Gisely C. Lopes

Application and analysis of the folin ciocalteu method for the determination of the total phenolic content from Limonium brasiliense L.

Limonium brasiliense is a common plant on the southern coast of Brazil. The roots are traditionally used for treatment of premenstrual syndrome, menstrual disturbances and genito-urinary infections. Pharmaceutical preparations obtained from its roots and used for these purposes were marketed in Brazil in the 1980s and 1990s. Currently, the Brazilian Drug Agency (National Health Surveillance Agency, ANVISA) has canceled the registration of these products, and their use was discontinued because of a lack of studies to characterize the plant raw material and ensure the effectiveness and safety of its use. The aim of the present study was to develop and validate an analytical method to determine the content of total polyphenols (TP) in an extract from L. brasiliense roots, by the UV/Vis spectrophotometric method. L. brasiliense roots were extracted in acetone:water (7:3, v/v-10% w/v). The crude extract was used to develop a method for TP assay. The method was validated according to national and international guidelines. The optimum conditions for analysis time, wavelength, and standard substance were 30 min, 760 nm, and pyrogallol, respectively. Under these conditions, validation by UV/Vis spectrophotometry proved the method to be linear, specific, precise, accurate, reproducible, robust, and easy to perform. This methodology complies with the requirements for analytical application and to ensure the reliability of the results.

Antioxidant and antifungal activities of extracts and condensed tannins from Stryphnodendron obovatum Benth.

The antioxidant activity of stem-bark extracts from Stryphnodendron obovatum Benth., including fractions and isolated compounds, was evaluated by DPPH in thin-layer chromatography. All the fractions and isolated compounds showed antioxidant activity. Antifungal activity was determined by the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) against the yeasts Candida albicans, Candida parapsilosis, Candida krusei and Candida tropicalis. All extracts (CE, EtOAc and FW), subfractions (F1-F12) and the compounds I, II and III were inactive against the yeasts. Against C. parapsilosis and C. albicans, fractions F13-15 and F20 showed moderate antifungal activity, and fractions F16-19 and F21-22 showed good activity. Chemical isolation of the ethyl-acetate fraction resulted in the identification of three compounds: epigallocatechin, gallocatechin and epigallocatechin-(4b®8)-gallocatechin.

Statistical mixture design selective extraction of compounds with antioxidant activity and total polyphenol content from Trichilia catigua.

Statistical design mixtures of water, methanol, acetone and ethanol were used to extract material from Trichilia catigua (Meliaceae) barks to study the effects of different solvents and their mixtures on its yield, total polyphenol content and antioxidant activity. The experimental results and their response surface models showed that quaternary mixtures with approximately equal proportions of all four solvents provided the highest yields, total polyphenol contents and antioxidant activities of the crude extracts followed by ternary design mixtures. Principal component and hierarchical clustering analysis of the HPLC-DAD spectra of the chromatographic peaks of 1:1:1:1 water-methanol-acetone-ethanol mixture extracts indicate the presence of cinchonains, gallic acid derivatives, natural polyphenols, flavanoids, catechins, and epicatechins.

Quality control and TLC profile data on selected plant species commonly found in the Brazilian market

The use of thin-layer chromatography (TLC) is a commonplace practice and can be of significant help to different laboratories with quality control, especially those that work with plant extracts and phytotherapeutics. This study evaluated ten species of plants (Schinus terebinthifolius; Arctium lappa; Trichilia catigua; Camellia sinensis; Mikania glomerata; Croton echioides; Achyrocline satureioides; Heteropterys aphrodisiaca; Plantago major; Arctostaphylos uva-ursi) that are commonly sold by compounding pharmacies, using TLC with reference substances and pharmacopoeic physical and chemical tests (loss on drying, level of extractives, and total ash content). The results showed that the ten species showed losses on drying consonant with the literature. The level of extractives for two species and total ash for five species were also consonant with the literature, and those of the other species were established in this study. The semipurified extracts of the ten species were assayed by TLC, and the analysis with the use of reference substances proved to be effective, in addition to being practical, simple, versatile, and economically viable.

Determinação quantitativa de taninos em três espécies de Stryphnodendron por cromatografia líquida de alta eficiência

A method of separation and quantification by reverse-phase high-performance liquid chromatography (HPLC) was developed, using water (0.05% TFA):acetonitrile (0.05% TFA) as the mobile phase in a gradient system. Flavan-3-ols present in a semipurified extract from the stem bark of Stryphnodendron adstringens, Stryphnodendron polyphyllum and Stryphnodendron obovatum were analyzed. The HPLC was performed with the ethyl-acetate fraction (EAF) using a solid-phase extraction on cartridges C18-E with methanol:water (2:8), filtered through a membrane of 0.5 μm pore size; the column was Phenomenex® Gemini C-18 (5 μm) at 30 oC, with a flow rate of 0.8 mL/min. The analysis was done at 210 nm. Gallic acid and gallocatechin solutions were used as calibration standards. The proposed method was validated by resolution RE No. 899/2003 of the National Health Surveillance Agency. Quantitative analysis of the EAF showed high contents of flavan-3-ols in the stem bark of all three species. This study demonstrated that it is possible to determine the concentration of individual substances in tannin-rich plants. The system developed can be used as a chromatographic profile for the semipurified fraction of S. adstringens, S. polyphyllum, and S. obovatum.

