- Email: [email protected]
Antimycobacterial and hypolipemiant activities of Bidens odorata (Cavanilles)
Author’s Accepted Manuscript Antimycobacterial and hypolipemiant activities of Bidens odorata (Cavanilles) Karla Mariela Hernández-Sánchez, Leticia Garduño-Siciliano, Julieta Luna-Herrera, L. Gerardo Zepeda-Vallejo, Selene Lagunas-Rivera, G. Esthefania García-Gutiérrez, María Elena Vargas-Díaz
PII: DOI: Reference:
www.elsevier.com/locate/jep
S0378-8741(18)30706-2 https://doi.org/10.1016/j.jep.2018.04.028 JEP11323
To appear in: Journal of Ethnopharmacology Received date: 24 February 2018 Revised date: 18 April 2018 Accepted date: 19 April 2018 Cite this article as: Karla Mariela Hernández-Sánchez, Leticia Garduño-Siciliano, Julieta Luna-Herrera, L. Gerardo Zepeda-Vallejo, Selene Lagunas-Rivera, G. Esthefania García-Gutiérrez and María Elena Vargas-Díaz, Antimycobacterial and hypolipemiant activities of Bidens odorata (Cavanilles), Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2018.04.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antimycobacterial and hypolipemiant activities of Bidens odorata (Cavanilles). Karla Mariela Hernández-Sáncheza,b, Leticia Garduño-Sicilianoa, Julieta Luna-Herrerac, L. Gerardo ZepedaVallejob, Selene Lagunas-Riverad, G. Esthefania García-Gutiérreza, María Elena Vargas-Díazb* a
Departamento de Farmacia, Laboratorio de Toxicología de Productos Naturales, Escuela Nacional de Ciencias Biológicas. Instituto Politécnico Nacional. Av. Wilfrido Massieu esq. con M. Stampa, Col. Planetario Lindavista. Del. GAM, C.P. 77380, CDMX, México. b
Departamento de Química Orgánica, Laboratorio de Química de Productos Naturales, cDepartamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prol. de Carpio y Plan de Ayala, Del. Miguel Hidalgo C.P.11340, CDMX, México. d
CONACyT, Tecnológico Nacional de México/Instituto Tecnológico de Tuxtla Gutiérrez, Carretera Panamericana km. 1080, Tuxtla Gutiérrez, Chiapas C.P. 29050, México.
*Author to whom correspondence should be addressed: María Elena Vargas-Díaz. Email: [email protected] (VDE).
Abstract Ethnopharmacological relevance Bidens odorata Cavanilles is a medicinal and edible plant known as "mozote blanco, aceitilla, acahual, mozoquelite" which is traditionally used in Mexico as a diuretic, hypoglycaemic, anti-inflammatory, antipyretic, antitussive, to treat gastrointestinal disorders, kidney pain, and lung or respiratory diseases. Aim of the study This research study was aimed at phytochemical analysis of aerial extracts of B. odorata for antimycobacterial and lipidlowering activities. Materials and methods Compounds 1 ((((2R, 3R, 4S, 5S, 6R)-3,4,5-Tryhidroxy-6-(((E)-3-(4-hydroxyphenyl) acryloyl) oxy) tetrahydro-2H-pyran-2yl) methyl-4-hydroxybenzoate) and 2 (3,5-Dihydroxybenzoic acid) were isolated from B. odorata aerial shoots and their structural elucidation was carried out using 1 and 2D NMR, infrared spectroscopy (IR) and mass spectrometry (ESI-MS). The antimycobacterial activity of various extracts and compounds 1 and 2 was determined using the Microplate Alamar Blue Assay (MABA). The evaluation of the hypolipidemic effect of the ethanolic extract and the glycosylated compound 1 was tested in a murine model of hypercholesterolemia induced by diet and by Triton WR-1339. On the other hand, the LD50 of the ethanolic extract was evaluated in ICR mice by the OECD protocol TG 423. Results Antimycobacterial assay of hexane, CH2Cl2, EtOAc, ethanolic and aqueous extracts, as well as the new glycosidic compound (1) and benzoic acid derivative (2) isolated from B. odorata showed minimal inhibitory concentrations (MIC) of 100, 12.5, 12.5,12.5, ˃ 200, 3.125 and 50 μg/mL, respectively, against Mycobacterium tuberculosis H37Rv. Only hexane and CH2Cl2 extracts were observed to be active against Mycobacterium smegmatis mc2155 at a concentration of 50 and 100 μg/mL, respectively. The ethanolic extract showed lipid-lowering activity at doses of 100 and 1000 mg/kg, while glycosidic compound 1 was active at doses of 50 and 100 mg/kg. In addition, the LD50 of the ethanolic extract was ˃ 2000 mg/kg, meaning that this extract does not cause lethality or adverse effects, and no signs of organs alterations or tissue damage were observed. Conclusion The hexane, CH2Cl2, EtOAc, and ethanolic extracts of B. odorata, as well as their components 1 and 2, displayed antimycobacterial activity against M. tuberculosis. Moreover, the ethanolic extract and glycosidic compound (1) showed an
important lipid-lowering effect, without lethality or secondary effect. The results of this study support the documented traditional use for B. odorata. Graphical abstract
Keywords: Hypolipidemic, Phenolic compounds, Bidens odorata, Mycobacterium, Traditional Medicine.
1. Introduction The genus Bidens comprises a complex of about 240 closely related species (Beltrán, 2016), many of which have been studied chemical and biologically (Achika et al., 2014). However, Bidens odorata Cavanilles (syn. Bidens rosea, Bidens tripartita, Bidens bipinnata, Coreopsis odorata) is a plant little studied in the taxonomic family Asteraceae and genus Bidens. It is known in Mexico as “mozote blanco, aceitilla, acahual, mozoquelite” and it is one of the most common and widely distributed species (Rzedowski and Rzedowski, 2005), mainly inhabiting the mountain ranges of Mexico, as well as the southwestern region of United States and Central America (Vibrans, 1995). This plant is edible, and according to the books, “Plantas Medicinales del estado de Durango y zonas aledañas” (González et al., 2004) and “Plantas Medicinales de Aguascalientes” (García-Regalado, 2015), the aerial parts of Bidens odorata (leaves, flowers and stems) are used in the Mexican traditional medicine for treatment of gastrointestinal diseases, kidney pain, as anti-inflammatory, antipyretic and hypoglycemic disorders. Also, it is well recognized for its beneficial effects on the lung related disorders like pulmonary diseases and cough (Argueta et al., 1994; Martínez, 1996; Zavala-Mendoza et al., 2013; Digital Library of Mexican Traditional Medicine- Indigenous Medicinal Flora of Mexico, 2012). Although it is frequently prepared as decoction, teas or maceration. It is also documented that aqueous extract of this plant has a diuretic effect (Meléndez-Camargo et al., 2004.). Nevertheless, this species have not been investigated chemically for pharmacologically active compounds, and the biological studies confirming its curative properties are few. Therefore, the main objective of this study is to correlate the
ethnomedical use of B. odorata to treat lung diseases with respect to its antimicrobial activity. Considering that in Mexico like in the rest of the world, pulmonary tuberculosis is still one of the most devastating diseases (SSA, 2014; WHO, 2017). Additionally, in this report, we show that the species B. odorata collected in Tlaxcala, has lipid-lowering effect like that reported in a preliminary study of the species collected in the state of Coahuila (Moreno et al., 2017), and in other species of the same genus (Bidens pilosa) (Dimo et al., 2001; Liang et al., 2016). Besides, in Mexico, overweight, obesity and its complications are a health problem, occurring in 72.5% of the adults and 33.2% of the children (ENSANUT, 2016). Therefore, it is important to identify the compounds that show lipid-lowering effect.
