April 30, 2021 Archives • 2021 • vol.1 • 306-312 BIOLOGICAL ACTIVITIES OF ORGANIC FRACTIONS FROM Ficus obtusifolia AND Haematoxylum brasiletto Bello Martínez, Jorge1; Vidal Gutiérrez, Max2; Rosas Acevedo, Jose L.1; Avila Caballero, Luz P.3; Navarro Tito, Napoleón3; Torres Guzmán, Felix4 and Robles Zepeda, Ramón E.2 * 1Universidad Autónoma de Guerrero, Unidad de Ciencias de Desarrollo Regional, Calle Pino s/n Col. El Roble C.P. 39640. Acapulco, Guerrero, México. 2Universidad de Sonora, Departamento de Ciencias Químico Biológicas. Blvd. Luis Donaldo Colosio esq. Rosales s/n Col. Centro Hermosillo Sonora. México. 3Universidad Autónoma de Guerrero, Facultad de Ciencias Químico Biológicas, Av. Lázaro Cárdenas S/N Col. La Haciendita CU Sur, C.P. 39070. Chilpancingo, Guerrero, México. 4Universidad Autónoma de Guerrero, Unidad de Ciencias Naturales. Rancho Xalaco, Petaquillas Guerrero, México.. *[email protected] Abstract Ficus obtusifolia and Haematoxylum brasiletto are traditional Mexican medicine plants used to treat different types of diseases. Both species belong to genera recognized as a potential source of compounds with antioxidant and antiproliferative activity. This study focuses on identifying the organic fractions of hexane (Hx), dichloromethane (DCM), and ethyl acetate (EtOAc) of these two species for the potential isolation of antioxidant compounds and with antiproliferative activity. The antioxidant activity was evaluated by the DPPH and TEAC tests; for the quantification of phenolic compounds and flavonoids, the Folin-Ciocalteau and AlCl3 colorimetric tests were used, respectively. The antiproliferative activity was determined by the MTT assay on the A549, RAW264.7, and L929 cell lines. The DCM fraction of H. brasiletto was the sample that showed the highest antioxidant activity (299.7 mgEAG/g; DPPH IC50 of 60 µg/mL, and; TEAC IC50 of 1.9 µMTE/g) with antiproliferative activity (IC50 of 22.6 µg/mL and 23.8 µg/mL for A549 and RAW264.7, respectively). In F. obtusifolia only its EtOAc fraction showed antioxidant activity (DPPH IC50 of 55 µg/mL, and; TEAC IC50 of 1.8 µMTE/g), while its Hx fraction showed antiproliferative activity with an IC50 of 48.1 µg/mL and 42.2 µg/mL for A549 and RAW264.7, respectively. The results obtained show that the H. brasiletto EtOH extract, and its DCM fraction, showed potential as antioxidants and antiproliferative in vitro. Whereas of F. obtusifolia, only its Hx fraction showed antiproliferative potential in vitro. Keywords: Antioxidant activity; Antiproliferative activity; Haematoxylum brasiletto; Ficus obtusifolia http://pharmacologyonline.silae.it ISSN: 1827-8620 PhOL Bello Martínez, et al. 307 (pag 306-312) Introduction used in traditional Mexican medicine, mainly for heart conditions and high blood pressure (10). Despite the advances in cancer medicine and the However, some research has shown that H. different types of therapies available, such as brasiletto extracts induce antiproliferative activity in chemotherapy, radiotherapy, surgery, and human T-cell leukemia (11). In this research, we will immunotherapy (1), cancer continues to be one of study the biological activities of organic fractions of the main health problems worldwide. Cancer F. obtusifolia and H. brasiletto. incidence and mortality have been increasing in recent years; By 2012, 14.1 million new cases and 8.2 Methods million deaths had been registered, that figure was Chemical Reagents altered for 2018, where the number of new cases increased to 18.1 million, and the number of deaths The used solvents, methanol (MeOH), ethanol to 9.6 million (2,3). This reality demands a (EtOH), n-hexane (Hx), dichloromethane (DCM), and continuous investigation of new therapeutic ethyl acetate (EtOAc), were purchased from schemes, as well as of new chemotherapeutic Fermont chemicals (Monterrey, NL, Mexico). All the agents. reagents (Na2CO3, Quercetin, K2S2O8, NaNO2, AlCl3, NaOH, FeCl , Gallic acid (3,4,5-trihydroxy-benzoic Medicinal plants are the largest source of 3 acid), Folin-Ciocalteu's phenol reagent, Trolox (6- secondary metabolites with multiple applications, so hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic even today, some communities and certain people acid), ABTS (2,2'-Azinobis [3-etilbenzothiazoline-6- still depend on them to treat many diseases. Several sulfonic acid] -diammonium salt) and DPPH (2,2- secondary metabolites obtained from plants have diphenyl-1-picrilhidrazilo) were purchased from provided the basis for the synthesis of many Sigma-Aldrich, Co (St. Louis, MO, USA). Water was pharmaceutical products (4). In this way, medicinal purified by a Milli-Q instrument (Millipore, Bedford, plants' ethnobotanical knowledge represents an MA, USA). Dulbecco's Modified Eagle's Medium alternative for identifying secondary metabolites High Glucose, L-glutamine solution, L-arginine with antioxidant activity, whose importance lies in monohydrochloride, L-asparagine, sodium pyruvate, their ability to neutralize reactive oxygen species penicillin-streptomycin solution, doxorubicin (ROS) and the potential to reduce