OPTIMIZATION OF SOLVENT MIXTURES FOR EXTRACTION FROM BARK OF Schinus terebinthifolius BY A STATISTICAL MIXTURE-DESIGN TECHNIQUE AND DEVELOPMENT OF A UV-VIS SPECTROPHOTOMETRIC METHOD FOR ANALYSIS OF TOTAL POLYPHENOLS IN THE EXTRACT

A statistical mixture-design technique was used to study the effects of different solvents and their mixtures on the yield, total polyphenol content, and antioxidant capacity of the crude extracts from the bark of Schinus terebinthifolius Raddi (Anacardiaceae). The experimental results and their response-surface models showed that ternary mixtures with equal portions of all the three solvents (water, ethanol and acetone) were better than the binary mixtures in generating crude extracts with the highest yield (22.04 ± 0.48%), total polyphenol content (29.39 ± 0.39%), and antioxidant capacity (6.38 ± 0.21). An analytical method was developed and validated for the determination of total polyphenols in the extracts. Optimal conditions for the various parameters in this analytical method, namely, the time for the chromophoric reaction to stabilize, wavelength of the absorption maxima to be monitored, the reference standard and the concentration of sodium carbonate were determined to be 5 min, 780 nm, pyrogallol, and 14.06% w v-1, respectively. UV-Vis spectrophotometric monitoring of the reaction under these conditions proved the method to be linear, specific, precise, accurate, reproducible, robust, and easy to perform.

Estudo físico-químico, químico e biológico de extrato das cascas de Stryphnodendron polyphyllum Mart. (Leguminosae)

Two flavan-3-ols and one proanthocyanidins have been isolated from the stem bark of Stryphnodendron polyphyllum Mart., which is traditionally used in Brazil against various diseases. The structure was determined on the basis of spectroscopic data including 1-D (1H, 13C) and 2-D NMR (1H/1H COSY) and MS. The antibacterial activities of an acetone:water and semipurified extracts from the stem bark of Stryphnodendron polyphyllum Mart. were evaluated. Both the crude and semipurified extracts showed activity against Bacillus subtilis and Staphylococcus aureus. Quality control was determined using several pharmacopoeial assay.

Development of a UV/Vis spectrophotometric method for analysis of total polyphenols from Caesalpinia peltophoroides Benth

Caesalpinia peltophoroides is a domesticated tree found in Brazil. It was necessary to develop an analytical method to determine the content of total polyphenols (TP) in this herbal drug. The pre-analytical method was standardized for analysis time, wavelength, and the best standard to use. The optimum conditions were: pyrogallol, 760 nm, and 30 min respectively. Under these conditions, validation by UV/Vis spectrophotometry proved to be reliable for TP of the crude extract and semipurified fractions from C. peltophoroides. Standardization is required for every herbal drug, and this method proved to be linear, precise, accurate, reproducible, robust, and easy to perform.

Development and validation of an HPLC method for the determination of epicatechin in Maytenus ilicifolia (Schrad.) Planch., Celastraceae

A simple, reproducible and efficient high-performance liquid chromatography (HPLC) method was developed. Water (0.05% TFA):acetonitrile (0.05% TFA) was used as the mobile phase in a gradient system for the determination of epicatechin (EP) in leaves of Maytenus ilicifolia (Schrad.) Planch. The analysis was performed using an RP C-18 column (5 µm) as the stationary phase, with a flow rate of 0.8 mL/min, at a wavelength of 210 nm for detection and determination. The main validation parameters of the method were also determined. The calibration curve was found to be linear, with a range of 10-120 µg/mL (EP). The correlation coefficient of the linear regression analysis was within 0.9988, and the detection and quantification limits were 28.61 and 86.77 µg/mL, respectively. The content of EP was successfully determined, with satisfactory reproducibility and recovery. Recovery of the EP was 99.32%. The method was successfully applied to the determination of epicatechin in leaves of M. ilicifolia. The interlaboratorial evaluation showed the reproducibility of the method with a relative standard deviation of 14.62%.

Development and validation of a LC-method for the determination of phenols in a pharmaceutical formulation containing extracts from Stryphnodendron adstringens

A sensitive RP-HPLC method with UV detection successfully measured phenol(s) in an ointment containing 3% Stryphnodendron adstringens extract. Chromatography used acetonitrile (0.05% trifluoroacetic acid):water (0.05% trifluoroacetic acid) (v/v), flow rate 0.8 mL min-1. Quantitation was accomplished by the external-standard method. Linearity for 2.00 to 16.00 μg mL-1 (gallic acid) and 1.14 to 18.24 μg mL-1 (gallocatechin) was established. Intra- and inter-day precision levels were under 5%. LOD and LOQ were 0.231 and 0.770 μg mL-1 (gallic acid) and 0.151 and 0.504 μg mL-1 (gallocatechin), respectively. Determination of phenols was unaffected by product excipients.