2. Materials and methods 2.1 General Procedure and Equipment Used RMN spectra were collected on a Varian NMR System at 500 MHz using CDCl3, Methanol-d4 as solvents, or a combination of these and tetramethylsilane as the internal standard. Chemical shift values are reported in ppm (δ) (tetramethylsilane δ = 0 for 1H; chloroform-d δ = 77.0 for 13C, and methanol-d4 δ = 3.31, 4.78 for 1H and δ = 49.2 for 13C). H NMR spectra are reported as follows: δ (multiplicity, number of protons, coupling constant J, atom assignment).
1
13
C
NMR spectra are reported as follows: δ (atom assigned). Multiplicities are indicated by d (doublet), dd (doublet of doublets), and m (multiplet). Infrared spectra were recorded on a Perkin-Elmer Spectrum 2000 spectrophotometer. High Resolution Mass Spectra (HRMS) were determined using a Bruker microTOF-QII spectrometer by Electrospray Ionization Mass. Thin-layer chromatograms (TLC) were performed on precoated TLC sheets of silica gel Merck 60F-254. Spots on TLC were visualized under UV lamp or developed by spraying with cerium molybdate and cerium sulfate. Purification of compounds was performed by column chromatography on silica gel (Merck 230-400 mesh).
2.2 Collection of Plant Material and Crude Extract Preparation Bidens odorata was collected in Calpulalpan, Tlaxcala, Mexico (19°35'17.0''N, 98°32'25.5''W), in June 2016 (Supplementary material-Figure A1). The specimen was registered in the herbarium of the Escuela Nacional de Ciencias Biológicas (ENCB) with voucher No. 001/2016/BoTLX. The aerial parts were dried at room temperature for a week, then crushed. Crude extracts of the aerial parts were prepared by maceration with water or solvents of different polarity (3 L) (hexane, dichloromethane, ethyl acetate, and ethanol). The extracts were filtered and concentrated under reduced pressure using a rotary evaporator and kept in dark conditions and refrigerated until use. 2.3 Extraction and Isolation of Secondary Metabolites Aerial parts (1.75 kg) of the plant was macerated at room temperature using solvents of different polarity, starting with hexane (C6H8), followed by dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and ethanol (EtOH). For the aqueous extraction, 350 g of plant material was used. 2.3.1 Ethanolic Extract The ethanolic extract was monitored by 1H NMR and was extracted (20g in 3 L) using the technique of selective extraction (CH2Cl2:H2O/EtOAc:H2O). The resulting organic phases were combined and concentrated under reduced pressure, obtaining 700 mg of a brown precipitate, which was analyzed by thin layer chromatography (TLC) using
CH2Cl2:EtOAc:EtOH (10:80:10) as eluent, and was also monitored by 1H NMR. Consecutively, 350 mg of the brown precipitate was subjected to column chromatography (CC) using a system EtOAc:EtOH (80:20). The fractions obtained were compared using 1H NMR. Fraction 8 (200 mg) was analyzed by
13
C NMR, expose the presence of a glycosylated
phenolic compound (1). To carry out the unequivocal structural assignment, the 2D NMR spectra were obtained and complemented with the mass spectrometry analysis, using the electrospray ionization method (ESI-MS) and the IR spectrum was also obtained. 2.3.2 Aqueous Extract Aqueous extract (10 g) was extracted selectively with EtOAc (3.5 L). A brown-yellow precipitate was obtained with EtOAc and monitored by TLC then washed with CH2Cl2 and EtOH, and these fractions were analyzed again by TLC using Hex: EtOAc (1:1) as eluent. Then the fraction of CH2Cl2 was selected to be separated by CC with Hex: EtOAc (1:1). From this separation, fraction 4 was selected, which was analyzed by 1H and 13C NMR, and an ESI-QTOF-MS [M-H] analysis was carried out and showed the presence of compound 2. Crude extracts and isolated compounds 1 (EtOH extract) and 2 (aqueous extract) were evaluated for antimycobacterial activity against mycobacteria. Only the ethanolic extract and compound 1 were tested in lipid-lowering models, as described below. 2.4 Bacterial Species Used in Microplate Alamar Blue Assay The following mycobacterial strains used in this study were obtained from the American Type Culture Collection (ATCC): Mycobacterium tuberculosis H37Rv (ATCC 27294) and Mycobacterium smegmatis mc2155 (ATCC 700084).
