Role of the flavonoid-rich fraction in the antioxidant and cytotoxic activities of Bauhinia forficata Link. (Fabaceae) leaves extract

Bauhinia forficata Link. is utilised as an antidiabetic in Brazilian folk-medicine; furthermore, its antioxidant properties suggest a potential usefulness in the prevention of diabetes complications associated with oxidative stress. The contribution of a flavonoid-rich fraction (FRF), HPLC-PDA-ESI-MS characterised, to the antioxidant and cytotoxic properties of B. forficata hydro-alcoholic leaves extract was evaluated for the first time. Both extract and FRF showed radical-scavenging activity and reducing power with a strong relationship with the flavonoid content found; hence, flavonoids are mainly responsible for the primary antioxidant activity of B. forficata extract. The extract significantly decreased FO-1 cell viability at the higher concentrations. FRF did not exert any effect; thus, flavonoids do not appear to be responsible for the cytotoxicity of the extract. The extract resulted virtually non-toxic against both Artemia salina and normal human lymphocytes, demonstrating potential selectivity in inhibiting cancer cell growth. Finally, no antimicrobial activity was observed against the bacteria and yeasts tested.


Introduction
Bauhinia forficata Link. (Fabaceae) is a small tree reaching up to 9 m height, native to the tropical area of Asia, Paraguay and Argentina, and is well adapted to the Brazilian climate. The leaves (7 -12 cm £ 13 cm) are alternate, simple, petiolate, bilobate to about one-third, or, more frequently to half of their length, rounded at the base, pubescent beneath; due to the characteristic shape of the leaves, this species is commonly known as 'Pata de Vaca' (cow's hoof) (Miyake et al. 1986;Da Silva et al. 2000). B. forficata is the Bauhinia species most used as an antidiabetic herbal remedy in popular medicine in Brazil, where it is defined also as 'vegetable insulin'. The leaves and stem barks of this plant are used in different phytopreparations to lower blood glucose levels (Peroza et al. 2013). Its hypoglycemic properties have been attributed to the flavonoid kaempferitrin (kampferol-3,7-O-(a)-Ldirhamnoside), which has been shown to occur only in B. forficata leaves and, consequently, can be used as a chemotaxonomical marker (Da Silva et al. 2000). It has been reported that B. forficata and other plant extracts used in traditional medicines for diabetes also have significant antioxidant activity, beneficial in the prevention of diabetes complications associated with oxidative stress (Khalil et al. 2008). Previous studies reported that hydro-alcoholic extracts of B. forficata have a significant antioxidant activity in vitro, and hypothesised a possible correlation with their content in some flavonoid derivatives (Ferreres et al. 2012). Lack of scientific data to support such claims prompted this study, which was therefore aimed at establishing, through different in vitro models, the contribution of the flavonoid-rich fraction (FRF) to the antioxidant properties of B. forficata leaves crude extract. Plants used in folk medicine have been considered an important source of compounds with pharmacological activities, including antitumour. B. forficata is traditionally used in Brazil for treating cancer; nonetheless few studies concerning the cytotoxic properties of this plant have been carried out (Lim et al. 2006;Gomes de Melo et al. 2011;Silva et al. 2014). In this work, the cytotoxic properties both of B. forficata leaves crude extract and FRF against two different tumour cell lines have been evaluated.
Furthermore, since B. forficata is traditionally used for its antiseptic action, it seemed interesting to extend the study to the evaluation of the antimicrobial activity against bacteria and yeasts.
2. Results and discussion 2.1. Phytochemical investigations 2.1.1. Determination of total phenolic, total flavonoid and condensed tannin content The total phenolic, total flavonoid and condensed tannin contents of B. forficata extract, HF and FRF are reported in Table 1. The total flavonoid content of the crude extract was superimposable to that of FRF (62.59^0.32 mg QE/g extract and 61.72^0.90 mg QE/g extract, respectively).

