Plants of the Rutaceae family are widespread throughout the world and contain approximately 2,000 types of species across 160 genera. Most species are trees or shrubs, with a few being herbs, frequently aromatic . According to the Plant List , the genus Erythrochiton contains 15 plant species, among which 7 are widely accepted: E. brasiliensis Nees & Mart., E. fallax Kallunki, E. giganteus Kaastra & A.H.Gentry, E. gymnanthus Kallunki, E. hypophyllanthus Planch. & Linden, E. odontoglossus Kallunki, and E. trichanthus Kallunki. These species are widespread in tropical countries such as Colombia, Mexico, Nicaragua, Costa Rica, and Brasil . The generic name Erythrochiton is derived from the Greek expression meaning “red tunic”, due to the red cylindrical calyx of plant . Erythrochiton genus plant extracts are used in traditional medicine as antifungals and vermifuges, as well as to relieve toothache [5,6,7].
One of the lesser known plants of this genus is Erythrochiton brasiliensis Nees & Mart. (syn. Pentamorpha graveolens Scheidw.). This is a tree, 0.5–12 m high, growing in some South American countries ; it also can be found in botanical gardens within Europe. Despite E. brasiliensis being used in traditional medicine, little is known about its phytochemical composition. Sargenti et al.  isolated β-sitosterol, isophytol, waxes, stachydrine, homostachydrine, free amino acids and N-methylpipecolic acid from several parts of the plant (bark, trunk, leaves, flowers and fruits); Johne and Hartling  isolated the furoquinoline alkaloid γ-fagarin.
In order to fully characterize the chemical profile of E. brasiliensis, samples from different botanical gardens in Europe were collected and their composition analyzed by HPLC-MS. The effect of the growing location on the total content of phenols, tannins and flavonoids, as well as the determination of their antioxidant properties were evaluated. The characterization of phenols is of particular interest from a phytochemical viewpoint because these natural substances display a wide spectrum of biological activities (anticancer, antiatherosclerosis, anti-inflammatory, antiviral and antibacterial) [10,11].
Although DPPH radical spectrophotometric test is widely used to evaluate the antioxidant properties of medicinal plant extracts [12,13,14], the electron paramagnetic resonance (EPR) technique is more suitable for the assessment of radical scavenging properties of cloudy and colored materials [15,16]. The importance of dietary antioxidant components for illness prevention is attracting increasing attention in research. Total polyphenol content and antioxidant activity of herbal extracts are of particular interest to food industry, which is looking for alternatives to conventional food preservatives and dietary supplements . Cytotoxicity of E. brasiliensis extract on African green monkey kidney (Vero) cells was also tested to evaluate the safety profile. Additionally, the influence of the plant extract on the occurrence of the cytopathic effect (CPE) in the HHV1-infected Vero cells was determined.
2 Materials and methods
2.1 Plant material
The plant material was collected from five different botanical gardens in four European countries: Botanische Gärten der Universität Bonn, Bonn, Germany (coded Ebo); Botanischer Garten, Jena, Germany (Eje); Botanická zahrada Liberec, Liberec, Czech Republic (Eli); University of Warsaw Botanical Garden, Warsaw, Poland (Epo); Botanic Garden Meise, Belgium (Ebe). Leaves were cut in the Spring 2015 and dried at room temperature. Afterwards, leaves were crushed, grinded, and sieved through 0.2 cm sieves. Ebo, Eje, Eli, Epo, and Ebe specimens were stored in the Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, Poland.
1,1-Diphenyl-2-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), Folin-Ciocalteu reagent, hide powder, gallic acid and quercetin were obtained from Sigma-Aldrich. Anhydrous sodium carbonate, methanol, hydrochloric acid, pyrogallol, methenamine, aluminum chloride, acetone and ethyl acetate, were bought from POCH (Poland). Water for extraction was purified with the Millipore simplicity system (Millipore Corp., Bedford, USA). Acetonitrile, formic acid, water and methanol for LC-MS analysis were HPLC-grade and purchased from J.T. Baker (USA).