2.4.1 Antimycobacterial assay The assay to determine the minimum inhibitory concentration (MIC) of crude extracts of B. odorata and compounds 1 and 2 against strains of Mycobacterium tuberculosis and Mycobacterium smegmatis was carried out in 96-well microplates using the following protocol described by Jimenez-Arellanes et al., in 2003. Briefly, extracts were tested at concentrations ranging from 200 to 6.25 µg/mL and pure compounds from 50 to 1.56 µg/mL. Mycobacterium tuberculosis H37Rv and Mycobacterium smegmatis mc2155 were grown in Middlebrook 7H9 broth supplemented with 0.2% of glycerol and 10% of OADC (oleic acid, albumin, dextrose, and catalase) The inoculum was adjusted according to the turbidity of tube No. 1 of the McFarland nephelometer, then 1: 10 was diluted with the same medium to obtain 6 x 106 colony-forming units (UFC)/mL. Appropriate positive and negative controls were included with each test. Following addition of the mycobacterial inoculum the plates were incubated for 5 days in case of M. tuberculosis and 2 days in case of M. smegmatis. Plates were developed by adding alamar blue, and visual and fluorometric readings were recorded in a plate fluorometer (Fluoroskan Ascent-Thermo). The minimal inhibitory concentration (MIC) was defined as the lowest concentration of the compound that prevented change of color from blue to pink in relation to positive and negative controls. 2.5 In Vivo assay
For animal studies, ICR mice of 25+5 g of body weight of both male and female sex were used (PROPECUA, S.A. de C.V., Mexico City). The animals were kept for 7 days in plastic boxes, according to the conditions of animal housing (controlled temperature 24+2 °C, relative humidity 60-65%, light/dark cycle 12 x 12 h) for adaptation. The animals were fed with a standard diet (Lab Rodent Diet 5001, PMI Nutrition International, Inc., Bienwood, MO, USA) and water on demand. After the conditioning period, the mice were marked and weighed, and distributed randomly. The animal experiments and study design were approved by the Laboratory Animal Care Committee of Instituto Politécnico Nacional (IPN) and conducted in compliance with the Official Mexican standard NOM-062-ZOO-1999 regarding technical specifications for production, care, and use of laboratory animals (Norma Oficial Mexicana 062-ZOO-1999). 2.5.1 Assessment of Acute Toxicity Evaluation of the acute toxicity of the ethanolic extract of the aerial parts of B. odorata was carried out following the protocol 423 described by Organization for Economic Co-operation and Development and Test Guideline (OECD TG 423, 2008) (Supplementary material). 2.5.2 Evaluation of Hypolipidemic Activity 2.5.2.1 Induction of hyperlipidemia by Hypercholesterolemic Diet Five groups of eight male ICR mice each were established. The mice in the hyperlipidemic groups were fed for 6 days with a hyperlipidemic diet (1% cholesterol, 0.5% sodium cholate, 5% butter, 30% sucrose, 10% casein; food standard Rodent Lab 5001, 53.5% (Hisashi, 1986; Pahua-Ramos et al., 2012) and water ad libitum). Simultaneosly, mice used as negative controls were fed standard food, and positive controls were fed with the same hyperlipidemic diet but no treatment. The lipid-lowering activity of the ethanolic extract of B. odorata was determined according to the methodology described by Pahua-Ramos et al., 2012 (with modifications, Supplementary material). 2.5.2.2 Induction of hyperlipidemia with Triton WR 1339 (Tyloxapol) Five groups of eight male ICR mice were established, and hyperlipidemia was induced by a single intraperitoneal dose of Triton WR 1339 (Tyloxapol 400 mg/kg) (Mendieta, et al., 2014). The mice were treated with ethanolic extract or phenolic compound (1) 1 hour before and 22 and 48 hours after the injection of Tyloxapol. Group I (negative control) did not receive Tyloxapol; group II (positive control) was only administered with Tyloxapol; groups III, IV, and V were administered with Tyloxapol intraperitoneally and treated by intragastric administration of 10, 100, 1000 mg/kg of the ethanolic extract or compound 1. Two hours after the last administration of the extract, blood samples were obtained. All blood samples were allowed to clot and centrifuged at 13000 rpm for 15 minutes. Subsequently, the recovered serum was analyzed for total cholesterol (TC), triglycerides (TG) and high-density lipoprotein (HDL) in the Selectra II Vita Lab autoanalyzer using commercial kits. With the values obtained from this analysis the concentration of low-density lipoprotein (LDL) was calculated using the Friedewald formula [LDL= (CT-HDL) - (TG x .45) (Friedewald, 1972). 2.6 Statistical Analysis The results from the hypolipidemic evaluation of the ethanolic extract of the aerial parts of B. odorata and of compound 1 were analyzed by the one-way ANOVA test, with subsequent comparative analysis by Tukey's method; the data obtained underwent normality tests by the Shapiro-Wilk, Anderson-Darling, and Jarque-Bera methods. Statistically significant difference was considered to be p<0.05. All data were analyzed and graphed with the software Past version 3.09
3. Results and Discussion 3.1 Phytochemical Study of Bidens odorata The aerial parts of B. odorata collected in Calpulalpan, Tlaxcala, were dried, fragmented, and extracted consecutively with solvents of different polarities, starting with hexane (10g/0.57% yield) and CH2Cl2 (9.8g/0.56% yield), followed by EtOAc (5.6g/0.32% yield), ethanol (30.9g/1.76% yield), and finally aqueous (15g/4.28% yield). An EtOH extract of the aerial part was subjected to solvent partitioning to obtain three fractions: dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and water fractions. In the phytochemical study, compound 1 was isolated from the ethyl acetate fraction (Fig.1). This is first time this compound has been isolated. On the other hand, maceration of vegetative matter (350 g) yielded 15g of aqueous extract, of which 10g was separated by selective extraction with EtOAc. The fraction was partitioned with EtOH and CH2Cl2. The latter fraction was chromatographed from which was isolated 3,5-Dihydroxybenzoic acid (2) (Figure 2). Structural examination of both compounds was performed mainly by 1D and 2D NMR spectroscopy, mass spectrometry (ESI-MS) and Infrared Spectroscopy (IR). NMR, Mass and IR spectra data are detailed in the Supplementary material- Figure A2A14.
Figure 1. Glycosidic compound (1) Figure 2. 3,5-Dihydroxybenzoic acid (2)
3.2 Characterization of the isolated compound (Spectral Data) Glycosidic compound (1) ((2R, 3R, 4S, 5S, 6R)-3,4,5-Tryhidroxy-6-(((E)-3-(4-hydroxyphenyl) acryloyl) oxy) tetrahydro-2H-pyran-2-yl) methyl-4hydroxybenzoate, was obtained (200 mg/28.5% yield) as a white solid (mp=200 °C). 1H NMR (500 MHz, CD3OD) (ppm): spectrum showed downfield signals of AA’BB’-aromatic ring protons at δ 7.95 (d, 2H, J= 8.9 Hz, H-21,25), 7.46 (d, 2H, J=8.6 Hz, H-13,17), 7.13 (d, 2H, J= 8.9 Hz, H-22,24), 6.82 (d, 2H, J= 8.6 Hz, H-14,16), along with vinylic protons signals at δ 7.63 (d, 1H, J=15.9 Hz, H-11), 6.34 (d, 1H, J=15.9, H-10), the spectrum also exhibit signals of anomeric proton at δ 5.04 (d, 1H, J= 7.6Hz, H-1), and other signals belonging for a β-D-glucopyranoside at δ 4.57 (dd, 1H, J=11.9, 2.1Hz, H-6a), 4.34 (dd, 1H, J= 11.9, 6.9 Hz, H-6b), 3.79 (d, 1H, J= 9.4, 2.1Hz, H-5), 3.52 (m, 2H, H-2,3), 3.44 (m, 1H, H4).