Identification of phenolic compounds by HPLC-DAD-ESI-MS analysis
The HPLC-PDA chromatograms of B. forficata extract and FRF were obtained at 280 nm wavelength; a total of 16 and 10 compounds, which are listed in Table 2, were positively identified on the basis of the complementary information provided by photodiode array and ESI-MS detectors along with literature data. For gallic acid, catechin, chlorogenic acid, rutin, isoquercetin, naringin, kaemferol-3-glucoside, quercetin and kaempferol the identification was confirmed with the use of authentic pure standards. The leaves hydro-alcoholic extract of B. forficata was characterised by the presence of two phenolic acids, belonging to the class of benzoic and cinnamic acids (gallic acid and chlorogenic acid), a flavan-3-ol (catechin), and  , LOD ¼ below the limits of detection thirteen flavonoid compounds; in addition, in the FRF gallic acid and nine flavonoids were detected. The achieved results suggest that kaempferol and quercetin derivatives represent the most abundant flavonoids identified, and this agrees with previous literature data ( Cechinel Filho 2009;Ferreres et al. 2012;Sayago et al. 2013). It is worth noting that kaempferitrin, used as a chemotaxonomical marker of B. forficata, was identified both in the extract and FRF. Gallic acid, chlorogenic acid and catechin were identified in this work for the first time. From a quantitative point of a view, both in the hydro-alcoholic extract and FRF, flavonoids represented the most abundant fraction (77.67 and 43.11 mg/g, respectively), whereas phenolic acids are present in a lower concentration (15.44 and 6.75 mg/g). Myricetin-3-O-rhamnoside was the main flavonoid in both extracts, followed by kaempferol-3-O-glucoside.

Antioxidant activity
Due to the complex nature of phytochemicals and their interactions, it is obvious that no single testing method is capable of providing a comprehensive picture of the antioxidant profile of a sample; it is thus important to evaluate the effectiveness of antioxidants by using several analytical methods and different substrates. In this study, three in vitro assays were used to screen the antioxidant potential of B. forficata extract and fractions: DPPH test and reducing power to evaluate primary (chain breaking) antioxidant effectiveness, metal chelating activity to determine secondary antioxidant properties. The results of the DPPH test show that both B. forficata extract and FRF have a noticeable free radical-scavenging effect, resulting superimposable to that of the standard butylated hydroxytoluene (BHT) starting from the concentration of 0.5 mg/mL (95%, 88% and 94%, respectively). The activity of the HF is weak for all tested concentrations (Table 1). Linear regression analysis revealed a positive relationship with total phenolic content (r 2 ¼ 0.5678), and a very strong relationship with total flavonoids (r 2 ¼ 0.9999). A weak correlation with condensed tannins was found (r 2 ¼ 0.3232). The obtained results are in agreement with those of Ferreres et al. (2012), which previously reported the free radical-scavenging properties of B. forficata leaves hydro-alcoholic extract (methanol: water 1:1).
In the reducing power assay B. forficata extract exhibited strong activity, which, in any case, was lower than BHT; the FRF resulted less active than the extract. The activity of the extract and FRF increased in a dose-dependent manner. The HF did not display any activity (Table 1). A positive relationship between reducing power and total phenolic content (r 2 ¼ 0.5837), and a very strong relationship with total flavonoid content (r 2 ¼ 0.9995) was found. The correlation with condensed tannins was weak (r 2 ¼ 0.3383). B. forficata extract displayed a moderate, dose-dependent, Fe 2þ chelating activity reaching 60% inhibition at the highest concentration assayed. The Fe 2þ chelating activity of HF resulted very weak at all tested concentrations. The FRF resulted even less active than HF (Table 1). No correlation with total phenolic, total flavonoid and condensed tannin content was found.
The results of the HPLC analysis showed that flavonoids represent the most abundant phenolic components both in the hydro-alcoholic extract and FRF; in particular, high amounts of the flavonol glycosides myricetin-3-O-rhamnoside (myricitrin), kaempferol-3-O-glucoside and quercetin-3-arabinoside were found, and the radical-scavenging properties of these compounds have been previously reported (Lu et al. 2000;Hayder et al. 2008;Delazar et al. 2008).
As showed by the coefficient of determination (r 2 ), a very strong relationship between both radical-scavenging activity and reducing power with the total flavonoid content was found; hence, flavonoids play the chief role in the overall primary antioxidant activity of B. forficata crude extract.
The antioxidant activity of flavonoids in vitro depends upon the arrangement of functional groups about the nuclear structure: the number and location of the hydroxyl moieties, the presence of 2,3-double bond in conjugation with a 4-oxo function in the C-ring, 3-and 5hydroxy groups, 3,5,7-trihydroxy, ortho-catechol group (3 0 ,4 0 -OH), and the glycosylation model (C-glycosides or O-glycosides) and position. (Rice-Evans et al. 1996). Cai et al. (2006) reported that flavonols, which contain more hydroxyl groups, such as quercetin and myricetin, have a very high ability to scavenge ABTS and DPPH radicals. Besides, gallic acid occurred in the hydro-alcoholic extract and FRF as the most abundant and only phenolic acid, respectively. Phenolic acids are considered to be efficient hydrogen donors due to their characteristic carboxylic group, which is easily ionised; it was demonstrated that both the number of hydroxyl groups and their meta position with respect to the carboxylic group correlated positively with antioxidant capacity against DPPH. Hence, the strong antioxidant capacity of gallic acid was related to the four hydroxyls (Palafox-Carlos et al. 2012).
Concerning metal chelating activity, no correlation with total phenolics and flavonoids was found; thus, it can be hypothesised that flavonoids are not responsible of the secondary antioxidant properties of B. forficata crude extract, but other classes of phytochemicals are involved in the observed effect.

