, potassium or calcium lactobionates, yields of 74, 77 and 84% were obtained, respectively. In the salts purification step, purity levels of approximately 95% were achieved. The structural identities of the lactobionate salts were determined by high resolution mass spectrometry, in addition to the 13C and 1H NMR analysis. The characterization demonstrates the selectivity of the enzymatic reaction of GFOR/GL of Z. mobilis, in the production of lactobionic acid and its salts.
Currently, there is an increasing interest on the development of topical formulations containing rosmarinic acid (RA) due to its well-documented antioxidant activity. This study aimed to develop and ...validate a stability-indicating ultra-fast liquid chromatography (UFLC) method for the determination of RA in nanoemulsions, porcine skin and nasal mucosa intended to be applied in permeation/retention studies and for development of topical nanoemulsions. Chromatographic separation was carried out using a C18 column packed with 2.6 μm particle size in isocratic conditions using as mobile phase water:acetonitrile (83:17, v/v), acidified with 0.1% trifluoracetic acid (v/v), with a total time of analysis of 3.5 min and detection at 330 nm. RA analysis was specific in the presence of both non-biological (blank nanoemulsion and receptor fluid) and biological matrices (porcine ear skin and porcine nasal mucosa). No interference of degradation products of RA was verified after different stress conditions such as acidic, alkaline, oxidative, light exposure (UV-A and UV-C) and thermal demonstrating the method stability-indicating property. The analytical (0.1–10.0 μg·mL−1) and bioanalytical (0.5–10.0 μg·mL−1) linearity was proved by analysis of the calibration curves of RA and no matrix effect was observed. The method was sensitive, precise and accurate, and showed recovery higher than 85%. The method was considered robust as evaluated by a Plackett-Burman experimental design. In the validated conditions, the RA was determined in the nanoemulsions obtained by spontaneous emulsification procedure (1.007 ± 0.040 mg·mL−1), porcine ear skin (1.13 ± 0.19 μg·cm−2) and nasal mucosa (22.46 ± 3.99 μg·cm−2) after retention/permeation studies. Thus, a highly sensitive, simple, fast and stability-indicating method was developed for RA analysis during the development of topical nanoemulsions and bioanalytical assays in complex matrices.
•A simple and fast analytical and bioanalytical UFLC method for RA was validated.•The method proved to be stability-indicating using different stress conditions.•The method was selective, linear, precise, accurate and robust with low matrix effect.•The method was able to estimate RA incorporated in pharmaceutical nanoemulsions.•The method was applied to assay RA in skin and nasal mucosa samples.
Equisetum giganteum L., commonly called “giant horsetail”, is an endemic species of Latin America. Its aerial parts have been widely used in ethnomedicine as a diuretic and in herbal medicine and ...food supplements as a raw material. The phenolic composition of E. giganteum stems was studied by liquid chromatography coupled to diode array detection (LC–DAD) and liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS), which identified caffeic acid derivatives, flavonoids and styrylpyrones. The most abundant glycosilated flavonoids in this sample were kaempferol derivatives. Other rare phenolic components, namely, quercetin-3-O-(caffeoyl)-glucoside and 3-hydroxyhispidin-3,4′-di-O-glucoside, were reported for first time in the Equisetum genus. An LC-UV method for the simultaneous quantification of flavonoid aglycones in E. giganteum obtained after hydrolysis was developed and validated. The method exhibited excellent linearity for all analytes, with regression coefficients above 0.998, LOD≥0.043μgmL−1, LOQ≥0.158μgmL−1 and recovery rates of 96.89–103.33% and 98.22–102.49% for quercetin and kaempferol, respectively. The relative standard deviation for the intra- and inter-day precision was≤3.75%. The hydrolysis process was optimized by central composite rotational design and response surface analysis. The second-order response models for the aglycones contents were as follows: quercetin (μgg−1)=24.8102+55.2823×HCl+0.776997×Time−7.23852×HCl2−7.46528E−04×Time2−0.229167×HCl×Time; kaempferol (μgg−1)=−9.66755+974.822×HCl+11.8059×Time−130.612×HCl2−0.0125694×Time2 −3.22917×HCl×Time, with estimated optimal conditions of 1.18M HCl and 205min of hydrolysis. The results obtained with these new methods were compared to those from a spectrophotometric assay used to determine the total flavonoids in the Equisetum arvense monograph (Horsetail, British Pharmacopoeia 2011). For all four species analyzed (E. giganteum, E. arvense, E. hyemale and E. bogotense), the calculated aglycone content was higher using the optimized hydrolysis conditions. Additionally, the LC method was more appropriate and specific for quantitative analysis.