African green monkey kidney (Vero) cell line was obtained from the European Collection of Authenticated Cell Cultures (ECACC, No. 84113001, UK), and Human Herpesvirus type 1 (HHV1) from the American Type Culture Collection (ATCC, No. VR-260, USA). DMEM (Dulbecco’s modified Eagle medium) was purchased from Biowest (France), whereas FBS (fetal bovine serum) from Cytogen (Poland). DMSO and DMF were purchased from POCH (Poland); phosphate-buffered saline (PBS) from Biomed (Poland); and MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) from Sigma (No. M2128, Poland). Penicillin and streptomycin were obtained from Polfa-Tarchomin (Poland) and SDS from Applichem (Germany).
2.3 Determination of total tannin content (TTC) and total phenol content (TPC)
The total tannin content (TTC) was determined by Folin-Ciocalteu and hide powder method according to European Pharmacopoeia (8th edition) . The absorbance was measured at 760 nm using a GENESYS 10S UV-Vis spectrophotometer (Thermo Fisher Scientific). TTC was expressed as mg of pyrogallol equivalents per g of dry matter (PYE·g–1 DW) and was calculated by Eq. (1):(1)
where A1 is the absorbance of total polyphenols in the sample, A2 is the absorbance of polyphenols not adsorbing on hide powder, A3 is the absorbance of a standard pyrogallol solution, m1 the mass (g) of sample and m2 the mass (g) of pyrogallol.
The total polyphenol content (TPC) was expressed as mg of gallic acid equivalents per g of dry matter (mg GAE·g–1 DW) and was determined using a calibrated curve of gallic acids (standard concentration = 0.25 –2.5 mg·mL–1) by Eq. (2):(2)
where y is the absorbance of sample and x is the number of equivalents of gallic acids.
2.4 Determination of total flavonoid content (TFC)
The total flavonoid content (TFC) was determined by the method described in Polish Pharmacopoeia 6 . Acetone (20 mL), hydrochloric acid (281 g·L–1, 2 mL) and methenamine (5 g·L–1, 1 mL) were added to each sample (0.5 g), and mixture was heated under reflux for 30 min. After filtration, the filtrate was extracted with acetone (2 × 20 mL). The extracts were combined, filtered into a 100-mL volumetric flask and filled up to the volume with acetone. Then, the obtained solution (20 mL) was transferred to a separating funnel, diluted with water (20 mL), and extracted with ethyl acetate (3 × 15 mL). The organic layers were separated, combined and washed with water (2 × 40 mL). The organic layer was filtered into a 50-mL volumetric flask and filled up to the volume with ethyl acetate.
The stock solution (10 mL) prepared as described above was added with an aqueous solution of aluminum chloride (20 g·L–1, 2 ml) and filled up to 25 mL with a mixture of acetic acid (1.02 kg·L–1) and methanol [1:19 (v/v)]. The comparative solution was a mixture of stock solution (10 mL) and acetic acid:methanol [15 mL, 1:19 (v/v)]. After incubation at room temperature for 30 min, the absorbance was measured at 425 nm. TFC content was expressed as mg of quercetin equivalents per g of dry matter (mg QuE·g–1 DW) and calculated by Eq. (3):(3)
where A is the absorbance of sample at 425 nm and m the mass (g) of the dry extract.
2.5 Preparation of extracts for the analysis of composition and biological activity
Raw material (300 mg) was suspended in methanol [1:20 (w/v)] and pH adjusted to 2 with 1M HCl. The suspension was placed in an ultrasonic bath (35kHz, 300W) and sonicated for 1 h at 60˚C. After neutralization with 5% aq. NaHCO3, the extract was evaporated to dryness, weighed and dissolved in methanol (2 mL) to obtain the following stock solutions: 0.475 mg·mL–1 (Ebo), 0.670 mg·mL–1 (Eje), 0.750 mg·mL–1 (Epo), 0.575 mg·mL–1 (Ebe) and 0.504 mg·mL–1 (Eli). Stock solutions were used for antioxidant and chromatographic analyses.
