Total Phenolic and Flavonoid Content and Biological Activities of Extracts and Isolated Compounds of Cytisus villosus Pourr.

The aim of this study was to evaluate the total phenolic and flavonoid content, and the in vitro antioxidant, anti-inflammatory, antibacterial, antifungal, antimalarial, cytotoxicity, and antiprotozoal activities of the Algerian plant Cytisus villosus Pourr. (Syn. Cytisus triflorus L’Hérit.). Additionally, the radioligand displacement affinity on opioid and cannabinoid receptors was assessed for the extracts and isolated pure compounds. The hydro alcoholic extract of the aerial part of C. villosus was partitioned with chloroform (CHCl3), ethyl acetate (EtOAc), and n-butanol (n-BuOH). The phenolic content of the C. villosus extracts was evaluated using a modified Folin–Ciocalteau method. The total flavonoid content was measured spectrometrically using the aluminum chloride colorimetric assay. The known flavonoids genistein (1), chrysin (2), chrysin-7-O-β-d-glucopyranoside (3), and 2″-O-α-l-rhamnosylorientin (4) were isolated. The antioxidant activities of the extracts and isolated compounds were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DDPH) and cellular antioxidant activity (CAA) assays. The plant extracts showed moderate antioxidant activity. EtOAc and n-BuOH extracts showed moderate anti-inflammatory activity through the inhibition of induced nitric oxide synthase (iNOS) with IC50 values of 48 and 90 µg/mL, respectively. The isolated pure compounds 1 and 3 showed good inhibition of Inducible nitric oxide synthase (iNOS) with IC50 values of 9 and 20 µg/mL, respectively. Compounds 1 and 2 exhibited lower inhibition of Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) with IC50 values of 28 and 38 µg/mL, respectively. Furthermore, the extracts and isolated pure compounds have been shown to exhibit low affinity for cannabinoid and opioid receptors. Finally, n-BuOH extract was a potent inhibitor of Trypanosoma brucei with IC50 value of 7.99 µg/mL and IC90 value of 12.61 µg/mL. The extracts and isolated compounds showed no antimicrobial, antimalarial nor antileishmanial activities. No cytotoxic effect was observed on cancer cell lines. The results highlight this species as a promising source of anti-inflammatory and antitrypanosomal agents.

folk medicine as a diuretic and in the treatment of mild hypertension, heart failure, cardiac edema, and wounds. Cytisus species have been found to exhibit bioactive properties, including antioxidant, anti-inflammatory, anxiolytic, antiparasitic, and antidiabetic activities [29][30][31]. The therapeutic properties of Cytisus are related to their high concentration of phenolic compounds, including flavonoids and caffeic acids [32]. In continuation of previous works on Algerian plants [33,34], herein, we extended our study to evaluate the antioxidant, anti-inflammatory, antiprotozoal, antimalarial, antimicrobial, cytotoxicity, and radioligand displacement affinity on opioid and cannabinoid receptors activities of extracts and isolated pure compounds of Cytisus villosus Pourr. (Syn. Cytisus triflorus L'Hérit.).

Plant Material
The aerial parts of Cytisus villosus Pourr. were collected from the Collo region, in Northeastern Algeria during its flowering stage in April 2010. A voucher specimen (UM-10232015) has been deposited in the culture collection of the Department of BioMolecular Sciences, University of Mississippi.

Determination of Total Phenolic and Total Flavonoid Content
Folin-Ciocalteu reagent, gallic acid, and quercetin standards were obtained from Sigma-Aldrich (Poznan, Poland). Aluminum chloride hexahydrate, methanol, and sodium carbonate were obtained from Sigma-Aldrich (Poznan, Poland). The total phenolic was measured using spectrophotometry with a modified Folin-Ciocalteu method [35]. Total phenol content, expressed as milligrams of gallic acid equivalent (GAE) per gram of extract (GAE mg/g), was calculated on the basis of a standard calibration curve of gallic acid (Y = 0.1157x + 0.087, R 2 = 0.9749). Total flavonoid content of the plants fractions crud extracts was determined by colorimetric method [36,37]. The concentration of total flavonoid content in the test samples was calculated from the calibration plot (Y = 1.2308x + 0.0151, R 2 = 0.9775) and expressed as mg quercetin equivalent (QE)/g of dried extract. The extracts were dissolved in dimethyl sulfoxide (DMSO) to make a stock solution of 20 mg/mL.

