Isolation of flavonoids from Anemopaegma arvense ( Vell ) Stellf . ex de Souza and their antifungal activity against Trichophyton rubrum

Anemopaegma arvense (Vell) Stellf. ex de Souza belongs to the family Bignoniaceae, and is popularly known as catuaba. To evaluate the cytotoxic and antimicrobial activity of A. arvense, fraction F3 and flavonoids 1 (quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside) (rutin) and flavonoid 2 (quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside) were isolated from the leaves of this plant. Fraction F3 and flavonoids 1 and 2 exhibited no antibacterial activity. Furthermore, no cytotoxic activity of fraction 3 or flavonoids 1 and 2 was observed against the tumor cells tested. However, analysis of the antifungal activity of flavonoids 1 and 2 revealed minimum inhibitory concentrations of 0.5 and 0.25 mg/mL, respectively, against the Trichophyton rubrum strains tested (wild type and mutant). This study demonstrates for the first time the antifungal activity of isolated flavonoids, validating the same activity for A. arvense.


INTRODUCTION
The family Bignoniaceae comprises about 800 plant species that are found mainly in the Neotropical region (Gentry, 1980).In Brazil, several plants of this family are used in folk medicine as astringent and against fever, rheumatism, diarrhea, cancer and microbial infections (Pio Côrrea, Penna, 1969;Fenner et al., 2006).Anemopaegma arvense (Vell) Stellf.ex de Souza is a species of the family Bignoniaceae, which is popularly known as "catuaba".Commercially available formulations of this plant are used as aphrodisiac (Manabe et al., 1992).The major components identified in A. arvense are flavonoids, catuabins, alkaloids, tannins, and resins (Charam, 1987;Zanolari et al., 2005;Tabanca et al., 2007).Flavonoids are becoming the subject of anti-infective research and many groups have isolated and identified the structures of flavonoids with antifungal, antiviral and antibacterial activity (Cushnie, Lamb, 2005).
There is an urgent need to develop new and more effective antifungal drugs because of the increased resistance of fungi to the drugs currently used in clinical practices (Rahalison et al., 1994).Plant secondary metabolites represent a good source of novel antimicrobial molecules.Furthermore, there has been an almost exponential rise in cancer-related mortality over recent years, which has led to an increase in the search for new medicines, including those derived from natural products, able to treat the various types of the diseases (Patocka, 2003;Aziz, 2004;Diwanay et al., 2005).
We investigated the cytotoxic and antimicrobial activity of chromatographic fraction F3 and flavonoids isolated from A. arvense in order to provide better understanding of the biological activities of this plant.

Identification of flavonoids
The identity of the flavonoids was confirmed based on 1 H and 13 C NMR spectral data and by comparison with the literature (Jaramillo et al., 2011).The position of the interglycosidic linkage was provided by 13 C NMR and was confirmed by HMBC and HMQC experiments. 1 H NMR (300 MHz) and 13 C NMR (75 MHz) spectra were recorded with a Bruker Avance DPX 300 spectrometer in DMSO-d 6 using TMS as internal standard.HPLC analysis was performed using a Shimadzu LC10ADvp system equipped with a Supelco LC18 column (Supelcosil TM RP-18, 250 x 4.6 mm i.d., 5 µm) and coupled to a diode array detector, monitored at 340 nm.The following gradient program was used: MeOH:H 2 O (0-32 min: 10-66% MeOH; 32-35 min: 66-10% MeOH; 35-40 min: 10% MeOH).The flow rate was 1.0 mL/min and the sample injection volume was 20 μL at a concentration of 1 mg/mL.

Trichophyton rubrum
The clinical strain of T. rubrum (ATCC MYA3108) was kindly provided by Dr. Nilce M. Martinez-Rossi.The TruMDR2 mutant strain was obtained by disruption of the TruMDR2 gene of strain MYA3108 (Fachin et al., 2006).Standard techniques of manipulation and growth as described previously (Fachin et al., 2001) were used.Susceptibility of the MYA3108 (wild type) and TruMDR2 (mutant) strains was tested by determining the minimum inhibitory concentration (MIC) of fraction F3 and of flavonoids 1 and 2 using the M38-A microdilution technique proposed by the Clinical and Laboratory Standards Institute (CLSI, 2002).Fraction F3 and flavonoids were diluted in 10% DMSO and the final concentration of DMSO in the antifungal assay was less than 1%.Colonies obtained by growth of the strains on Sabouraud agar plates at 28 °C for 15 days were harvested by sterile scraping and mixed with sterile saline and the solution was filtered through glass wool.
The resulting mixture was transferred to a sterile tube and adjusted spectrophotometrically at a wavelength of 530 nm, ranging from 70 to 75% transmittance.These conidial suspensions were diluted 1:50 in RPMI 1640 (Sigma, St. Louis, MO, USA) buffered with MOPS, corresponding to twice the density needed for the test of approximately 3-5 × 10 5 CFU/mL.Growth, solvent and sterility controls were included.Microtiter plates were incubated at 28 °C for 7 days.The MIC100 was defined as the lowest concentration of the fraction or flavonoid that resulted in the complete inhibition of fungal growth.The range of concentrations tested was 2.5-0.019mg/mL and 0.500-0.019mg/mL for fraction F3 and the flavonoids (1 and 2), respectively.The assays were carried out in triplicate in three independent experiments.Fluconazole and griseofulvin were used as positive controls.