Ebo sample was subjected to cytotoxicity and antiviral assays. A suspension of powdered plant material (10.0 g) in methanol [200 mL, 1:20 (w/v)] was heated under reflux.
The extract was filtered and evaporated to dryness under reduced pressure. Dry extract was dissolved in DMSO (50 mg·mL–1) and filtered through syringe filter (0.2 μm pore diameter) to obtain the stock solution for cytotoxicity and antiviral tests.
2.6 Determination of antioxidant activity by electron paramagnetic resonance (EPR)
EPR measurements were carried out using a Miniscope MS200 spectrometer (Magnettech GmbH, Germany) with the following parameters: central field 334 mT, sweep range 8 mT, sweep time 30 s, microwave power 10 mW, modulation amplitude 0.1 mT.
The free radical-scavenging activity of extracts was evaluated with the DPPH assay  based on the reducing ability of antioxidants toward DPPH radical. DPPH solution for EPR measurements was prepared by dissolving DPPH (49.7 mg) in methanol (100 mL, final concentration = 1.26 mM). Sample solution (15-30 μL) was mixed with a methanolic solution of DPPH (30 μL). After mixing, the reaction mixture was kept in the darkness for 30 min, then its EPR spectrum was recorded. Trolox was used as the reference substance and was kept in the darkness for 4 h before measurements. Appropriate DPPH mixtures with methanol were used as standards. The intensities were taken as the double integrals of spectra.
The percent inhibition of the EPR spectrum was calculated according to Eq. (4):(4)
where I0 is the area of the EPR spectrum of DPPH solution (control sample), and I is the area of the EPR spectrum of the mixture of DPPH solution with sample solution.
The percent inhibition of EPR signal intensity of DPPH and the mmol number of Trolox were in linear relationship according to Eq. (5):(5)
where y is the mass of scavenged DPPH and x is the mass of Trolox.
2.7 HPLC-fingerprint analysis
Reverse phase HPLC analysis was performed on a Dionex (Sunnyvale, US) system consisting of gradient 7580 pump, UVD 340S detector, and Jet Stream II plus column thermostat (WO Indus-trial Electronics) controlled by Chromeleon software. The Luna C18 column (250 × 4.6 mm i.d., 5 μm particle size) and C18 precolumn (4 × 3 mm i.d., 5 μm particle size) were purchased from Phenomenex. The extracts were dosed before and after acid hydrolysis. Sample injection was made through a Rheodyne injector valve with a 20 μL sample loop. Deionized water with 0.1% trifluoroacetic acid (solvent A) and pure acetonitrile (solvent B) were used as mobile phase. Gradient elution was carried out as follows: 15% B initially, maintained for 10 min, raised to 20% in 20 min, maintained for 5 min, increased to 30% in 32 min, maintained for 13 min, raised to 90% in 55 min, maintained for 10 min and returned to 15% in 75 min.
2.8 HPLC-DAD/ESI-Q-TOF-MS for qualitative determination of phenols
HPLC-MS analysis was performed using an Agilent LC-Q-TOF/MS system composed of the following parts: LC system (1260), binary pump (G1312C), degasser (G1322A), column oven (G1316A), PDA detector (G1315D), autosampler (G132B), quadrupole–time-of-flight tandem mass detector (G6530B) and ESI ionization source. Zorbax reverse phase Stable Bond chromatographic column (150 × 2.1 mm i.d., 3.5 μm particle size) was purchased from Agilent Technologies.