13
C NMR (125 MHz, CD3OD) (ppm): spectrum showed 17 signals assigned for 22 carbon atoms in the molecule,
which were assigned to two carbonyls, one ester and one α, β-unsaturated ester at δ 168.95 (C-19,9), four quaternary aromatic carbons at δ 162.58 (C-23), 161.35 (C-15), 146.89 (C-20), 127. 15 (C-12) and four CH aromatic carbon signals were observed at δ 132.66 (C-21,25), 131.26 (C-13,17), 117.20 (C-22,24), 116.93 (C-14,16). Signals at δ 146.89 (C-11), 114.95 (C-10) belong to vinylic carbons. This spectrum also showed six carbons for a β-D-glucopyranoside moiety at δ 101.54 (C-1) (signal of anomeric carbon), 77.90 (C-3), 75.69 (C-5), 74.82 (C-2), 71.80 (C-4), 64.65 (C-6). IR spectrum exhibited absorption bands due to hydroxyl (3370 cm-1), α, β-unsaturated carbonyl (1689 cm-1) and aromatic rings (1606 cm-1). Molecular formula was determined as C22H22O10 by ESI-QTOF-MS [M-H]- m/z 445.1183, C22H22O10, calcd: 445.1213). The unequivocal assignment of 1H and
13
C was established using 2D NMR experiments, such as COSY,
TOCSY, ROESY, HSQC and HMBC, which helped to assign the glucose and ester carbonyl signals and differentiate the signals from the aromatic protons. 3.3 Toxicity Test 3.3.1 Acute Toxicity LD50 for EtOH extract of B. odorata was reached to 2000 mg/kg in both sexes of ICR mice. It is considered a Category 5 substance according to OECD TG 423. None of the four doses of ethanolic extract analyzed produced toxicity during the 14 days of the study. In mice of both sexes, there were no alterations in body weight or change in water or food consumption. There were no changes in the behavior of the animals, nor were there any differences at the macroscopic level in the organs (heart, lungs, stomach, intestines, gallbladder, liver, and kidneys). 3.4 Evaluation of Hypolipidemic Activity 3.4.1 Induction of hyperlipidemia by hypercholesterolemic diet The mice showed no signs of toxicity during the experiment. Table 1 shows the results obtained after administration of ethanolic extract of the aerial parts of B. odorata. The hypercholesterolemic diet induced significant increases (p <0.05) in total cholesterol (TC), low-density lipoprotein (LDL-C), and triglycerides (TG) compared to the group of normocholesterolemic mice. On the other hand, the groups treated with different doses (10, 100, 100 mg/kg) of the ethanolic extract of the aerial part of B. odorata showed significant reduction in the concentrations of TC, LDL-C, and TG. It is important to mention that the doses of 100 and 1000 mg/kg were found to be most effective, because they rendered concentrations of HDL-C and TG similar to those of the normolipid group. Therefore, it was found that there is a doseeffect relationship for the ethanolic extract. (Supplementary material-Figure A15-A18). Table 1. Effect of the administration of ethanolic extract (e.e.) of the aerial parts of B. odorata on the concentration of TC, C-LDL, C-HDL and TG in male ICR mice on hypercholesterolemic diet. The data is represented by means ± se ANOVA p <0.05 * significant difference vs the hyperlipidemic control, × significant difference vs the normal control, post hoc Tukey's. Treatment
Normocholesterolemic Hypercholesterolemic (Diet) Hypercholesterolemic
B. odorata e.e (mg/kg) -----------
TC (mmol/L)
C-LDL (mmol/L)
C-HDL (mmol/L)
TG (mmol/L)
3 ± 0.05*
1.10 ± 0.09*
1.46 ± 0.06*
1.34 ± 0.10*
-----------
7.44 ± 0.12
6.46 ± 0.12
0.80 ± 0.03
2.52 ± 0.15
10
3.48 ± 0.11*
2.54 ± 0.12*
1.08 ± 0.07*
1.40 ± 0.08*
(Diet) + ethanolic extract (e.e)
100
3.02 ± 0.12*
1.42 ± 0.07*
1.60 ± 0.07*
0.96 ± 0.02*×
1000
3.10 ± 0.10*
1.26 ± 0.11*
1.62 ± 0.08*
1.24 ± 0.07*
3.4.2 Induction of hyperlipidemia with Triton WR 1339 In Table 2 shows the lipid-lowering effect of the ethanolic extract of parts B. odorata in Triton WR 1339-induced hyperlipidemia mouse model. The extract at a dose of 100 mg/kg reduced total cholesterol levels significantly (p<0.05) compared to the hyperlipidemic group. For the LDL-C parameter, all doses were effective compared to the hyperlipidemic control, but it is important to mention that the 100 mg/kg dose also exhibited a significant decrease in the concentration of LDL-C with respect to the normal control. HDL-C showed a significant increase for the 100, 1000 mg/kg dose, which was different from the hypercholesterolemic control and normocholesterolemic groups. Mice treated with the ethanolic extract at all three doses showed a decrease in triglyceride concentration; again, the 100, 1000 mg/kg dose showed greater effectiveness with respect to TG concentration in the hyperlipidemic control and normocholesterolemic groups. (Supplementary material-Figure A19-A22). Table 2. Effect of the administration of ethanolic extract (e.e) of aerial parts of Bidens odorata on the concentration of TC, LDL-C, HDLC and TG in male ICR mice treated with Tyloxapol. The data is represented by means ± se. ANOVA p <0.05 * significant difference vs the hyperlipidemic control, × significant difference vs the normal control, post hoc Tukey's.
Treatment
Normocholesterolemic Hypercholesterolemic (Tyloxapol) Hypercholesterolemic (Tyloxapol) + Ethanolic extract (e.e)
B. odorata e.e (mg/kg) -----------
TC (mmol/L)
C-LDL (mmol/L)
C-HDL (mmol/L)
TG (mmol/L)
6.72 ± 0.13*
3.56 ± 0.06*
3.58 ± 0.14*
2.48 ± 0.09*
-----------
10.32 ± 0.46
8.54 ± 0.13
1.88 ± 0.06
3.06 ± 0.20
10
9.66 ± 0.20
6.44 ± 0.14*
2.54 ± 0.10*
2.14 ± 0.11*
100
7.38 ± .15*
2.68 ± 0.15*×
4 ± 0.17*
1.64 ± 0.09*×
1000
9 ± 0.15
5.48 ± 0.24*
3.6 ± 0.10*×
1.98 ± 0.11*
The ethanolic extract of the aerial parts of B. odorata showed high lipid-lowering activity, particularly at the 100 and 1000 mg/kg dose, with a significant effect on decreasing cholesterol, triglycerides, and low-density lipoproteins, and increasing HDL. These results show that the species B. odorata collected in Tlaxcala has lipid-lowering activity similar to that collected in Coahuila and evaluated by Moreno et al., 2017. Additionally, in the present study it was determined that metabolite 1 is one of those responsible for the lipid-lowering activity of this species. Therefore, the compounds extracted from this species behave as potent hypolipidemic agents, perhaps as antiatherosclerotics since the reduction of LDL cholesterol is essential in preventing the formation of atherosclerotic plaque. Additionally, the decrease in TG concentration is important, because it is also considered a risk factor for the development
of cardiovascular disease. Another interesting result was that B. odorata extracts induced an increase of high-density lipoprotein (HDL), which plays a major role in the transport of excess cholesterol in cells, through a reverse transport system. HDL is also involved in the protection of membranes against oxidative damage. In addition, clinical and epidemiological studies have shown that increasing the concentration of HDL potentially contributes to antiatherogenesis, possibly through an ability to inhibit LDL oxidation and protect endothelial cells from the cytotoxic effects of oxidized LDL (Assmann and Nofer, 2003; Shamala, et al., 2016). Monitoring of the ethanolic extract of the aerial part of B. odorata by TLC and NMR allowed us to find a greater proportion of glycosidic compound (1). It has been described that the metabolites structurally related to compound 1, are antioxidant and have protective function against cardiovascular and coronary diseases, since they have demonstrated ability to decrease LDL oxidation, inhibit lipoperoxidation and suppress the progressive increase of atherosclerotic plaque (Shamala, et al., 2016). It was interesting to know if compound 1 was responsible for lipid-lowering activity by the model induced with Triton WR 1339. Table 3 shows the results of the lipid-lowering effect of compound 1 (EtOH extract) (Supplementary material-Figure A23-A26). Table 3. Effect of the administration of compound 1 on the concentration of TC, C-LDL, HDL-C and TG in male ICR mice treated with tyloxapol for 3 days. The data is represented by means ± se. ANOVA p<0.05 * significant difference vs the hyperlipidemic control, post hoc Tukey's.
