Potential of Baccharis alnifolia Meyen & Walpan (Chilka) from northern Chile used as a medicinal infusion. Potential of Baccharis alnifolia Meyen & Walpan (Chilka) from northern Chile used as a medicinal infusion

: Baccharis alnifolia Meyen & Walp. is a native and medicinal, Chilean Altiplano herb that is used as a poultice and also consumed as an infusion to relieve inflammation. This study evaluated the antioxidant and cytotoxic effect of the ethanolic extract of B. alnifolia and their infusion. It was reported that the ethanol extract contains 4.42 mg GAE/g of dry weight and the infusion contains 35.86 mg GAE/L of total polyphenols. Also, it determined the antioxidant capacity using Ferric reducing antioxidant power (FRAP), Oxygen Radical Absorbance Capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC) assay. Moreover, seven metabolites including between phenolic acids and flavonoids were identified for the first time in both extracts of the medicinal plant Baccharis alnifolia. The UHPLC-DAD chromatograms revealed the majority presence of ferulic acid and luteolin, both could be responsible for the antioxidant and cytotoxic activity. Furthermore, we realized a preliminary screening of cytotoxicity in different tumor cell lines, finding that these extracts have cytotoxic potential on kidney cells.


INTRODUCTION
The northern of Chile, ecoregion of Puna, presents unique characteristics and stretches out from southern-central Peru to northern Argentina and Chile with an altitude ranging from 3000 to 5000 meters above sea level. This region is characterized by low relative humidity, high solar radiation levels, lack of oxygen and a wide variation of temperature during the day and the night (CABRERA, 1968). In this habitat grow many vegetal products that have developed a great variety of bioactive compounds as an adaptation to environmental stress, including the biosynthesis of secondary metabolites with relevant pharmacological activities (TIMMERMANN & HOFFMANN, 1985;KLEIER & LAMBRINOS, 2005). Vegetation is dominated by an alternation of grasses and shrubs that are used by the Andeans inhabitants as a source of food, medicine, forage, and building (TORRES-CARRO et al., 2017). One of the most abundant shrubs is the Chilka, a plant known by this name for the communities of the Andes. This common name is attributed to several species belonging to the genus Baccharis (B. alnifolia Meyen & Walp., B. boliviensis (Wedd.) Cabrera var. Boliviensis, B. salicifolia (Ruiz et Pav.) Pers., B. calliprinos Grise, and Lejia (or Lekia), identified as Baccharis antelices Phil. spp. santelices and B. tola Phil. spp. Tola (ORTIZ et al., 2019). For this research, we choose the Baccharis alnifolia Meyen & Walp., because according to our Soto et al. knowledge, there is little or almost no information about this species (BELTRÁN et al., 2006). In Chile, it grows from 2800 to 3800 meters above sea level in rugged and flat areas of Precordillera. B. alnifolia serves occasional fodder when there is a shortage of food. Also, it serves to build roofs and as firewood.
In traditional medicine, it is used to prepare poultices against blows, bruises and also against tonsillitis as infusion tea (GONZÁLEZ & MOLINA, 2017). This research aimed to realize a comprehensive study about the antioxidant potential and anticancer potential screening of Baccharis alnifolia Meyen & Walp in its leaves, as well as in its infusion.

Ethanolic extraction
Dried and powdered aerial parts of B. alnifolia (1 g) were extracted with absolute ethanol for 30 minutes in the dark in an ultrasonic bath (100 mL, three times) in order to obtain an extract for the UHPLC, antioxidant and cytotoxic analyses. The extract was immediately concentrated under vacuum, and a resulting brown gum was obtained (127 mg).

Infusion extraction
B. alnifolia infusion was prepared from the whole herb (stem and leaves), using 0.5 g of plant brewed for 5 minutes in 250 mL water at 100°C. Infusion was left to cool at room temperature and tested for bioactive compounds on the same day. All measurements were performed in triplicate (LARRAZABAL et al., 2018).