Antimicrobial activity
B. forficata extract, HF and FRF did not display any antimicrobial effect against all the tested strains (MICs . 625 mg/mL). The obtained results do not appear to support the ethnopharmacological utilisation of this species as an antiseptic.

Cytotoxic activity
It is clear how the potential selectivity of a medicinal plant in inhibiting cancer cell growth, while not affecting normal cells, is a primary goal in anticancer drug research; hence the cytotoxic effects of B. forficata extract were evaluated in non cancerous cells by in vivo and in vitro systems, Artemia salina and human lymphocytes, respectively. The extract did not display any toxicity neither in the A. salina lethality assay (LC 50 . 1000 mg/mL) nor against normal human lymphocytes, at all the tested concentrations; for this reason, the cytotoxic effect of the FRF was not investigated.
The results obtained by testing the cytotoxic activity against FO-1 cells show that, after 24 h of treatment, the B. forficata extract significantly reduced cancer cell growth at the higher concentrations tested, resulting approximately of 50.58% at the dose of 50 mg/mL. After 48 h exposure to the extract, an inhibition of 41.67% at the dose of 50 mg/mL was observed (Figure 1). Both at 24 and 48 h the extract resulted less active than paclitaxel. FRF did not show any activity at all tested concentrations (data not shown). Concerning CACO-2 cell line, both B. forficata extract and FRF did not affect the cell viability in the range of doses assayed (data not shown).
The cancer protective properties of polyphenols have been well documented; numerous studies have established that polyphenols can either scavenge the constitutive ROS or paradoxically generate additional amounts of ROS to inhibit the proliferation of cancer cells. Due to the polyphenolic structure, flavonoids have been found to possess both anti-and prooxidant action; it seems to depend on dose, cell type, and also culture conditions (Sak 2014). In spite of this, FRF obtained from B. forficata leaves, which showed strong antioxidant properties, did not exert any cytotoxic effect against the tumour cell lines tested; thus it can be assumed that flavonoids are not responsible for the cytotoxic activity of B. forficata crude extract, and that other different phytochemicals might act additively and/or synergistically to contribute to the observed effect. Our results are in agreement with those of other works, in which a weak relationship between antioxidant potential and cytotoxic activity of flavonoids was found (Cao et al. 2013).
Further investigations will be necessary to evaluate the role of flavonoids in the cytotoxic effect of B. forficata against other different tumour cell lines.