Treatment
Compound 1 (mg/kg)
TC (mmol/L)
C-LDL (mmol/L)
C-HDL (mmol/L)
TG (mmol/L)
Normocholesterolemic
-----------
2.88 ± 0.22*
0.96 ± 0.16*
1.48 ± 0.11*
1.24 ± 0.08*
Hypercholesterolemic (Tyloxapol)
-----------
6.12 ± 0.79
3.83 ± 0.76
0.58 ± 0.16
3.77 ± 0.60
25
4.72 ± 0.39
1.9 ± 0.18*
1.35 ± 0.26
3.47 ± 0.71
50
3.62 ± 0.57*
1.07 ± 0.29*
1.52 ± 0.28*
2.24 ± 0.44
100
2.56 ± 0.14*
0.64 ± 0.10*
1.43± 0.09*
1.31 ± 0.11*
Hypercholesterolemic Tyloxapol + Compound 1
3.5 Antimycobacterial Activity One of the common uses of B. odorata reported in traditional medicine is its use as an antitussive agent and to treat lung diseases. Since there are no previous reports in the literature related to the activity of B. odorata on these conditions, present work was undertaken to determine antimycobacterial activity of its different extracts. This study also describes for the first
time the antimycobacterial properties of compound 1 isolated from this species. According to Jimenez-Arellanes et al., 2013, in the ethnobotanical literature the definition of tuberculosis has not been described, even though numerous plant species have been described to treat symptoms related to the disease (such as cough with fever and lung pain, among others). Herein, the results of our antimycobacterial evaluation are shown in Table 4, revealing that hexane, CH2Cl2, EtOAc, and ethanolic extracts having antibacterial activity against Mycobacterium tuberculosis H37Rv; and only the extracts of hexane and dichloromethane had effect against Mycobacterium smegmatis mc2155. On the other hand, the ethanolic extract was subjected to fractionation by selective extractions, from which a purified compound of phenolic nature was obtained that presented an excellent activity with MIC of 3.125 g/mL. It should be mentioned that the aqueous crude extract did not display any antimycobacterial activity, but when it was subjected to fractionation it was possible to isolate 3,5hydroxybenzoic acid (compound 2), which unlike the aqueous extract, did show biological activity against M. tuberculosis H37Rv (Supplementary material-Figure A27). In this context, the antibacterial activity observed with the extracts of less polarity agrees with the results reported by other researchers (Jimenez-Arellanes, et al., 2010). This review includes 75 species of the medicinal flora of Mexico, which were evaluated in in vitro models against mycobacteria, being the extracts of less polarity that showed better bioactivity, mainly due to their lipophilic nature. However, the ethanolic extract and compounds 1 and 2, despite their polar nature, showed outstanding activity against M. tuberculosis H37Rv at a low concentration (Table 4). So far, few compounds with chemical structure similar to compound 1 have been studied for its antimycobacterial activity. For instance, Sashida in 1991 and Wang in 1997 reported the isolation of compound 1,6-di-O-pcoumaroyl--D-glycopyranoside from B. pilosa. This compound presented a mild antimicrobial activity against Klebsiella pneumoniae, Sthaphylococcus aureus and Mycobacterium aurum (125 μg/mL) and slightly better activity against Mycobacterium tuberculosis H37Ra (63 μg/mL). Table 4. a H37Rv: drug-sensitive Mycobacterium tuberculosis. b (1) Hexane extract; (2) CH2Cl2 extract; (3) EtOAc extract; (4) Ethanol extract; (5) Aqueous extract; (6) Compound 1; (7) Compound 2. MIC: minimum inhibitory concentration. The assays with a MIC >50, ˃ 200 µg/mL were considered inactive. MIC 2 (µg/mL) * Extract/Fraction
1b
2
3
4
5
6
7
100
12.5
12.5
12.5
˃ 200
3.125
50
50
100
˃ 200
˃ 200
˃ 200
˃ 50
˃ 50
Microorganism Mycobacterium tuberculosis H37Rv
a
Mycobacterium smegmatis 2
mc 155
4. Conclusions Bidens odorata is a species widely distributed and used in the traditional Mexican medicine, however there are few reports in this plant. In the present study, it was demonstrated that its ethanolic extract and the isolated glycoside compound (1) had an important effect on decreasing TC, LDL-C, and TG and increasing HDL-C, which could be potential lipid-lowering agents. The toxicological results demonstrated that ethanolic extract does not present toxic effects in mice. Extracts of different polarity of the aerial parts of this plant showed antimycobacterial properties, particularly the isolated compound 1, gave MIC of 3.125 μg/mL. These results show that B. odorata can be an excellent source of active molecules with antimycobacterial and hypolipidemic properties. The antimycobacterial activity found in this study contributes to finding
potential natural alternatives for the treatment of tuberculosis, thus alleviating the emerging drug-resistance problem shown by some tuberculosis strains. The above results found in this study supports the documented traditional use of B. odorata. Funding This work was supported by SIP-IPN (20181909 and 20171699), JLH, LGZV, LGS and MEVD are COFAA, EDI and SNI fellows. KMHS was CONACyT (335863) fellow. References Achika, J.I; Gerald, I.; Adedayo, A. 2014. A review on the phytoconstituents and related medicinal properties of plantas in the Asteraceae family. Journal of Applied Chemistry. 7(8),1-8. Argueta, A., Cano, L., Rodarte, M. 1994. Atlas de las plantas de la medicina tradicional mexicana I. Instituto Nacional Indigenísta, Ciudad de México, México. 1786 pp., http://www.medicinatradicionalmexicana.unam.mx/atlas.php (accesed 2017). Assmann, G., Nofer, J., 2003. Atheroprotective effects of high-density lipoproteins. Annu. Rev. Med. 54: 321–341. DOI: 10.1146/annurev.med.54.101601.152409 Beltrán, H. 2016. The Asteraceae (Compositae) from Laraos district (Yauyos, Lima, Peru). Revista Peruana de Biología. 23(2), 195-220. DOI: http://dx.doi.org/10.15381/rpb.v23i2.12439 Digital Library of Mexican Traditional MedicineIndigenous http://www.medicinatradicionalmexicana.unam.mx/index.php (accesed 2017).