► Twelve phenolic compounds were identified by LC-DAD and LC–ESI-MS/MS in E. giganteum. ► The acid hydrolysis of flavonoid glycosides was statistically optimized. ► A LC–UV method was validated for flavonoid aglycones determination. ► The method was suitable for the assay of flavonoid aglycones in Equisetum species.
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.
A RP-LC method was developed and validated to quantify ecdysterone in extractive solution from subterraneous parts of
Pfaffia glomerata. The analysis was performed using a RP-18 column with ...acetonitrile:water isocratic elution and the detection was carried out by UV at 242
nm. The standard curve for ecdysterone was linear over the range of 5.2–41.6
μg/ml (
R
2
=
0.9995). The extractive solution showed linear response in the range of 25.05–175.35
μg/ml (
R
2
=
0.9977). This method showed excellent repeatability (relative standard deviation, R.S.D.
<
2.0%), intermediary precision (R.S.D.
=
2.13%) and accuracy (101.04; R.S.D.
=
1.51%). The limit of detection (LOD) was 0.036
μg/ml and the limit of quantification (LOQ) was 0.110
μg/ml, demonstrating the sensitivity of the method. This assay can be readily utilized as quality controlled method for
P.
glomerata preparations.
Objectives
The present study was designed to verify if quercetin (QCT), a flavonoid with antioxidant and antiviral activity, and 3‐O‐methylquercetin (3OMQ), a quercetin C3‐methoxylated derivative, ...present differences in their behavior against complexation with β‐cyclodextrin (β‐CD) and the corresponding permeation/retention trhough porcine ear skin, when incorporated into hydroxypropyl methylcellulose (HPMC) or chitosan (CS) hydrogels.
Methods
The influence of β‐CD on the skin permeation/retention of QCT and 3OMQ from hydrogels is comparatively evaluated for both flavonoids using porcine ear skin in Franz cells model. The properties of the two flavonoids using the semi‐empirical method Recife Model was studied.
Key findings
Quercetin presented higher skin retention compared with its C3‐methoxy derivative 3OMQ. The best permeation/retention of QCT was observed when it was incorporated into CS hydrogel containing 5% β‐CD, whereas, for 3OMQ, the HPMC hydrogel containing 5% β‐CD was the best formulation. The flavonoids complexation with β‐CD in water occurred preferentially with the insertion of the B ring through the secondary OH rim.
Conclusions
The dynamic molecular modeling revealed that the methyl group at C3 in 3OMQ molecule determined significant difference in its complexation with β‐CD, in comparison to its analogous QCT and that difference is coincident with the permeation behavior of these flavonoids, denoting a possible relationship with their molecular dynamics.
Mate (Ilex paraguariensis A. St. Hil.) is a raw material used to prepare popular beverages in South America, whose healthy effects have been pointed out. The leaves and stems of mate contain a ...complex assemblage of saponins, mainly ursolic acid and oleanolic acid glycosides, and their quantification presents difficulties such as weak cromophorous sites and interference of other compounds. On the other hand, the sugar moieties are a chief character that distinguishes them. We developed and validated a high-performance liquid chromatography method using a refractometer detector for quantifying the major saponins in extractive solution of mate leaves. The validation indicates the suitability of the method, presenting linearity in the concentration range between 102.5 to 854.0 microg/mL to matesaponin 1 and between 103.0 to 515.0 microg/mL to matesaponin 3. The repeatability coefficient of variation was 4.70% and 6.61% for matesaponin 1 and matesaponin 3, respectively. The inter-day coefficient of variation was 6.77% and 7.79% for matesaponin 1 and matesaponin 3, respectively. The analysis of plants from Brazilian States Mato Grosso do Sul and Rio Grande do Sul showed the predominance of matesaponins 1, 2, and 3 in the corresponding extractive solution. The sum of these three saponins ranged between 3 and 10 mg/g for mature leaves in the majority of sampled plants.