Cellular Antioxidant Activity (CAA) Assay
The cellular antioxidant activity was measured in HepG2 cells as described by Wolfe and Rui [39,40]. The antioxidant activity was expressed in terms of CAA units. The area under the curve (AUC) of fluorescence versus time plot was used to calculate CAA units as described by Wolfe and Rui [39,40].
2.6. Anti-Inflammatory Activity 2.6.1. Anti-Inflammatory Activity Assay for the Inhibition of iNOS The extracts and isolated compounds of C. villosus were evaluated in terms of their interaction with cellular targets related to inflammation and metabolic disorders, such as iNOS and NF-κB. The inhibition of intracellular NO production as a result of iNOS activity was assayed in mouse macrophages (RAW 264.7cells) [41]. Cytotoxicity of test samples to macrophages was also determined in parallel to check if the inhibition of iNOS was due to cytotoxic effects.

Reporter Gene Assay for the Inhibition of NF-κB
Reporter gene assay for the inhibition of NF-κB Activity was performed as described earlier [42]. In brief, cells transfected with NF-κB luciferase plasmid construct were plated in 96-well plates at a density of 1.25 × 105 cells/well. After 24 h, cells were treated with the test compounds and, after incubating for 30 min, phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Burlington MA, USA) (70 ng/mL) was added and further incubated for 6−8 h. Luciferase activity was measured as described above. Percent decrease in luciferase activity was calculated relative to the vehicle control. Parthenolide (Sigma-Aldrich, Burlington MA, USA) was used as a positive control.

Antiprotozoal Assay
The in vitro antileishmanial and antitrypanosomal assays were done on cell cultures of L. donovani promastigotes, axenic amastigotes, THP1-amastigotes, and Trypanosoma brucei trypomastigotes by Alamar Blue assays [43]. The conditions for seeding the THP1 cells, exposure to the test samples, and evaluation of cytotoxicity were the same as described in parasite-rescue and transformation assay [44]. IC 50 and IC 90 values were computed from the dose response curves using XLfit software (XLfit 5.3.1, IDBS analytical, Boston MA, USA). DFMO (difluoromethylornithine) was used as the positive control. The antiprotozoal activity of C. villosus extracts and isolated compounds were evaluated in vitro against L. donovani promastigotes, axenic amastigotes, and intracellular amastigotes in THP1 cells. The extracts and some isolated compounds were also evaluated against T. brucei trypomastigote forms. All the extracts and compounds were simultaneously tested against THP1 cell for determination of general cytotoxicity. The extracts and isolated compounds were also evaluated for their antimalarial activity against chloroquine-sensitive (D6, Sierra Leone) and chloroquine-resistant strains (W2, Indochina) strains of Plasmodium falciparum [45]. Furthermore, they were tested for cytotoxicity against the Vero cell line.

Antimicrobial Assay
Extracts and pure compounds were tested for their antimicrobial activity against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium intracellulare. The antifungal activities were evaluated against a panel of pathogenic fungi, including Candida albicans, C. glabrata, C. krusei, Aspergillus fumigatus, and Cryptococcus neoformans, associated with opportunistic infections. Ciprofloxacin (MP Biomedicals Inc, Aurora OH, USA) for antibacterial bioassays and Amphotericin B (MP Biomedicals Inc, Aurora OH, USA) for fungal bioassays were used as positive controls, respectively [45].

Cytotoxicity Assays
Each assay was performed in 96-well tissue culture-treated microplates. Cytotoxic activity was determined against four human cancer cell lines (SK-MEL, KB, BT-549, andSKOV-3,) and two noncancerous kidney cell lines (LLC-PK1 and Vero). All cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Each assay was performed in 96-well tissue culture-treated microplates [46]. Cells were seeded at a density of 25,000 cells/well and incubated for 24 h. Samples at different concentrations were added and cells were again incubated for 48 h. At the end of incubation, the cell viability was determined using neutral red dye according to a modification of the procedure of Borenfreund et al. [46,47]. IC 50 values were determined from dose−response curves of percent growth inhibition against test concentrations. Doxorubicin was used as a positive control, while DMSO was used as the negative (vehicle) control.

Radioligand Displacement for Cannabinoid and Opioid Receptor Subtypes
The evaluated extracts and isolated compounds of C. villosus were run in competition binding with cannabinoid receptor subtypes, cannabinoid receptor 1 (CB 1 ) and cannabinoid receptor 2 (CB 2 ), and were tested against the opioid receptor subtypes (µ, κ, and δ) as previously described [48].

