Total polyphenols from Syzygium cumini ( L . ) Skeels fruit extract

A precise, accurate and low cost spectrophotometric method was developed and validated for routine determination of total polyphenols, as pyrogallic acid equivalents, from the percolated and lyophilized extract of Syzygium cumini (L.) Skeels fruits. Validation was assessed experimentally and data were rigorously treated by statistical analysis. Analytical parameters were: linearity, interval (range), precision and recovery/accuracy, limit of detection (LOD, μg mL-1) and limit of quantification (LOQ, μg mL-1). The visible spectrophotometric method presented linearity (r2 = 0.9979 ± 0.0010) over the concentration range 0.25-7.5 μg mL-1 of standard pyrogallic acid, precision < 2.918171%, recovery/accuracy ranging from 96.228693 to 107.17701%, LOD = 0.21 μg mL-1 and LOQ = 0.64 μg mL-1.


InTroducTIon
Syzygium cumini (L.) Skeels (Synonym: S. jambolanum, E. jambolana) (Myrtaceae), popularly known in Brazil as "jambolão" (jambolan or java plum in English), is a native tree of the tropics, originally from India and SE Asia.It is widespread in some states of North, Northeast and Southeast Brazil (Grover et al., 2001;Migliato et al., 2006;Migliato et al., 2007, Rey-nertson et al., 2008) and is used as a popular treatment against various diseases.In Brazil, the bark, fruits, seeds and leaves of this plant are used for the treatment of diabetes and administered in various pharmaceutical preparations (e.g., aqueous or alcoholic extract, decoctions or crude plant juice) (Braga et al., 2007).Syzygium cumini seeds have already shown hypoglycemic and antioxidant activities.A decoction of the bark is also used for dysentery and diarrhea.Moreover, Syzygium cumini has been shown to have sedative and anticonvulsant effects and a potent central nervous system depressant effect (Pepato et al., 2004).
According to Shafi and co-workers (2002), plant extracts of Syzygium species have known antibacterial activity.In addition, Chandrasekaran and Venkatesalu (2004) showed that aqueous and methanol extracts inhibited the growth of some of the fungal microorganisms implicated in skin diseases, such as C. albicans, T. rubrum, T. mentagrophytes and M. gypseum.
The aim of this research work was to validate a routine and low cost spectrophotometric method (mainly to be used as a quality control tool) to quantify total polyphenols, as pyrogallic acid equivalents, from the percolated and lyophilized extract of fruits from Syzygium cumini (L.) Skeels.

Solvents, reagents and reference standard
All reagents and solvents were of analytical grade and they were purchased from Merck (Germany), Sigma-Aldrich (Brazil) and LabSynth (Brazil), and used without any further purification (sodium carbonate PA-ACS, ethanol 99.5%, Folin-Ciocalteau 2 N).Pyrogallic acid > 99%, from Riedel-de Haën (Sigma-Aldrich) was used as reference standard.

Plant Material
The fruits of Syzygium cumini (L) Skeels were collected during December 2006 to January 2007, in the Medicinal and Toxic Plant Garden of FCF/UNESP, Araraquara -SP, Brazil.On the same opportunity, the voucher was deposited under the number 19586 in SJRP Herbarium, Department of Botany of IBILCE-UNESP of São Jose do Rio Preto Campus, by Dr. Neusa Taroda Ranga.

Preparation of Syzygium cumini (L.) Skeels extract
Extract was obtained by percolation using Syzygium cumini fruits.The extract was prepared with 10% w/v of Syzygium cumini fruits in ethanol:water (1:1).Percolated extract was concentrated under reduced pressure at 40 ºC and lyophilized until total solvent elimination.

Total polyphenols determination
Chemical analysis was performed at samples protected from light during dilution.Water free of carbon dio-xide was used for the experiments.Lyophilized Syzygium cumini extract was weighed, 0.750 g, and transferred to a 250 mL volumetric flask completed with water.
The amount of 5 mL of the above mentioned dilution was filtered and transferred to a 25 mL volumetric flask.To a 5 mL of the last solution was added 2 mL of Folin-Ciocalteau 2 N and diluted to 50 mL with sodium carbonate solution 10% w/v.Absorbance (A1) was achieved at 757.0 nm after 3 min of the addition of the last reagent.Water was considered the blank (Farmacopéia, 1988).

analysis of polyphenols not-absorbed by skin powder
An amount of 20 mL of the lyophilized extract solution in water was filtered and added of 0.2 g skin powder.
Vigorous agitation during 60 min was used to assure homogenization and, then, dispersion was filtered.5 mL of the filtered solution was diluted in 25 mL of water.An amount of 5 mL of the last solution was added of 2 mL Folin-Ciocalteau 2 N and diluted to 50 mL with sodium carbonate solution 10% w/v.Absorbance (A2) was registered at 757.0 nm after 3 min of the addition of the last reagent.Water was considered the blank (Farmacopéia, 1988).
reference standard stock solution 50 mg of standard pyrogallic acid was diluted in 50 mL of water, following, 5 mL of this solution was transferred to a 100 mL volumetric flask completed with water.To 5 mL of the last solution, 2 mL of Folin-Ciocalteau 2 N were assed, being then diluted to 50 mL with sodium carbonate solution 10% w/v.Absorbance (A3) was obtained at 757.0 nm, 3 min after of the addition of the last reagent and within 15 min of the standard pyrogallic acid dilution.Water was considered the blank.

analytical curve and linearity
Standard pyrogallic acid was diluted in the range of EQUATION 1 -Total polyphenols (TP, in g) from Syzygium cumini fruit extract.m: mass of the sample (g); A1: absorbance of total polyphenols from the extract; A2: absorbance of polyphenols not-absorbed by skin powder from the extract; A3: absorbance of standard.
Total polyphenols from Syzygium cumini (L.) Skeels fruit extract 123 concentrations from 0.25 to 7.5 µg.mL -1 .The absorbance measurements were obtained at 757.0 nm using Spectrophotometer Hewlett Packard with a 1 cm quartz cuvette.Mean of eight replicates of each concentration value was employed to construct the analytical curve (Causon, 1997;Beer et al., 2003;Rolim et al., 2005).