Bacteria
The following strains were used as test organisms: Staphylococcus aureus (ATCC 6538), Staphylococcus epidermides (ATCC 2228), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853).The antimicrobial activity of fraction F3 and flavonoids 1 and 2 was evaluated using the microdilution method according to CLSI M7-A7 ( 2006).The test strains were incubated in BHI medium for 24 h at 37 o C. The crude extract, fraction F3 and flavonoids were diluted in 10% DMSO and the final concentration of DMSO in the antibacterial assay was less than 1%.The crude extract and fraction were assayed at concentrations of 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039, and 0.019 mg/mL.The flavonoids were assayed at a concentration range of 0.500 to 0.039 mg/mL.The assays were carried out in triplicate in three independent experiments.Ampicillin and chloramphenicol were used as positive controls.

Cytotoxic activity
The following cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Gaithersburg, MD, USA) supplemented with 15% fetal calf serum (Life Technologies Inc.) at 37 °C in a humidified atmosphere of 5% CO 2 : HeLa (human cervix tumor), 3T3 (mouse embryonic fibroblasts), and U343MG-a (human glioblastoma cell line).Penicillin (100 U/mL) and streptomycin (0.1 mg/mL) were added to the medium to prevent bacterial growth.A stock solution (20 mg/mL) was prepared by dissolving fraction F3 and flavonoids 1 and 2 in 10% DMSO (v/v).The final concentration of these compounds (0.2, 0.02 and 0.002 mg/mL, respectively) was obtained by direct dilution in the culture medium.The final concentration of DMSO in the control and experimental groups was 1%.The cells (10 5 cells/well) were seeded into a 96-well plate 24 h prior to the beginning of the experiment.Actinomycin D (Sigma) was used as positive control.The cells were incubated for 48 h with the fraction or flavonoids and analyzed by the MTT assay (Mosmann, 1983;Rubinstein et al., 1990).