Medicinal
Flora
of
Mexico.
2012.
Dimo, T., Azay, J., Tan, P.V., Pellecuer, J., Cros, G., Bopelet, M., Jacques-Serrano, J. 2001.Effects of the aqueous and methylene chloride extracts of Bidens pilosa lea fon fructose-hypertensive rats. Journal of Ethnopharmacology. 76, 215-221. Gutierrez, J.P., Rivera-Dommarco, J., Shamah-Levy, T., Villalpando-Hernandez, S., Franco, A., Cuevas-Nasu, L., Romero-Martinez, M., Hernandez-Avila, M., Gomez-Acosta, L.M., Gaona-Pineda, E.B., Gomez-Humaran, I., Saturno, P., Avila, M.A., Mauricio-Lopez, E.R., Martinez, J., García Lopez, D.E. Encuesta Nacional de Salud y Nutricion 2016. Resultados Nacionales. Cuernavaca, Mexico: Instituto Nacional de Salud Publica (MX), 2016. Friedewald, W.T., Levy, R.I., Fredrickson, D.S. 1972. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifugate. Clinical Chemistry. 8(6), 499-502. García-Regalado, G. Plantas Medicinales de Aguascalientes. Universidad Autónoma de Aguascalientes. 2015, 498 pp., http://www.uaa.mx/direcciones/dgdv/editorial/docs/plantas_medicinales_aguascalientes.pdf (accesed 2017). http://www.medicinatradicionalmexicana.unam.mx/flora2.php?l=4&t=Aceitilla&po=tepehuan_del_sur&id=6126&clave_region=13 (accesed 2017). Garduño, L., Salazar, M., Salazar, S., Morelos, M.E., Labarrios, F., Tamariz, J., Chamorro, G.A. 1997. Hypolipidaemic activity of αasarone in mice. Journal of Ethnopharmacology. 55,161-163. DOI: https://doi.org/10.1016/S0378-8741(96)01492-4 González, E. M., López, E. I. L., González, E. M.S., Tena, F. J. A. 2004. Plantas medicinales del Estado de Durango y Zonas aledañas. CIIDIR-Durango, Instituto Politécnico Nacional. 144pp. Hisashi, M., Takeshi, C., Yasushi, K., Johji, Y., Tokunosuke, S., Hajime, F. Hitoshi, K. 1986. Effects of Crude Drugs on Experimental Hypercholesterolemia. I. Tea and Its Active Principles. Journal of Ethnopharmacology. 17, 213-224. DOI: https://doi.org/10.1016/03788741(86)90110-8 Jiménez, A. A., Cornejo, G.J., León, D. R. 2010. Las plantas mexicanas como fuente de compuestos antimicrobianos. Revista Mexicana de Ciencias Farmacéuticas. 41(1), 22-29. Jimenez-Arellanes, A., Meckes, M., Ramírez, R., Torres, J., Luna-Herrera, J. 2003. Activity against Multidrug-resistant Mycobacterium tuberculosis in Mexican Plants Used to Treat Respiratory Diseases. Phyther Resp. 17, 903-908. DOI: 10.1002/ptr.1377 Jiménez-Arellanes, M.A., Yépez-Mulia, L., Luna-Herrera, J., Gutiérrez-Rebolledo, G.A., García-Rodríguez, R.V. 2013. Actividad antimicobacteriana y antiprotozoaria de Moussonia deppeana (Schldl and Cham.) Hanst. Revista Mexicana de Ciencias Farmacéuticas. 42 (2), 31-35. Liang, Y.C., Yang, M.T., Lin, C.J., Chang, C.L.T., Yang, W.C. 2016. Bidens pilosa and its active compound inhibit adipogenesis and lipid accumulation via down-modulation of the C/EBP and PPARγ pathways. Sci. Rep. 6, 24285. DOI: 10.1038/srep24285. Martínez, M. 1996. Las plantas medicinales de México. 7ª Reedición, México. 656 pp. Meléndez- Camargo, M. E., B., Berdeja, and G., Miranda. 2004. Diuretic effect of the aqueous extract of Bidens odorata in the rat. Journal of Ethnopharmacology. 95 (2-3), 363–366. DOI: 10.1016/j.jep.2004.08.005
Mendieta, A., Jiménez, F., Garduño-Siciliano, L., Mojica-Villegas, A., Rosales-Acosta, B., Villa-Tanaca, L., Chamorro-Cevalllos, G., Medina-Franco, J., Meurice, N., Gutiérrez, R., Montiel, L., Cruz, M.C., Tamariz, J. 2014. Synthesis and highly potent hypolipidemic activity of alpha-asarone- and fibrate -based 2-acyl and 2-alkyl phenols as HMG-CoA reductase inhibitors. 22, 5871-5882. DOI: http://dx.doi.org/10.1016/j.bmc.2014.09.022 Moreno-Pena, D.P., Cordero-Pérez, P., Leos-Rivas, C., Bucio, L., Viveros-Valdez, J.E., Muñoz-Espinosa, L.E., Galindo-Rodríguez, S.A., Rivas-Morales, C. 2017. Evaluation of hypocholesterolemic activity of extracts of Bidens odorata and Brickellia eupatprioides. Pak. J. Pharm. Sci. 2, 613- 617. Norma Oficial Mexicana 062-ZOO-1999. Technical specifications for the reproduction, care and use of laboratory animals. Revised in 2017. Organization for Economic Co-operation and Development (OECD). 2008. Guidelines for the Testing of Chemicals. Assay TG 423. Revised in 2017. Pahua-Ramos, M.E., Ortiz-Moreno, A., Chamorro-Cevallos, G., Hernández Navarro, M.D., Garduño-Siciliano, L., NecoecheaMondragón,H., Hernández-Ortega, M. 2012. Hypolipidaemic Effect of Avocado (Persea americana Mill) Seed in a Hypercholesterolemic Mouse Model. Plants Food Hum Nutr. 67, 10-16. DOI: 10.1007/s11130-012-0280-6 Rzedowski, G., C. de, J. Rzedowski. 2005. Flora fanerogámica del Valle de México. 2a. ed., 1a reimp., Instituto de Ecología, A.C. y Comisión Nacional para el Conocimiento y Uso de la Biodiversidad, Pátzcuaro (Michoacán). 1406 pp. Sashida, Y., Ogawa, K., Kitada, M., Karikome, H., Mimaki, Y., Shimomura, H. 1992. New aureone glucosides and new phenylpropanoid glucosides from Bidens pilosa. Chem. Pharm. Bull. 39, 709-711. DOI: https://doi.org/10.1248/cpb.39.709 Shamala, S., Baskaron, G., Mohd, Y.S., Zuki, A.B. 2016. Phytochemical investigation, hypocholesterolemic and anti-atherosclerotic effects of Amaranthus viridis leaf. RSC Adv. 6, 32685-32696. DOI: 10.1039/C6RA04827G Secretaría de Salud. 