Water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) were used as the mobile phase. A gradient eluition was performed as follows: 1% B at start, increased to 55% in 75 min, increased to 95% in 2 min, maintained for 6 min. HPLC runs were performed using a flow rate of 0.2 mL·min–1, a sample injection volume of 20 μL and a sample concentration of 10 mg·mL–1, and were monitored by UV detection at 210, 254, 280, 320 and 365 nm.
MS analysis of the eluate was conducted in both positive and negative modes, in a method-dependant MS/ MS protocol. The two highest peaks were fragmented to obtain the MS/MS spectra and excluded for the following 0.3 min. The apparatus was freshly tuned before analysis by the addition of an external reference ion mixture suitable for the on-line calibration.
Detailed MS parameters were as follows: mass range m/z = 40–1500, gas and sheath gas temperatures = 350 and 400 ºC, gas and sheath gas flows = 12 mL·min–1, nebulilzer pressure = 35 psig, fragmentor voltage = 130 V, nozzle voltage = 1000 V, capillary voltage = 4000 V, skimmer voltage = 65 V, fixed collision voltages (CID) = 15 and 25 V, injection volume = 10 μL. MassHunter software was used to record and analyze the spectra. All compounds were identified based on their fragmentation pattern and comparison with scientific literature data [21,22,23,24].
2.9 Cytotoxicity evaluation
Microculture tetrazolium assay was used to evaluate cell viability. This test is based on the ability of succinate dehydrogenase enzymes to reduce the yellow water soluble substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into an insoluble, purple formazan. Formazan crystals are dissolved in an SDS/DMF/PBS solution (14% SDS, 36% DMF, 50% PBS), and after 24 h the solution absorbance is measured. Because MTT reduction may only occur in viable, metabolically active cells, the level of enzyme activity is a measure of cell viability.
Vero cells were cultivated in DMEM containing L-glutamine, 10% FBS, penicillin (100 U·mL–1) and streptomycin (100 μg·mL–1). The cells were grown at 37 °C under 5% CO2 in the air (CO2 incubator, LabLine-Thermo Scientific, USA) to produce a monolayer. Vero cells were suspended in a medium containing 10% FBS and suspension (1.5·105 cells·mL–1) was seeded in a 96-well plate (BD Falcon). After 24 h incubation the extract in a culture medium containing 2% FBS at a concentration of 1.95–1000 μg·mL–1 was added, then further incubated for 72 h. Control cells were only supplemented with a medium containing 2% FBS. DMSO cytotoxicity at the concentration of stock solution after dilution was also evaluated. All samples were incubated at 37 °C under 5% CO2 in the air. After 72 h, all culture media were removed from the plates, and the cells were washed with PBS. The cell medium (100 μL) containing 10% MTT solution (5 mg·mL–1) was added to each well and the plates were incubated at 37 °C for the next 4 h. Then, SDS/DMF/PBS solution (100 μL) was added to each well. After an overnight incubation, the absorbance was measured at 540 and 620 nm using a microplate reader (Epoch, BioTek Instruments, USA). Data assessment was performed by Gen5 software (ver. 2.01.14; BioTek Instruments, Inc.). The cytotoxicity was expressed as CC50 (the sample concentration required to inhibit 50% of cell proliferation) and CC10 (concentration inhibiting 10% of cell proliferation), calculated from the dose–response curve (nonlinear regression analysis). Its assessment was based on comparison with untreated cells .
2.10 Antiviral assay
After cytotoxicity assessment, non-toxic concentrations were chosen for antiviral assay. The titer of HHV-1 used in antiviral assays was 105±0·53 CCID50·mL·1 (CCID50 = 50% cell culture infectious dose, i.e., the virus dose causing cytopathic effect in 50% of infected cells). The Vero cells seeded in 48-well plates (BD Falcon) were pre-incubated with HHV-1 (100·CCID50) for an hour (except for cell control), then test samples were added. The plates were incubated (37 °C, 5% CO2) until the final stage of cytopathic effect was observed in virus control. Then, the plates were observed under microscope for possible inhibition of cytopathic effect in the cells infected with HHV-1 and treated with tested extract.
