Antioxidant Activity and Total Phenols in Different Extracts of Four Staphylea L. Species

Staphylea L. is a deciduous ornamental shrub that possesses significant cytotoxic and antibacterial activity, although the chemical composition of its extracts and the identity of the structures responsible for these biological activities are not yet known. In this study we have determined the total phenolic content in chloroform and ethyl acetate extracts of four Staphylea species: Staphylea colchica Stev., S. elegans Zab., S. holocarpa Hemsl. and S. pinnata L.. The antioxidant potential (DPPH radical and peroxynitrite scavenging activity) of these extracts was also determined and a correlation between the phenolic content and antioxidant activities of the ethyl acetate extracts has been found. Ethyl acetate extracts were more active and one of them, obtained from S. colchica Stev., possessed the highest activity.


Introduction
Staphylea L., (bladdernut, Staphyleaceae), belongs to a well-known group of deciduous ornamental shrubs that are widely distributed throughout the Northern Hemisphere.In Slovakia they are cultivated for their nice bright green foliage and small white and whitish flowers.S. pinnata L. grows naturally in this territory and is designated a protected species.A decoction prepared from the fruit of S. bumalda DC. has been used in Traditional Chinese Medicine as a cough remedy.The fresh roots are supposed to possess a blood refreshing effect after delivery.The dried fruit is also used as a folk anti-diarrhoeal medicine.Indians used the infusion from S. trifolia L. for its antirheumatic, dermatological, sedative and gynecological activities.The seeds were considered sacred and were used in gourd rattles for dream and medicine dances [1].
In 2000 Jantová et al. found that the plants possesses significant cytotoxic and antibacterial activity [2,3], but the chemical composition of the extracts was not elucidated.Only a limited number of papers have described the various flavonoid, amino-containing, triterpene, steroid, megastigmane, saccharide, organic and fatty acid compounds found therein [4].For this reason we have focused on establishing a relationship between the total phenolic content of the non-polar (chloroform) and polar (ethyl acetate) extracts of four Staphylea species (Staphylea colchica Stev., S. elegans Zab., S. holocarpa Hemsl.and S. pinnata L.) and their biological activity, measured via a determination of their antioxidant activities (DPPH and peroxynitrite scavenging activity).

Total phenolic content
These measurements were performed using a modified Folin-Ciocalteu colorimetric method according the procedure described in the Experimental section.The results, presented in Table 1, show that the total phenolic content varies among different Staphylea L. species and in the different extracts.As might be expected, the ethyl acetate extracts are generally richer in phenols than the chloroform ones.Thus, the phenolic content in ethyl acetate extracts from the various species tested varied from 10.6 to 2.3 gallic acid equivalents (GAE) in the following order: S. colchica (SCE) > S. holocarpa (SHE) > S. elegans (SEE) > S. pinnata (SPE).Chloroform extracts contain less phenolic compounds, from 4.2 to 2.9 GAE, but in the same order: S. colchica (SCCH) > S. holocarpa (SHCH) > S. elegans (SECH) > S. pinnata (SPCH).

DPPH radical scavenging activity
The antioxidant potential of the ethyl acetate extracts correlates with the phenolic content.The most active one was the extract prepared from S. colchica (SCE).The determination of this activity in the chloroform extracts was not possible, probably due to some complex interaction occurring after the addition of the reagent.Thus, it would appear that peroxynitrite scavenging activity measurements are a better technique to evaluate the antioxidant potential of Staphylea L. extracts.

Peroxynitrite scavenging activity
To obtain an extended picture of the antioxidant activity in the mentioned species and their extracts, the protective activity against peroxynitrite-induced tyrosine nitration has also been investigated.A correlation between the phenolic content and the intensity of this biological activity was again observed in the ethyl acetate extracts, which were more active than the chloroform ones in the range from 26.8 CE (SCE) to 2.1 CE (SPCH).

Conclusions
This study reports for the first time the antioxidant activity and total phenolic content of several Staphylea L. extracts.A relationship between the biological activities and the total phenolic content was found [5].Chemical elucidation of contents of the ethyl acetate extract from S. colchica Stev., as the most promising species based on the findings of this study, should be the main objective of further investigations.This should lead to the structural identification of the active metabolites and the determination of their antioxidant profile in pure form.

Preparation of extracts
The dried, minced leaves (100 g) of four Staphylea L. species were macerated in sequence at oneweek intervals with petroleum ether, chloroform, ethyl acetate and water (300 mL).Extracts were filtered and evaporated under vacuum.The residues were dried, weighed and the ethyl acetate (E) and chloroform (CH) extracts were analyzed.The presence of selected secondary metabolites was determined using specific reagents on silica gel chromatographic plates.

Quantitative determination of total phenols
The total content of phenols in the crude chloroform and ethyl acetate extracts was determined using a modified Folin-Ciocalteu colorimetric method with gallic acid as a standard.The extract solution in DMSO (700 µL) was transferred to a 10 mL volumetric flask, the Folin-Ciocalteu reagent (400 µL) was added and after 3 min, each flask was made up to the mark with sodium carbonate (Na 2 CO 3 ) solution (75 g/L).After 2 hours, the suspension was centrifuged (5000 r.p.m., 5 min) and the absorbance of the solution was measured at 760 nm.The total phenolic content was expressed as a gallic acid equivalent (GAE) in g/100 g of dry extract.Data are reported as mean ± SD for at least three replicates [6].

Evaluation of antioxidant activity by scavenging of DPPH radical
The free radical scavenging activity of the extracts was determined with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay.The extract solution (30 µL) in DMSO (1 mg/mL) was made up to 2.0 mL with 0.1 mM DPPH in methanol.After 5 min, the absorbance was measured at 517 nm.The reduction of DPPH was calculated relative to the measured absorbance of the control.The obtained data were compared to the calibration curve of catechin.The results were expressed as an antioxidant activity equivalent of catechin (CE) in mmol/100 g of dry extract (mean ± SD of at least three measurements) [7].

Evaluation of antioxidant activity by scavenging of peroxynitrite
Peroxynitrite solutions (10.0 mM, 8 µL) in 0.1 mM NaOH were drawn and mixed rapidly in the injector of the HPLC autosampler with 1.0 mM tyrosine solution in 0.05 mM KH 2 PO 4 -Na 2 HPO 4 buffer (42 µL, pH = 6.0) containing the tested extract (0.1 mg/mL) and DMSO (in a 1:1 ratio with water).The reaction mixture was injected directly into a HP 1100 HPLC system (Agilent Technologies, Germany) equipped with an autosampler, quaternary pump and DA detector.The separation was carried out on a Supelcosil ABZ + Plus column (250 × 4.6 mm, 5 µm particle size, column temperature 25 °C); the mobile phase consisted of 90% 40 mM HCOOH and 10% CH 3 CN (v/v) at a flow rate of 1 mL/min.The chromatograms were detected at 276 nm ± 20 nm.The obtained results were compared to the calibration curve of catechin.Data were expressed as an antioxidant activity equivalent of catechin (CE) in mmol/100 g of dry extract (mean ± SD of at least three measurements) [8].

Table 1 .
Total phenolic content and antioxidant activities of Staphylea L. extracts.