Precision and recovery/accuracy
Precision was evaluated as the assessment of the closeness of the results obtained in a series of measurements of a multiple sampling of the same sample, and recovery/ accuracy represented the degree of match between the individual results found and a theoretical value accepted as reference (Brittain, 1998;Brasil, 2003;Shabir, 2003).
Precision and accuracy were calculated according to Equations 4 and 5, respectively:

reSuLTS and dIScuSSIon
Beyond the regulatory requirements, performance and reliability of analytical procedures are essential to the quality control of botanical raw materials.Based on the validation characteristics and necessities of guidelines presented on official and scientific literature, each analytical procedure must be validated with respect to parameters which are relevant to its performance.Parameters like linearity, range, specificity, precision, accuracy, limit of detection and limit of quantification are required to the experimental establishment of the validation process (Baby et al., 2006).
The method used to quantify total polyphenols, as pyrogallic acid equivalents, from Syzygium cumini (L.) Skeels fruit extract has offered linearity, over the concentration range of 0.25 to 7.5 µg.mL -1 .Through the visual analysis of the analytical curve, the plotted data of absorbance measurements at 757.0 nm versus range of the concentrations generated a straight line.The spectrophotometry used in this research work presented advantages for routine utilization as: simplicity, rapidity, sensitivity, equipment convenience and relative low cost of reagents.
Besides the visual analysis of the analytical curve, the least-squares fit method was employed to achieve the linearity, as statistical tool, reported at Table I.The regression line with corresponding coefficient of linear correlation (r 2 ) were y = 0.1744 ± 0.0055)x + 0.032 (± 0.0034) and r 2 = 0.9979 ± 0.0010), where: y is absorbance and x is total polyphenols concentration (µg.mL -1 ).Statistical data were exposed with their respective standard deviation ± SD).
The results of the precision assay (RSD, %) were reported at Table II for the total polyphenols and the polyphenols not-absorbed by the skin powder for the S. cumini (L.) Skeels extract.
The methods demonstrated a RSD inferior than 5.0%, indicating an adequate precision for all assays (Brasil, 2003).
EQUATION 2 -Detection limit.LOD is estimated detection limit (µg.mL -1 ); σ is mean standard deviation; S is slope of the analytical curve.
EQUATION 3 -Quantification limit.LOQ is estimated quantification limit (µg.mL -1 ); σ is mean standard deviation; S is slope of the analytical curve.
EQUATION 4 -Precision.RSD (%) is precision; SD is standard deviation; C is mean of calculated concentrations.
EQUATION 5 -Recovery/Accuracy.C spiked is total polyphenols from extract spiked with standard; C extract is total polyphenols from extract; TC is theoretical concentration of the standard reference (pyrogallic acid).
Recovery/accuracy data were described at Tables III and IV.It was observed a greater percentage of recovery/ accuracy for the lowest concentrations of pyrogallic acid, indicating a positive correlation with that one of the extract.
The proposed method was considered accurate, as the recovery found in it totalizes the concentrations studied and showed adequate to the quantitative analysis of the extract and the standard pyrogallic acid.
After the validation of the methodology, the determination of the total polyphenols present on the extract was 27.706%.
LOD and LOQ were estimated according to the standard curve deviation and the slope of the regression line, presented at Table V. Limit values were found to be 0.21 and 0.64 µg.mL -1 , respectively, for LOD and LOQ.
Agreeing with the study interval and the accuracy obtained for the theoretical concentrations of the standard pyrogallic acid, LOQ should present a value >0.50 µg.mL -1 , which corroborated the estimated value of 0.64 µg mL -1 .0.036485 a Theoretical concentrations; SD: standard deviation; σ: mean standard deviation; S: slope from the regression line; LOD: estimated limit of detection, µg.mL -1 (LOD = 3.3 σ/S); LOQ: estimated limit of quantification, µg mL -1 (LOQ = 10 σ/S) concLuSIon A routine and economical spectrophotometric method was developed and validated for assay of total polyphenols, as pyrogallic equivalents, present in the Syzygium cumini (L.) Skeels extract.Method advantages were the absence of sample extraction, rapidity, direct responses and results, equipment convenience and relative low cost of reagents.The experimental results, in addition to statistical analysis, have proven that analytical parameters were precise, accurate and sensitive according to adequate linearity, recovery, repeatability and reproducibility, LOD and LOQ.

TaBLe IV -
Recovery/accuracy for polyphenols not-absorbed by the skin powder presented in the Syzygium cumini (L.) Skeels extract

TaBLe II -
Precision, as RSD%, for the total polyphenols, as pyrogallic acid equivalents, and for the polyphenols not-absorbed by the skin powder

TaBLe III -
Recovery/accuracy for the total polyphenols, as pyrogallic acid equivalents, presented in the Syzygium cumini (L.)

TaBLe V -
Estimative of limits of detection and quantification from standard curve deviation