Statistical Analysis
All the experiments for determination of total phenolics, total flavonoids, and antioxidant properties using DPPH and cellular antioxidant assay (CAA) were conducted in triplicates. The values are expressed as the mean ± standard deviation (SD). Analysis of variance and significance of difference among means were tested by one-way ANOVA and least significant difference (LSD) on mean values. Correlation coefficients (R) and coefficients of determination (R 2 ) were calculated using Microsoft Excel 2007.

Chemistry
Phytochemical study of the hydro ethanolic extract of the aerial part of C. villosus led to the isolation of four known flavonoids (1) genistein, (2) chrysin, (3) chrysin-7-O-β-d-glucopyranoside, and (4) 2"-O-α-l-rhamnosylorientin ( Figure 1). The structures of the known compounds were identified by comparison of their spectroscopic data with those reported in the literature [33]. The spectroscopic data for the isolated compounds can be seen the Supplementary Materials Figures S1-S12.

Chemistry
Phytochemical study of the hydro ethanolic extract of the aerial part of C. villosus led to the isolation of four known flavonoids (1) genistein, (2) chrysin, (3) chrysin-7-O-β-D-glucopyranoside, and (4) 2"-O-α-L-rhamnosylorientin ( Figure 1). The structures of the known compounds were identified by comparison of their spectroscopic data with those reported in the literature [33]. The spectroscopic data for the isolated compounds can be seen the Supplementary Materials Figures S1-S12  Table 1 shows the total phenolic content in the extracts of the C. villosus aerial parts. Total phenolic content was measured for the CHCl3, EtOAc, and n-BuOH extracts. Among the extracts of C. villosus, the highest phenolic content was found in the n-BuOH extract (363.00mg GAE/g dried extract) followed by EtOAc (208.00 mg GAE/g dried extract) and CHCl3 extract (56.00 mg GAE/g dried extract). The total flavonoids content in the C. villosus extracts are shown in Table 1. Similarly, the highest amount of flavonoid content was found in the n-BuOH extract (21.16 mg QE/g dried extract).   Table 1 shows the total phenolic content in the extracts of the C. villosus aerial parts. Total phenolic content was measured for the CHCl 3 , EtOAc, and n-BuOH extracts. Among the extracts of C. villosus, the highest phenolic content was found in the n-BuOH extract (363.00mg GAE/g dried extract) followed by EtOAc (208.00 mg GAE/g dried extract) and CHCl 3 extract (56.00 mg GAE/g dried extract). The total flavonoids content in the C. villosus extracts are shown in Table 1. Similarly, the highest amount of flavonoid content was found in the n-BuOH extract (21.16 mg QE/g dried extract).

2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay
The antioxidant activity of extracts and isolated compounds of C. villosus was evaluated in terms of their free radical scavenging capacity (DPPH) assay ( Figure 2). The CHCl 3 and EtOAc extracts of C. villosus showed moderate antioxidant activity with IC 50 values of 0.459 and 0.425 mg/mL, respectively. The n-BuOH extract showed highly antioxidant activity against DDPH compared to EtOAc and CHCl 3 extracts with an IC 50 value of 0.164 mg/mL (Table 1). The antioxidant activity of extracts and isolated compounds of C. villosus was evaluated in terms of their free radical scavenging capacity (DPPH) assay ( Figure 2). The CHCl3 and EtOAc extracts of C. villosus showed moderate antioxidant activity with IC50 values of 0.459 and 0.425 mg/mL, respectively. The n-BuOH extract showed highly antioxidant activity against DDPH compared to EtOAc and CHCl3 extracts with an IC50 value of 0.164 mg/mL (Table 1).

Cellular Antioxidant Activity (CAA) assay
The antioxidant activity of C. villosus extracts and isolated pure compounds was also evaluated using the cellular antioxidant assay (CAA). The results are shown in Table 2. The extracts of C. villosus showed weak inhibition of intracellular oxidative stress (29% to 36% inhibition of ROS generation at 250 µg/mL). Similarly, the tested isolated compounds were not effective except for compound 4 from n-BuOH extract (Figure 2). Compound 4 showed weak inhibition of intracellular oxidative stress (28% at 250 µg/mL) ( Table 2).

Cellular Antioxidant Activity (CAA) assay
The antioxidant activity of C. villosus extracts and isolated pure compounds was also evaluated using the cellular antioxidant assay (CAA). The results are shown in Table 2. The extracts of C. villosus showed weak inhibition of intracellular oxidative stress (29% to 36% inhibition of ROS generation at 250 µg/mL). Similarly, the tested isolated compounds were not effective except for compound 4 from n-BuOH extract (Figure 2). Compound 4 showed weak inhibition of intracellular oxidative stress (28% at 250 µg/mL) ( Table 2).