2014. Prevención y Control de la Tuberculosis: Programa Sectorial de Salud 2013-2018. México. Vibrans, H. 1995. Bidens pilosa L. y Bidens odorata Cav. (Asteraceae: Heliantheae) en la vegetación urbana de la Ciudad de México. Acta Botánica Mexicana. 32, 85-89. Wang, J., Yang, H., Lin, Z.W., Sun, H.D. 1997. Flavonoids from Bidens pilosa var. radiata. Phytochemistry. 46, 1275- 1278. DOI: https://doi.org/10.1016/S0031-9422(97)80026-X WHO. 2017. Tuberculosis [Internet]. Available from: http://www.who.int/mediacentre/factsheets/fs104/es/ (Accesed, 2017). Zavala-Mendoza, M., Alarcón-Aguilar, F.J, Pérez-Gutiérrez, S., Escobar-Villanueva, M.C., Zavala-Sánchez, A. 2013. Composition and Antidiarrheal Activity of Bidens odorata Cav. Evidence- Based Complementary and Alternative Medicine. 1, 1-7. DOI: 10.1155/2013/170290
PII: DOI: Reference:
www.elsevier.com/locate/jep
S0378-8741(18)30706-2 https://doi.org/10.1016/j.jep.2018.04.028 JEP11323
To appear in: Journal of Ethnopharmacology Received date: 24 February 2018 Revised date: 18 April 2018 Accepted date: 19 April 2018 Cite this article as: Karla Mariela Hernández-Sánchez, Leticia Garduño-Siciliano, Julieta Luna-Herrera, L. Gerardo Zepeda-Vallejo, Selene Lagunas-Rivera, G. Esthefania García-Gutiérrez and María Elena Vargas-Díaz, Antimycobacterial and hypolipemiant activities of Bidens odorata (Cavanilles), Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2018.04.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antimycobacterial and hypolipemiant activities of Bidens odorata (Cavanilles). Karla Mariela Hernández-Sáncheza,b, Leticia Garduño-Sicilianoa, Julieta Luna-Herrerac, L. Gerardo ZepedaVallejob, Selene Lagunas-Riverad, G. Esthefania García-Gutiérreza, María Elena Vargas-Díazb* a
Departamento de Farmacia, Laboratorio de Toxicología de Productos Naturales, Escuela Nacional de Ciencias Biológicas. Instituto Politécnico Nacional. Av. Wilfrido Massieu esq. con M. Stampa, Col. Planetario Lindavista. Del. GAM, C.P. 77380, CDMX, México. b
Departamento de Química Orgánica, Laboratorio de Química de Productos Naturales, cDepartamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prol. de Carpio y Plan de Ayala, Del. Miguel Hidalgo C.P.11340, CDMX, México. d
CONACyT, Tecnológico Nacional de México/Instituto Tecnológico de Tuxtla Gutiérrez, Carretera Panamericana km. 1080, Tuxtla Gutiérrez, Chiapas C.P. 29050, México.
*Author to whom correspondence should be addressed: María Elena Vargas-Díaz. Email: [email protected] (VDE).
Abstract Ethnopharmacological relevance Bidens odorata Cavanilles is a medicinal and edible plant known as "mozote blanco, aceitilla, acahual, mozoquelite" which is traditionally used in Mexico as a diuretic, hypoglycaemic, anti-inflammatory, antipyretic, antitussive, to treat gastrointestinal disorders, kidney pain, and lung or respiratory diseases. Aim of the study This research study was aimed at phytochemical analysis of aerial extracts of B. odorata for antimycobacterial and lipidlowering activities. Materials and methods Compounds 1 ((((2R, 3R, 4S, 5S, 6R)-3,4,5-Tryhidroxy-6-(((E)-3-(4-hydroxyphenyl) acryloyl) oxy) tetrahydro-2H-pyran-2yl) methyl-4-hydroxybenzoate) and 2 (3,5-Dihydroxybenzoic acid) were isolated from B. odorata aerial shoots and their structural elucidation was carried out using 1 and 2D NMR, infrared spectroscopy (IR) and mass spectrometry (ESI-MS). The antimycobacterial activity of various extracts and compounds 1 and 2 was determined using the Microplate Alamar Blue Assay (MABA). The evaluation of the hypolipidemic effect of the ethanolic extract and the glycosylated compound 1 was tested in a murine model of hypercholesterolemia induced by diet and by Triton WR-1339. On the other hand, the LD50 of the ethanolic extract was evaluated in ICR mice by the OECD protocol TG 423. Results Antimycobacterial assay of hexane, CH2Cl2, EtOAc, ethanolic and aqueous extracts, as well as the new glycosidic compound (1) and benzoic acid derivative (2) isolated from B. odorata showed minimal inhibitory concentrations (MIC) of 100, 12.5, 12.5,12.5, ˃ 200, 3.125 and 50 μg/mL, respectively, against Mycobacterium tuberculosis H37Rv. Only hexane and CH2Cl2 extracts were observed to be active against Mycobacterium smegmatis mc2155 at a concentration of 50 and 100 μg/mL, respectively. The ethanolic extract showed lipid-lowering activity at doses of 100 and 1000 mg/kg, while glycosidic compound 1 was active at doses of 50 and 100 mg/kg. In addition, the LD50 of the ethanolic extract was ˃ 2000 mg/kg, meaning that this extract does not cause lethality or adverse effects, and no signs of organs alterations or tissue damage were observed. Conclusion The hexane, CH2Cl2, EtOAc, and ethanolic extracts of B. odorata, as well as their components 1 and 2, displayed antimycobacterial activity against M. tuberculosis. Moreover, the ethanolic extract and glycosidic compound (1) showed an
important lipid-lowering effect, without lethality or secondary effect. The results of this study support the documented traditional use for B. odorata. Graphical abstract
Keywords: Hypolipidemic, Phenolic compounds, Bidens odorata, Mycobacterium, Traditional Medicine.