•A bioanalytical HPLC method for coumestrol quantification in porcine ear skin permeation studies was validated.•The method proved to be suitable to quantify coumestrol in biological and ...non-biological matrices: porcine ear skin layers (stratum corneum, epidermis and dermis), hydrogel formulation and adhesive tape.•The chemical structure of degradation products of coumestrol against UV light and alkalin media are suggested using UPLC-QTOF/HDMS stability-indicating method.
Coumestrol is present in several species of the Fabaceae family widely distributed in plants. The estrogenic and antioxidant activities of this molecule show its potential as skin anti-aging agent. These characteristics reveal the interest in developing analytical methodology for permeation studies, as well as to know the stability of coumestrol identifying the major degradation products. Thus, the present study was designed, first, to develop and validate a versatile liquid chromatography (HPLC) method to quantify coumestrol in a hydrogel formulation in different porcine skin layers (stratum corneum, epidermis, and dermis) in permeation tests. In the stability-indicating test coumestrol samples were exposed to stress conditions: temperature, UVC light, oxidative, acid and alkaline media. The degradation products, as well as the constituents extracted from the hydrogel, adhesive tape or skin were not eluted in the retention time of the coumestrol. Hence, the HPLC method showed to be versatile, specific, accurate, precise and robust showing excellent performance for quantifying coumestrol in complex matrices involving skin permeation studies. Coumestrol recovery from porcine ear skin was found to be in the range of 97.07–107.28μg/mL; the intra-day precision (repeatability) and intermediate precision (inter-day precision), respectively lower than 4.71% and 2.09%. The analysis using ultra-performance liquid chromatography coupled to a quadrupole time-of-flight high definition mass spectrometry detector (UPLC-QTOF/HDMS) suggest the MS fragmentation patterns and the chemical structure of the main degradation products. These results represent new and relevant findings for the development of coumestrol pharmaceutical and cosmetic products.
There is a growing interest in the pharmaceutical field concerning isoflavones topical delivery systems, especially with regard to their skin care properties and antiherpetic activity. In this ...context, the present work describes an ultra-fast liquid chromatography method (UFLC) for determining daidzein, glycitein, and genistein in different matrices during the development of topical systems containing isoflavone aglycones (IA) obtained from soybeans. The method showed to be specific, precise, accurate, and linear (0.1 to 5µgmL−1) for IA determination in soybean acid extract, IA-rich fraction obtained after the purification process, IA loaded-nanoemulsions, and topical hydrogel, as well as for permeation/retention assays in porcine skin and porcine esophageal mucosa. The matrix effect was determined for all complex matrices, demonstrating low effect during the analysis. The stability indicating UFLC method was verified by submitting IA to acidic, alkaline, oxidative, and thermal stress conditions, and no interference of degradation products was detected during analysis. Mass spectrometry was performed to show the main compounds produced after acid hydrolysis of soybeans, as well as suggest the main degradation products formed after stress conditions. Besides the IA, hydroxymethylfurfural and ethoxymethylfurfural were produced and identified after acid hydrolysis of the soybean extract and well separated by the UFLC method. The method’s robustness was confirmed using the Plackett-Burman experimental design. Therefore, the new method affords fast IA analysis during routine processes, extract purification, products development, and bioanalytical assays.
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•An analytical and bioanalytical UFLC method for isoflavone aglycones was validated.•Acid hydrolysis of soybeans was performed to obtain the isoflavone aglycones.•Extract impurities as hydroxymethylfurfural and ethoxymethylfurfural were detected.•A pure rich-fraction was produced and incorporated in nanoemulsions and hydrogels.•Permeation assays in porcine skin or porcine esophageal mucosa were performed.
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