Chemicals
Lymphoprepe was from Axis-Shield (Scotland). CellTiter-Bluew was supplied from Promega (USA). All the other cell culture reagents and medium were from Lonza (Milan, Italy). Unless indicated otherwise, chemicals were purchased from Sigma-Aldrich (Milan, Italy).

Plant material, extraction and fractionation procedure
The dried leaves of Bauhinia forficata Link. were purchased from herbalist's shop in Rio de Janeiro (Brazil). The identity of the plant was confirmed by microscopic observation of the leaves, referring to previous published works (Engel et al. 2008;Garibotti Lusa & Bona 2009). Furthermore, the flavonoid kaempferitrin, occurring only in B. forficata leaves and used as chemical marker for the identification of this species, was detected by using HPLC-DAD-ESI/ MS analysis (Da Silva et al. 2000). The dried and powdered B. forficata leaves were percolated according to the Brazilian Pharmacopeia method, using 80% ethanol (Atheneu 2001). The extractive solution was evaporated to dryness by rotary evaporator (408C). The yield of B. forficata extract, referred to 100 g of dried leaves, was 18.67%. The extract was suspended in distilled water (150 mL) and then partitioned between n-hexane (150 mL £ 4) and ethyl acetate (120 mL £ 6), successively. The supernatants obtained with each solvent were pooled and concentrated under reduced pressure to give n-hexane fraction (HF, 5.21% yield) and ethyl acetate fraction (FRF, 18.37% yield).

Phytochemical investigations
3.3.1. Determination of total phenolic, total flavonoid, and condensed tannin content The total phenolic, total flavonoid and condensed contents of B. forficata extract, HF and FRF were measured by using colorimetric assays as previously reported ) and expressed as mg gallic acid equivalents (GAE)/g extract (dw)^standard deviation (SD), mg quercetin equivalents (QE)/g extract (dw)^SD and mg catechin equivalents (CE)/g extract (dw)^SD, respectively. For the fractions, they were calculated on the basis of the yield with respect to dried extract.

Identification of phenolic compounds by HPLC-DAD-ESI-MS analysis
A quali-quantitative investigation of phenolic compounds of B. forficata extract and FRF has been carried out by using HPLC-DAD-ESI-MS as previously described (Germanò et al. 2012). As detection, a diode array detector and a quadrupolar mass analyser (LCMS-2020, Shimadzu), equipped with an ESI interface, in negative ionisation mode were employed. Data acquisition was performed by Shimadzu LabSolution software ver. 5.10.153. Each sample (10 mg) was dissolved in 1 mL of MeOH and filtered through 0.45 mm membrane filters (Whatman, Clifton, USA). Analyses were carried out on a Ascentis Express C18, 15 cm £ 4.6 mm i.d. with particle size of 2.7 mm (Supelco, Bellefonte, PA). The injection volume was 2 mL, mobile phase consisted of water/formic acid (99.9:0.1) (solvent A) and ACN/formic acid (99.9:0.1) (solvent B), the linear gradient profile was as follows: 0 min, 0% B, 5 min, 5% B, 15 min, 10% B, 30 min, 20% B, 60 min, 50% B, 70 min, 100%B, 71 min, 0%B. The flow-rate was 1 mL/min and it was split to 0.2 mL/min prior to MS detection. PDA and MS conditions were reported in a previous work (Germanò et al. 2012). Quantitative determination was carried out using calibration curves in a concentration range 1-50 mg/L of four standards, namely catechin, chlorogenic acid, isoquercitrin and kaempferol. The calibration curves with the external standards were obtained using concentration (mg/L) with respect to the area obtained from the integration of the PDA peaks at a wavelength of 280 nm for benzoic-like and flavan-3-ol -like compounds, 325 nm for cinnamic acid-like compounds, 354 nm for flavonol-glycoside-like compounds and 369 nm for flavonol-like compounds. For all standards, an excellent linearity (catechin: y ¼ 1425.4 £ 2 422.48, R 2 ¼ 0.999; chlorogenic acid: y ¼ 6021.6 £ 2 6409.1, R 2 ¼ 0.998; isoquercitrin: y ¼ 2428.66 £ 2 1897.1, R 2 ¼ 0.999; quercetin: y ¼ 803.66 £ þ 818.28, R 2 ¼ 1) was attained. The amount of the compound was finally expressed in mg/g of extract.