2.11 Statistical analysis
All experiments were performed in triplicate and data analyzed using the Statistica 12 software (StatSoft, USA). The Pearson’s correlation coefficient was appointed to check the strength of the association. Cluster analysis was performed based on Euclidean distance and Ward variance method.
The conducted research is not related to either human or animals use.
3 Results and discussion
Erythrochiton brasiliensis does not occur naturally in Europe; however, due to its decorative qualities, it is often cultivated in gardens. Raw materials for the research were collected from the botanical gardens in five European locations. Location positions are shown in Figure 1 and their coordinates listed in Table 1.
All botanical gardens are located at a similar latitude, with Meise and Warsaw being the two most distant places (1200 km away from each other). According to the classification of Köppen-Geiger, this area in Europe is dominated by two climates: temperate oceanic climate (Cfb) and temperate continental or humid continental climate (Dfb) . Leaves of E. brasiliensis tested in our study originated from Cfb (Meise, Bonn) and Dfb (Liberec, Warsaw), with Jena being located between Cfb and Dfb.
Analysis of raw material revealed significant differences in the content of polyphenols, tannins and flavonoids depending on the geographic origin of samples (Table 2). The highest TPC and TTC were found in plants from Belgium; the lowest contents in samples from Poland, presumably due to a different climate.
TPC, TTC and TFC in plant material from different locations had a normal distribution. Analysis of variance indicated statistical significance between the individual values and indicators.
The mean TPC, TTC and TFC values in the tested samples, approximated to the closest unit, were 36 (±7) mg GAE·g–1 DW, 15 (±4) mg PyE·g–1 DW, and 3 (±1) mg QuE·g–1 DW, respectively. There are currently no data in the literature about TPC, TTC and TFC of E. brasiliensis. In the absence of other data, a comparison is only possible with other species from the Rutaceae family. TPC measured for E. brasiliensis is comparable to that reported for the aqueous extracts of Citrus aurantium L. (44 mg GAE·g–1 DW) and is sensibly higher than that of Phellodendron amurense Rupr. and Zanthoxylum nitidum (Roxb.) (17 and 7 mg GAE·g–1 DW, respectively).
DPPH solution showed a reduction in the intensity of EPR signal after addition of E. brasiliensis extracts (Figure 2), indicating that the plant extracts have antioxidant properties. The values of scavenged DPPH and Trolox equivalents determined by EPR spectroscopy were in the range 1.23–4.14 and 0.50–1.44 mmol·g–1 for all dry extracts. The highest antioxidant activity was exhibited by E. brasiliensis grown in Belgium (Ebe). The highest correlation between TPC content and antioxidant activity (expressed as scavenged DPPH) was 0.88 (significance level p <0.05).
Cluster analysis was performed to investigate the statistical relationship between the content of active compounds and the collection place (Figure 3).
Dendrogram is a useful graphical tool that allows visualization of clusters and the correlation between samples and variables simultaneously. Hierarchical analysis showed that the samples closest to each other in terms of active substance content (Eje and Ebo) were also the closest in terms of geographic distance (Jena and Bonn, both in Germany).
The change in the content of active compounds depending on the plant geographic region is frequently reported. Kusznierewicz et al.  studied the phenolic content in extracts from cabbage grown in different parts of Europe (England, Germany, Belgium and Poland). The highest TPC and TFC were noticed for plants from Belgium, and the lowest in the plants from Poland. The same trend was observed for TPC, TFC and TTC of E. brasiliensis (highest in Belgium, lowest in Poland). This observation confirms the positive influence of climate and growing conditions on the phenolic content .
To gain an insight into the chemical composition of E. brasiliensis the reverse phase HPLC-fingerprint analysis of its extracts was performed. Analytical resolution of the components was difficult due to the close retention time of constituents. UV analysis confirmed the phenolic nature of the main components, but the retention times were much longer than those of simple phenols or flavonoids, suggesting the presence of their complex glycosidic derivatives. The extracts were subjected to acid hydrolysis in order to identify their aglycones.