UHPLC-DAD instrument
The Knauer Azura analytical UHPLC system is comprising of the Azura P 6.1 L pump, 3950 auto samplers, and Azura DAD 2.1 L diode array detector with high sensitivity Knauer . For the analysis 5 mg of the resin, the extract was dissolved in 2 mL of methanol, filtered (PVDF filter) and 10 µL was injected in the instrument.

Total polyphenol and flavonoid contents
Total polyphenolic contents (TPC) were determined by the Folin-Ciocalteau method as reported (PARRA et al., 2017). Determination of total flavonoid content (TFC) of the resin was performed as previously described (PARRA et al., 2017).

Antioxidant assays
To measure the antioxidant activity of nine plants, we used the FRAP, TEAC, and ORAC assays. Every analysis was performed in BioTek Synergy HTX Multi-Mode Microplate Reader (Winooski, VT, USA) using 96-well microplates. For all antioxidant assays, a standard curve of Trolox was used, and results were expressed as Trolox equivalents gram dry weight (mmol TE/g of DW). The FRAP and TEAC assays were performed according to PARRA et al. (2017), with modifications. For the FRAP assay, 25 μL of appropriately diluted extract sample was mixed on a 96well microplate with 175 μL of freshly prepared FRAP reagent. After the mixture, the sample was incubated for 15 min at 37 ºC, and then the absorbance of the ferrous complex was recorded at 593 nm. For TEAC assay, 20 μL of extract was added to 200 μL of diluted ABTS •+ solution (A 734nm = 0.700 ± 0.002). The reactive mixture was incubated for 7 min at 25 ºC, and the absorbance was immediately recorded at 734 nm. The ORAC assay was performed according to Ou with some modifications (OU et al., 2002). Fluorescein (8 nM), AAPH (75 mM) and dilution of sample extract were prepared in phosphate buffer solution (75 mM, pH 7.0). Extract sample (25 μL) was mixed with 100 µL of fluorescein in a Nunc 96 Microwell black plates (Thermo Scientific, UK) and incubated at 37 ºC for 30 min. Then, 75 µL of AAPH was added, and the fluorescein intensity was measured every minute for 120 min at excitation and emission wavelengths of 485 and 520 nm, respectively. The final ORAC values were calculated using the area under the curve (AUC) and the regression equation between Trolox and the net AUC.

In vitro cytotoxic screening
To measure cytotoxic effects, we assessed cell viability using MTT assay. Cells in the DMEM medium without serum were exposed to ethanolic extract and infusion (10 and 100 µg/mL) for a period of 24 hrs. Culture medium with extract treatments was discarded and replaced with 200 μl MTT medium and incubated for 2.5 h at 37 °C and 5% CO 2 . An amount of 200 μL of DMSO was used for solubilizing the formazan crystals. The absorbance was measured with a TECAN Infinite pro 200 plate reader at 560 nm. The viability percentage was calculated against a non-drug treated control (designated 100% viability) considered as a control vehicle (≤0.5% DMSO). For background absorption, some wells remained cell-free with DMSO as a blank control. The experimental data were expressed as means ± SD; the comparisons were made between controls and treated cultures using a one-way ANOVA followed by Dunnett's post-hoc test for multiple comparisons. p<0.05 was considered to indicate a statistically significant difference between values.