RESULTS AND DISCUSSION
The aglycons were identified as quercetin by 1 H and 13 C NMR.The 1 H NMR spectra indicated the presence of a rhamnosyl and a glucosyl group at δ 5.34 and δ 4.41 in flavonoid 1 and of a rhamnosyl and a galactosyl group at δ 5.30 and δ 4.36 in flavonoid 2. The relatively deshielded rhamnosyl protons suggested that they are not directly attached to the aglycone, but that there is a sugar-sugar linkage.The position of the sugar in the aglycons provided by HMBC was demonstrated by cross-peaks between H-1'' and C-3.The position of the interglycosidic linkage was provided by the 13 C-NMR downfield shift of C-6'' at δ 67.5 and δ 65.7 in flavonoids 1 and 2, respectively.This fact was confirmed by HMBC and HMQC experiments.On the basis of the spectroscopic data, flavonoids 1 and 2 were characterized as quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (rutin) and quercetin 3-O-α-L-rhamnopyranosyl-(1→6)β-D-galactopyranoside, respectively (Figure 1).HPLC analysis of fraction F3 revealed the presence of six flavonoids (Figure 2A).Flavonoids 1 and 2 isolated from A. arvense were detected at 24.1 and 23.2 min, respectively (Figure 2B and 2C).
Analysis of the bioactivity of A. arvense showed no cytotoxic activity of fraction F3 or of the isolated flavonoids at a dose of 0.2 mg/mL against the cell lines tested.In addition, at a dose of 2.5 mg/mL, the crude extract, fraction F3 and the flavonoids exhibited no antibacterial activity against the strains tested (data not shown).These results partially agree with those reported by Tabanca et al. (2007)  who isolated a new catuabin (catuabin A) and three known flavan-type phenylpropanoids (cinchonain Ia, cinchonain IIa, and candelin A1) from A. arvense.These compounds possessed no anti-inflammatory, cytotoxic, antimicrobial or antimalarial property, but exhibited antioxidant activity.
In the present study, fraction F3 exhibited antifungal activity against the mutant and wild-type strains of T. rubrum at concentrations of 1.25 and 0.625 mg/mL, respectively.Isolated flavonoid 1 showed weak antifungal activity, with an MIC of 0.5 mg/mL against the two fungal strains tested, whereas flavonoid 2 exhibited moderate antifungal activity, with an MIC of 0.25 mg/mL (Table I).Antifungal activity of ethanol extracts of Hyptis ovalifolia and Eugenia uniflora has been demonstrated by Souza et   Fluconazole and griseofulvin were used as positive controls.Fraction F3: concentration range of 2.5-0.019mg/mL.Flavonoids: concentration range of 0.500-0.019mg/mL.al. (2002).These extract completely inhibited the growth of the 30 dermatophytes tested.The MIC of the H. ovalifolia extract against T. rubrum strains was 0.25 mg/mL and the E. uniflora extract exhibited antifungal activity against 19 of the 30 isolates at a concentration of 0.5 mg/mL.Rocha et al. (2004) demonstrated antifungal activity of Clytostoma ramentaceum and Mansoa hirsuta (Bignoniaceae) when testing the low and medium polar fractions at concentrations of 0.1 to 0.3 mg/mL.Pacciaroni et al. (2008) isolated several flavonoids from the aerial parts of Heterothalamus alienus and tested these compounds against clinical isolates of dermatophytes.The flavanones showed very good fungicidal activity against standard (MIC: 31.2 μg/mL) and clinical isolates of T. rubrum and T. mentagrophytes (MIC: 31.2-62.5 and 31.2-125μg/mL, respectively).However, rutin, spathulenol (1) and two of the 3-acetylated flavanones were inactive or marginally active against the fungal strains (MIC > 250 µg/mL).
Methods for antimicrobial assessment of natural products and effective MIC values are not well established in the literature.Holetz et al. (2002), who screened hydroalcoholic extracts from 13 Brazilian plants using the microdilution technique, defined an MIC < 0.1 mg/mL as good antimicrobial activity, MIC of 0.1 to 0.5 mg/mL as moderate antimicrobial activity, and MIC of 0.5 to 1 mg/mL as weak antimicrobial activity.Extracts exhibiting MIC higher than 1 mg/mL are considered to be ineffective.Reports of activity in the field of antibacterial flavonoid research are widely conflicting, probably because of interand intra-assay variation in susceptibility testing (Cushnie, Lamb, 2005).
Membrane transporters, especially efflux transporters, affect the adsorption and bioavailability of drugs.MIC of flavonoids 1 and 2 was the same for the wild-type and mutant strain of T. rubrum (in which the ABC gene is disrupted).This finding may be explained by the fact that ABC transporters are not involved in the transport of flavonoids (Walgren et al., 2000).The TruMDR2 gene was disrupted in the mutant strain of T. rubrum and this strain has been shown to be susceptible to several compounds (Fachin et al., 2006).Therefore, despite relatively high MIC, flavonoid-based inhibitors of fungi may be an alternative for the treatment of multidrug-resistant strains since efflux pumps do not transport these compounds.
In the present study, we were able to isolate and identify two quercetin-derived glycosylated flavonoids from A. arvense, which showed antifungal activity.Quercetin is a substance widely distributed in the plant kingdom.However, this study describes for the first time the antidermatophyte activity of A. arvense, which could be attributed to the presence of quercetin.In fact, the antifungal activity of quercetin and its derivatives has been described in other medicinal plants.Semwal et al. (2009) demonstrated the antifungal activity of an ethanol extract of Boehmeria rugulosa leaves and of three new flavonoid glycosides against T. rubrum, Microsporum canis and Microsporum gypseum, with MIC of 100 µg/mL.Pereira et al. (2008) isolated the flavonoid rutin from the aerial parts of Solanum palinacanthum and evaluation of its antimicrobial activity showed an MIC of 35 μg/mL against the fungus Aspergillus ochraceus.
Human mycoses are not always treated effectively.The most important causes of treatment failure are the recurrence of infections, drug resistance of pathogens, and toxicity of currently available antifungal agents (Turel, 2011;Butts, Krysan 2012).Therefore, the continual search for new and more effective antifungal drugs, which should also be safer than currently used agents, is important (Zacchino, 2001).The increasing prevalence of multidrugresistant pathogens requires the identification of new antimicrobial agents as alternative therapies in difficultto-treat infections (Pereira et al., 2006).In conclusion, the flavonoids isolated from A. arvense were bioactive against T. rubrum and may be a promising target in studies on new antifungal agents.