Determination of Anti-Inflammatory Activity
The EtOAc and n-BuOH extracts of C. villosus showed weak inhibition of iNOS with IC 50 values of 48 and 90 µg/mL, respectively. Compounds 1 and 3 isolated from the EtOAc extract of C. villosus showed good inhibition of iNOS with IC 50 values of 9 and 20 µg/mL, respectively ( Table 3). The increase in transcriptional activity of NF-κB in PMA-treated cells was also not suppressed by the plant's extracts and isolated compounds with the exception of compounds 1 and 2, which showed moderate inhibition of NF-κB activity with IC 50 values of 28 and 38 µg/mL, respectively (Table 3).

Antiprotozoal Activity
The results for this assay are presented in Table 4. The EtOAc extract showed weak antitrypanosomal activity against T. brucei with IC 50 values of 19.48 µg/mL, while the n-BuOH extract was found to exhibit high antitrypanosomal activity against T. brucei with IC 50 values of 7.99 µg/mL and IC 90 values of 12.61 µg/mL. No significant activity was observed in vitro against Leishmania donovani (promastigotes, axenic amastigotes, and intracellular amastigotes in THP1 cells).

Antimicrobial Activity
The plant's extracts and isolated compounds showed no antimicrobial activity against all tested microorganisms. The results of antimicrobial assay are given in Table 5.

Anti-Malarial Activity
The results of the antimalarial activity assay are presented in Tables 6 and 7. No antimalarial activity was observed against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium.

Cytotoxicity
The results of Cytotoxicity assays are shown in Table 8. The tested extracts and isolated compounds of C. villosus were not active against any cell lines used in this study.

Radioligand Displacement for Cannabinoid and Opioid Receptor Subtypes
The affinity of the total extracts and isolated compounds towards cannabinoid and opioid receptors was tested. The results are shown in Table 9. Low affinity for cannabinoids was found in both extracts evaluated and no affinity for the compound 2. For opioids, both fractions tested revealed a preference toward δ-opioids with low displacement values. Table 9. Displacement radioligand assay for human opioid receptors (Subtypes δ, κ, and µ) and cannabinoid receptors (Subtypes CB 1 and CB 2 ) of C. villosus.