RESULTS AND DISCUSSION
Due to the scarce information available for this specie, we considered it very important to determine some chemical parameters that can give an orientation of the antioxidant potential related to the consumption of this medicinal plant. As outlined in the introduction there are no available data on bioactive compounds in medicinal plants like B. alnifolia. The first goal of our study was to establish a comprehensive overview of the antioxidant properties of leaves and herbal infusion of this species. For this, it assessed the total polyphenol content (TPC) and total flavonoid content (TFC) and compared to other plants. Moreover, Ferric reducing antioxidant power (FRAP), Oxygen Radical Absorbance Capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC) assay were used to evaluate the antioxidant capacity of B. alnifolia (Table 1). These methods are simple and widely used for the evaluation of antioxidant capacity (LÓPEZ-ALARCÓN & DENICOLA, 2013). Thus, the TPC values of this plant, 4.42 ± 0.06 mg GAE/g dry weight, were close to some fruits, grains, and seeds (CHIRINOS et al., 2013), but lower than other Baccharis reports (SIMIRGIOTIS et al., 2016). While the infusion sample (35.86 ± 0.58 mg GAE/L), also showed values close to those reported for the aerial parts of Baccharis tola infusion (ROJO et al., 2009). The FRAP value for the ethanolic extract was 13.29 ± 2.78 μmol Trolox equivalents/g of dry weight, is higher than that reported for some vegetables, legumes, and cereals that are between 2.2 and 10.3 μmol Trolox equivalents/g dry matter edible part (SAURA-CALIXTO & GOÑI, 2006). Whereas the infusion of B. alnifolia (619 ± 8.58 μmol Trolox equivalents/L) was much higher than the infusion of Acantholippia deserticola (Phil.) infusion Soto et al.
(69.2 ± 0.04 μmol Trolox equivalents/L), mint and chamomile commercially available (LARRAZABAL et al., 2018). The TEAC value (6.19 ± 0.03 µmol Trolox/g dry weight) were close to reported for different medicinal plants and were between 6.15 to 7.81 µmol Trolox/g dry weight (GAN et al., 2010). Moreover, the TEAC values for infusion sample also showed values close to those reported for Acantholippia deserticola (Phil.) infusion (LARRAZABAL et al., 2018), but it was lower to obtain Baccharis tola infusion and other plants from South American Andes (ROJO et al., 2009). The ORAC value for ethanolic extract was 170.64 ± 0.01 µmol Trolox/g dry weight, and its value for herbal infusion was 599.52 ± 0.15 µmol Trolox/L is a little higher than Aloysia triphylla infusion (ABDERRAHIM et al., 2011). Furthermore, we have determined some metabolites for ethanolic extract and infusion of B. alnifolia, among them four phenolic acids and three flavonoids, they were identified by UHPLC (Figure 1). The examination of the ethanolic extract chromatogram revealed the presence of caffeic acid (peak 4); vanillic acid (peak 5); p-cumaric acid (peak 8); ferulic acid (peak 9). Also identified one flavone, one flavanone, and one flavonol: luteolin (peak 14); naringenin (peak 18) and kaempferol (peak 19), respectively. All compounds  were reported and identified by co-elution with an authentic compound. For the infusion extract, the same compounds of the ethanolic extract were identified but with change in their concentrations (Table 2). When we analyzed the chromatograms, we observed that the caffeic and vanillic acids were reported in a lower amount than in ethanolic extract. However, the ferulic acid and luteolin increased their concentration by five times compared to the ethanol extract ( Table 2). The ferulic acid increase could be related with antioxidant and cytotoxic activity of Chilka infusion (JANICKE et al., 2011). In contrast, the increase of the signal of luteolin in the infusion could contribute to reducing inflammation, and it acts as potent neuroprotective (NABAVI et al., 2015). Also, we observed the appearance of new signals (peaks 11, 12, 16, 24 and 26) in the aqueous extraction. The increase exponential of the ferulic acid signal in B. alnifolia infusion also could be related to the antiinflammatory effect of this plant (ZHU et al., 2014). Other compounds that have increased their signal force are the peaks 9, 14, 15 and 16, it being the last one of the major compounds in the chromatogram (Figure 1). Compounds extracted from the infusion were identified by spiking experiment with standards.
To improve the knowledge about this species, we realize a preliminary cytotoxic screening was made over six cell lines at two concentrations (10 and 100 ug/mL) (Table 3). Neither the ethanolic extract or infusion presented cell viability lesser than ≤50% at 10 µg/mL in any cell line at 24 h exposure time, with only a slight activity of the infusion on HepG-2 cells (75.1 ± 2.9 µg/mL), meanwhile at 100 µg/ mL they showed activity over most of all cell lines, showing high activity at higher concentration. For    84.0 ± 6.9 2.3 ± 1.0 77.5 ± 9.5 3.0 ± 1.6 * The values were expressed as mean ± SD (n = 6).