Free radical-scavenging activity
The free radical-scavenging activity of B. forficata extract, HF and FRF was determined using the DPPH (1,1-diphenyl-2-picrylhydrazyl) method ). The extract was tested at different concentrations (0.0625 -1.5 mg/mL); the fractions were tested at concentrations corresponding to 0.0625 -1.5 mg/mL extract, calculated on the basis of the fractionation yield. BHT was used as reference standard. The results are expressed as mean radical-scavenging activity percentage (%)^SD and as 50% inhibitory concentration (IC 50 ), determined graphically by interpolation of the dose -response curve.

Measurement of reducing power
The reducing power of B. forficata extract, HF and FRF was determined as previously reported . Different amounts of extract (0.0625 -1.5 mg/mL) and fractions (doses corresponding to 0.0625 -1.5 mg/mL extract), were tested. Ascorbic acid and BHT were used as reference standards. The results are expressed as mean absorbance values^SD and as ascorbic acid equivalent (ASE/mL) ).

Ferrous ions (Fe 2þ ) chelating activity
The Fe 2þ chelating activity of B. forficata extract, HF and FRF was estimated using the method previously reported . Different concentrations of extract (0.0625-1.5 mg/mL) and fractions (doses corresponding to 0.0625 -1.5 mg/mL extract) were assayed. Ethylenediaminetetraacetic acid (EDTA) was used as reference standard. The results are expressed as mean values of inhibition of the ferrozine-(Fe 2þ ) complex formation (%)^SD and as IC 50 .

Antimicrobial activity
The following strains were used as indicators for the antimicrobial testing and were obtained from in-house culture collection of 'Scienze del Farmaco e Prodotti per la Salute' Department: Bacillus subtilis ATCC6683, Staphylococcus aureus ATCC 6538P, Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis (wild-type strain), Serratia marcescens (wild-type strain), Candida albicans ATCC 10231. The minimum inhibitory concentrations (MICs) of B. forficata extract, HF and FRF were determined according to the Clinical Laboratory Standards Institute guidelines . The MICs of the extracts on yeasts were determined according to National Committee for Clinical Laboratory Standards . As positive controls were used ofloxacin (range from 0.2 to 0.3 mg/mL) and tetracycline (2 -8 mg/mL) for antibacterial activity, amphotericin B (0.2 -0.3 mg/mL) and clotrimazole (2 -8 mg/mL) for antifungal activity. The results were obtained from the average of three independent experiments and modal results were calculated.
3.6. Cytotoxic activity 3.6.1. Artemia salina Leach lethality bioassay The Artemia salina Leach (brine shrimp) lethality bioassay was employed to predict the potential cytotoxic activity of B. forficata extract. Medium lethal concentration (LC 50 ) determination was carried according to the method previously reported . Three independent experiments were carried out, and LC 50 was determined using the probit analysis method. Extracts are considered active if the LC 50 is lower than 1000 mg/mL.

Cytotoxic activity in human normal lymphocytes
The cytotoxic effect of B. forficata extract in non-cancerous cells were evaluated using lymphocytes obtained from pooled peripheral blood samples collected from consenting healthy adult donors. Lymphocytes were isolated using Lymphoprepe, seeded in 96-well culture plates and incubated in RPMI 1640 supplemented with 10% foetal bovine serum (FBS) at 378C, 5%