Discussion
The antioxidant capacity of medicinal plants extracts and pure natural compounds can be tested using various methods. In the present study, the antioxidant activity of the studied species extracts and its isolated phenolics were evaluated in terms of their free radical scavenging capacity by DPPH assay. Their activity against intracellular oxidative stress was determined by CAA assay. Our results showed that the radical scavenging activity of the n-BuOH extract of C. villosus aerial parts was high compared to the EtOAc and CHCl 3 extracts. The n-BuOH extract was found to have the highest inhibition of intracellular oxidative stress with 36% inhibition at 250 µg/mL.
The relationship between total phenolic content and total flavonoid and antioxidant activity using DPPH assay of different extracts is shown in Figures 3 and 4, respectively. Regression analysis showed that phenolic compounds contributed to about 74% (R 2 = 0.744, p< 0.05) of radical scavenging properties in the extracts of C. villosus (Figure 4). Similarly, flavonoid compounds contributed to about 74% (R 2 = 0.736, p < 0.05) of antioxidant activity in the extracts (Figure 4). Figure 5 shows the comparison between total phenolic and total flavonoid contents (TPC and TF, respectively) and radical scavenging potential (DPPH) expressed in (IC 50 ) data in different extracts of C. villosus. Figure 5 also shows that the n-BuOH extract exhibited the highest radical scavenging potential (DPPH) expressed in (IC 50 ). Hence, a high phenolic content is an important factor to determinate the antioxidant activity. This result is in agreement with previous studies, reporting that the phenolic compounds significantly contribute to the antioxidant activity in different plant species [49].      In contrast to the antioxidant assays results, the n-BuOH extract from the C. villosus aerial parts showed weak anti-inflammatory activity for the inhibition of iNOS expression, with an IC50 value of 90 µg/mL. Whereas the EtOAc extract exhibited higher inhibition of iNOS with an IC50 value of 48 µg/mL. The increase in transcriptional activity of NF-κB in PMA-treated cells was not suppressed by the plant's extracts. Among all tested compounds, compound 1 from the EtOAc extract showed good inhibition of iNOS with an IC50 value of 9 µg/mL. This compound showed lower inhibition of NF-κB activity with an IC50 value of 28 µg/mL. Previous studies also indicated that genistein (1) acts as anti-inflammatory agent [50]. This isoflavone has been reported to have inhibitory effects on iNOS expression and to inhibit the activation of nuclear factor-κB (NF-κB) [51,52]. Hence, the EtOAc extract could be a good source of phenolics with anti-inflammatory activity. Our results also showed that n-BuOH extract exhibited potent antitrypanosomal activity against T. brucei with an IC50 value of 7.99 µg/mL and an IC90 value of 12.61 µg/mL. Compound 4 that was isolated from this extract didn't show an effect against T. brucei. Future examination of the polar components of C. villosus, shall determine the active components from the n-BuOH extract. In contrast to the antioxidant assays results, the n-BuOH extract from the C. villosus aerial parts showed weak anti-inflammatory activity for the inhibition of iNOS expression, with an IC 50 value of 90 µg/mL. Whereas the EtOAc extract exhibited higher inhibition of iNOS with an IC 50 value of 48 µg/mL. The increase in transcriptional activity of NF-κB in PMA-treated cells was not suppressed by the plant's extracts. Among all tested compounds, compound 1 from the EtOAc extract showed good inhibition of iNOS with an IC 50 value of 9 µg/mL. This compound showed lower inhibition of NF-κB activity with an IC 50 value of 28 µg/mL. Previous studies also indicated that genistein (1) acts as anti-inflammatory agent [50]. This isoflavone has been reported to have inhibitory effects on iNOS expression and to inhibit the activation of nuclear factor-κB (NF-κB) [51,52]. Hence, the EtOAc extract could be a good source of phenolics with anti-inflammatory activity. Our results also showed that n-BuOH extract exhibited potent antitrypanosomal activity against T. brucei with an IC 50 value of 7.99 µg/mL and an IC 90 value of 12.61 µg/mL. Compound 4 that was isolated from this extract didn't show an effect against T. brucei. Future examination of the polar components of C. villosus, shall determine the active components from the n-BuOH extract.
The opioid system consists of three receptors, mu, delta, and kappa, which are activated by endogenous opioid peptides (enkephalins, endorphins, and dynorphins). The endogenous cannabinoid system comprises lipid neuromodulators (endocannabinoids), enzymes for their synthesis and their degradation, and two well-characterized receptors, cannabinoid receptors CB1 and CB2 [53]. Evidence has suggested that the opioid system can regulate inflammatory responses in rodents [54]. Mastinou et al. [25], recently described the link between neuroinflammation and cannabinoid systems. The radioligand displacement affinity towards opioid and cannabinoid receptors were evaluated for the extracts and isolated compounds of C. villosus. The EtOAc extract exhibited low/moderate activity towards the CB1 and CB2 receptors (32.1% and 25.2% displacement) and moderate activity in the delta (δ) opioid receptor (31.3% displacement). Similarly, the n-BuOH extract was found to have moderate activity towards CB1, CB2, and delta (δ) opioid receptors (33.7%, 26.1% and 24.8% displacement, respectively). None of the isolated compounds showed activity towards cannabinoid nor opioid receptors.

Conclusions
In conclusion, we reported the phenolic and flavonoid content, antioxidant, anti-inflammatory, antibacterial, antifungal, antimalarial, antitrypanosomal, antileishmanial, and cytotoxicity activities, in addition to the affinity towards cannabinoid and opioid receptors, of C. villosus aerial parts extracts and their isolated compounds. Our results showed that the n-BuOH extract had the highest phenolic and flavonoid content. Furthermore, n-BuOH extract produced a potent antitrypanosomal activity that makes it a promising source for the extraction of bioactive components with high activity against human African trypanosomiasis. EtOAc extract was found to exhibit moderate anti-inflammatory activity against iNOS, while the n-BuOH extract showed lower inhibitory effect against iNOS. Among isolated compounds, genistein, which isolated from the EtOAc extract, showed the highest anti-inflammatory activity. Further explorations of EtOAc extract could afford more potent anti-inflammatory agents. Although the EtOAc and n-BuOH extracts showed moderate activity towards CB1, CB2, and δ opioid receptors, these results encourage further exploration of Cytisus species and its isolated compounds to study their cannabinoid and opioid receptors activities.
Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/9/11/732/s1, Figures S1-S12, spectroscopic data for the isolated compounds. Funding: The project described was partially supported by grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS), a component of the National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIGMS or the NIH. Furthermore, this investigation was conducted in a facility constructed with support from research facilities improvement program C06RR14503 from the NIH National Center for Research Resources (NCRR).