oxidative stress, hydrochloride, dimethyl sulfoxide (DMSO), trypsin– which is related with inflammatory diseases (5). EDTA and MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5- The ROS produced in the organisms due to the UV diphenyltetrazolium], were purchased from Sigma- radiation, chemical reactions, and metabolic Aldrich (St. Louis, MO, USA). Fetal bovine serum processes cause considerable damage such as lipid (FBS) was obtained from Gibco Life Technologies and protein peroxidation, DNA damage, cell (Grand Island, NY, USA). degeneration, aging, and cancer (6). Throughout Plant Samples various studies, plants have been proposed as the main source of antioxidants also can exert The plants were collected in two regions of the protective effects against oxidative stress in state of Guerrero, Mexico. F. obtusifolia was biological systems (7,8). Therefore, ROS damage collected in Chilpancingo, 99° 22’ 16’’ W; 17° 09’ 25’’ N may be reduced by the action of polyphenols and, H. brasiletto was collected in Mochitlán, produced in plants due to their ability to capture 99°21’19.03” W; 17°29’03,27” N. The plants were free radicals that they possess. authenticated by Professor María de los Angeles Venalonzo Martínez and one specimen of each plant Species of the genus Ficus, such as Ficus was deposited at the Universidad Autónoma de obtusifolia, have been shown to have different types Guerrero herbarium with a number of vouchers of biological activities, such as the antioxidant, UAGROHBH15 and UAGROFOH15, respectively. antiproliferative and antibacterial. This genus species are indicated by traditional medicine for Preparations of Plants Extracts respiratory problems, ulcers, diarrhea, and tumors The extract of F. obtusifolia was prepared from (9). On the other hand, Haematoxylum brasiletto is finely ground air-dried samples (1 kg) and the http://pharmacologyonline.silae.it ISSN: 1827-8620 PhOL Bello Martínez, et al. 308 (pag 306-312) heartwood of H. brasiletto in powder (1 kg). The the microplate reader (BIO-RAD iMark) using a extraction was carried out with EtOH by maceration wavelength of 730 nm (15). Measurements were at room temperature for ten days, with brief manual carried out by triplicate. The antioxidant activity was stirring twice a day. The EtOH extracts were expressed as IC50; The concentration required to evaporated under reduced pressure, the residue reduce 50% of the ABTS radical. The results were was partitioned by a successive liquid-liquid also expressed in micrograms equivalent to Trolox partition of an aqueous (3:2 H2O/MeOH) suspension (μgTE/mL), using the same procedure as for the with Hx (2:1, 1:1, 1:1, v/v), DCM (2:1, 1:1, 1:1, v/v) and samples, a linear regression was performed with the EtOAc (2:1, 1:1, 1:1, v/v) to produce the corresponding antioxidant Trolox at concentrations of 10 to 200 low, low-medium, and medium polarity fractions μg/mL. Subsequently, using the linear regression (12). equation, IC50's were calculated in μMTE/mL. Free Radical Scavenging Activity – DPPH Assay Estimation of Total Phenol Content A DPPH solution was prepared in MeOH until an The total phenolic content of plant extracts was absorbance of 0.7 was reached at 517 nm. 280 µL of determined using the Folin-Ciocalteu reagent, as this solution was placed in a 96-well plate, to which previously described (16). Briefly, 40 μL of plant 20 µL of the samples dissolved in MeOH was added, extract samples (1 mg/mL) were diluted in 300 μL of allowed to stand for 30 minutes at room H2O. 80 µL of Folin-Ciocalteu reagent and 120 μL of temperature, and read in the microplate reader 20% sodium carbonate solution (w/v) was added. (BIO-RAD iMark) using a wavelength of 517 nm. The The volume was brought up to 1 mL with H2O. After radical scavenging activity of DPPH was calculated two h the absorbance was measured at 760 nm in a using the following formula: spectrophotometer Genesis 20. Based on a standard curve realized previously, the results were % discoloration = (1 – (Abssample /AbsMeOH)) × 100 expressed as milligrams of Gallic acid equivalents Where Abssample and Absblank are the absorbance of (mg GAEq/g). the sample and the blank (MeOH), respectively. Estimation of Total Flavonoid Content Measurements were carried out by triplicate. The antioxidant activity was expressed as IC50; The The total flavonoid content was determined using concentration required to reduce 50% of the DPPH a colorimetric method, as previously described (17). radical (13,14). The results were also expressed in Briefly, 0.5 mL of the plant extract samples (1 micrograms equivalent to Trolox (μgTE/mL), using mg/mL) was diluted with 2 mL of H2O. Then, 0.15 mL the same procedure as for the samples, a linear of a 5% NaNO2 solution was added to the mixture. regression was performed with the antioxidant After 6 min, 0.15 mL of a 10% AlCl3 solution was Trolox at concentrations of 10 to 200 μg/mL. added, and the mixture was allowed to stand for 6 Subsequently, using the linear regression equation, min; 2 mL of 1 M NaOH was added, and the total IC50's were calculated in μMTE/mL.