Exemplary chromatograms of Ebe and Eje as the most potent and the least potent extracts (in terms of DPPH quenching) are shown in Figure 4. The extracts revealed a qualitatively similar but quantitatively different composition.
3.2 MS characterization of free phenolic compounds
Most secondary metabolites were revealed by MS analysis in the positive ionization mode; however, the majority of phenolic compounds were identified by the negative ionization mode (Table 3).
The extracts of Ebe samples were found to contain various phenolic compounds (Table 3). Flavonoid vitexin was also present as the major compound in Ebe, Eje and Epo.
Thirteen acids were identified: benzoic acid and its derivatives (p-hydroxybenzoic, m-hydroxybenzoic, o-hydroxybenzoic, protocatechuic, vanillic and isovanillic), trans-cinnamic acid derivatives (sinapinic, coumaric, caffeic, ferulic and isoferulic) and quinic acid. The biological activity of E. brasiliensis may be mainly attributed to vitexin, which is the major component of the plant extract and is known for its wide spectrum of biological activities (anticancer, antioxidant, anti-inflammatory, antinociceptive, antihypertensive, antispasmodic, antidepressant and antiviral) .
3.3 Cytotoxic and antiviral activity
We investigated the in vitro cytotoxicity and antiviral activity of E. brasiliensisextract. The IC50 value of the Ebo extract on Vero cells was 175.64 (± 3.43) μg·mL–1, whereas IC10 was 72.5 (± 17.67) μg·mL–1 (Figure 5). Tested samples had no influence on the occurrence of cytopathic effect in HHV-1 infected Vero cells.
This is the first report describing the in vitro cytotoxicity of E. brasiliensis. However, it is possible to compare it with some other species from the Rutaceae family. Leaf extracts of Clausena excavata Burm. f. showed an IC50 >200 μg·mL–1on Vero cell lines using MTT assay. Ethanol and aqueous extracts of Glycosmis pentaphylla displayed similar citotoxicity with IC50 values of 443 and 521 μg·mL–1, respectively. Furthermore, leaf extracts of G. pentaphylla (Retz.) showed specific cytotoxicity towards MCF-7 and MDA-MB-231 breast cancer cells, inducing apoptosis through the activation of caspase-3/7 . Therefore, it would be advisable to measure the cytotoxicity of E. brasiliensis on cancer cell lines to evaluate the anticancer potential of this plant. According to the classification of cytotoxicity for natural ingredients (Table 4) , which was previously used to assess the cytotoxicity of Lansium domesticum and Mutellina purpurea [33, 34], the tested extract can be classified as potentially harmful.
The chemical profile, in vitro cytotoxicity and antiviral activity of E. brasiliensis extract were described for the first time. E. brasiliensis leaves contain over ten polyphenols, with a composition strongly dependent on climatic conditions. The total content of polyphenols, tannins and flavonoids correlates well with the antioxidant activity. Future studies will involve the fractionation of the extracts and the evaluation of biological activity of each single compound.
The authors sincerely thank the botanical gardens employees, who prepared the material for research: Anett Krämer (Botanische Gärten der Universität Bonn, Bonn, Germany), Frank Van Caekenberghe (Botanic Garden Meise, Belgium), Joanna Bogdanowicz (University of Warsaw Botanic Garden, Warsaw, Poland), Dr. Miloslav Studnička (CSc., Botanická zahrada Liberec, Liberec, Czech Republic) and Stefan Arndt (Botanischer Garten Jena, Jena, Germany). This research was supported by the grant for young scientist at the Medical University of Lublin (MNmb 261, 2015-2017).
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About the article
Published Online: 2017-12-29
Conflict of interest: Authors state no conflict of interest.