1. Introduction The genus Bidens comprises a complex of about 240 closely related species (Beltrán, 2016), many of which have been studied chemical and biologically (Achika et al., 2014). However, Bidens odorata Cavanilles (syn. Bidens rosea, Bidens tripartita, Bidens bipinnata, Coreopsis odorata) is a plant little studied in the taxonomic family Asteraceae and genus Bidens. It is known in Mexico as “mozote blanco, aceitilla, acahual, mozoquelite” and it is one of the most common and widely distributed species (Rzedowski and Rzedowski, 2005), mainly inhabiting the mountain ranges of Mexico, as well as the southwestern region of United States and Central America (Vibrans, 1995). This plant is edible, and according to the books, “Plantas Medicinales del estado de Durango y zonas aledañas” (González et al., 2004) and “Plantas Medicinales de Aguascalientes” (García-Regalado, 2015), the aerial parts of Bidens odorata (leaves, flowers and stems) are used in the Mexican traditional medicine for treatment of gastrointestinal diseases, kidney pain, as anti-inflammatory, antipyretic and hypoglycemic disorders. Also, it is well recognized for its beneficial effects on the lung related disorders like pulmonary diseases and cough (Argueta et al., 1994; Martínez, 1996; Zavala-Mendoza et al., 2013; Digital Library of Mexican Traditional Medicine- Indigenous Medicinal Flora of Mexico, 2012). Although it is frequently prepared as decoction, teas or maceration. It is also documented that aqueous extract of this plant has a diuretic effect (Meléndez-Camargo et al., 2004.). Nevertheless, this species have not been investigated chemically for pharmacologically active compounds, and the biological studies confirming its curative properties are few. Therefore, the main objective of this study is to correlate the
ethnomedical use of B. odorata to treat lung diseases with respect to its antimicrobial activity. Considering that in Mexico like in the rest of the world, pulmonary tuberculosis is still one of the most devastating diseases (SSA, 2014; WHO, 2017). Additionally, in this report, we show that the species B. odorata collected in Tlaxcala, has lipid-lowering effect like that reported in a preliminary study of the species collected in the state of Coahuila (Moreno et al., 2017), and in other species of the same genus (Bidens pilosa) (Dimo et al., 2001; Liang et al., 2016). Besides, in Mexico, overweight, obesity and its complications are a health problem, occurring in 72.5% of the adults and 33.2% of the children (ENSANUT, 2016). Therefore, it is important to identify the compounds that show lipid-lowering effect.
2. Materials and methods 2.1 General Procedure and Equipment Used RMN spectra were collected on a Varian NMR System at 500 MHz using CDCl3, Methanol-d4 as solvents, or a combination of these and tetramethylsilane as the internal standard. Chemical shift values are reported in ppm (δ) (tetramethylsilane δ = 0 for 1H; chloroform-d δ = 77.0 for 13C, and methanol-d4 δ = 3.31, 4.78 for 1H and δ = 49.2 for 13C). H NMR spectra are reported as follows: δ (multiplicity, number of protons, coupling constant J, atom assignment).
1
13
C
NMR spectra are reported as follows: δ (atom assigned). Multiplicities are indicated by d (doublet), dd (doublet of doublets), and m (multiplet). Infrared spectra were recorded on a Perkin-Elmer Spectrum 2000 spectrophotometer. High Resolution Mass Spectra (HRMS) were determined using a Bruker microTOF-QII spectrometer by Electrospray Ionization Mass. Thin-layer chromatograms (TLC) were performed on precoated TLC sheets of silica gel Merck 60F-254. Spots on TLC were visualized under UV lamp or developed by spraying with cerium molybdate and cerium sulfate. Purification of compounds was performed by column chromatography on silica gel (Merck 230-400 mesh).
2.2 Collection of Plant Material and Crude Extract Preparation Bidens odorata was collected in Calpulalpan, Tlaxcala, Mexico (19°35'17.0''N, 98°32'25.5''W), in June 2016 (Supplementary material-Figure A1). The specimen was registered in the herbarium of the Escuela Nacional de Ciencias Biológicas (ENCB) with voucher No. 001/2016/BoTLX. The aerial parts were dried at room temperature for a week, then crushed. Crude extracts of the aerial parts were prepared by maceration with water or solvents of different polarity (3 L) (hexane, dichloromethane, ethyl acetate, and ethanol). The extracts were filtered and concentrated under reduced pressure using a rotary evaporator and kept in dark conditions and refrigerated until use. 2.3 Extraction and Isolation of Secondary Metabolites Aerial parts (1.75 kg) of the plant was macerated at room temperature using solvents of different polarity, starting with hexane (C6H8), followed by dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and ethanol (EtOH). For the aqueous extraction, 350 g of plant material was used. 2.3.1 Ethanolic Extract The ethanolic extract was monitored by 1H NMR and was extracted (20g in 3 L) using the technique of selective extraction (CH2Cl2:H2O/EtOAc:H2O). The resulting organic phases were combined and concentrated under reduced pressure, obtaining 700 mg of a brown precipitate, which was analyzed by thin layer chromatography (TLC) using
CH2Cl2:EtOAc:EtOH (10:80:10) as eluent, and was also monitored by 1H NMR. Consecutively, 350 mg of the brown precipitate was subjected to column chromatography (CC) using a system EtOAc:EtOH (80:20). The fractions obtained were compared using 1H NMR. Fraction 8 (200 mg) was analyzed by
13
C NMR, expose the presence of a glycosylated
phenolic compound (1). To carry out the unequivocal structural assignment, the 2D NMR spectra were obtained and complemented with the mass spectrometry analysis, using the electrospray ionization method (ESI-MS) and the IR spectrum was also obtained. 2.3.2 Aqueous Extract Aqueous extract (10 g) was extracted selectively with EtOAc (3.5 L). A brown-yellow precipitate was obtained with EtOAc and monitored by TLC then washed with CH2Cl2 and EtOH, and these fractions were analyzed again by TLC using Hex: EtOAc (1:1) as eluent. Then the fraction of CH2Cl2 was selected to be separated by CC with Hex: EtOAc (1:1). From this separation, fraction 4 was selected, which was analyzed by 1H and 13C NMR, and an ESI-QTOF-MS [M-H] analysis was carried out and showed the presence of compound 2. Crude extracts and isolated compounds 1 (EtOH extract) and 2 (aqueous extract) were evaluated for antimycobacterial activity against mycobacteria. Only the ethanolic extract and compound 1 were tested in lipid-lowering models, as described below. 2.4 Bacterial Species Used in Microplate Alamar Blue Assay The following mycobacterial strains used in this study were obtained from the American Type Culture Collection (ATCC): Mycobacterium tuberculosis H37Rv (ATCC 27294) and Mycobacterium smegmatis mc2155 (ATCC 700084).
2.4.1 Antimycobacterial assay The assay to determine the minimum inhibitory concentration (MIC) of crude extracts of B. odorata and compounds 1 and 2 against strains of Mycobacterium tuberculosis and Mycobacterium smegmatis was carried out in 96-well microplates using the following protocol described by Jimenez-Arellanes et al., in 2003. Briefly, extracts were tested at concentrations ranging from 200 to 6.25 µg/mL and pure compounds from 50 to 1.56 µg/mL. Mycobacterium tuberculosis H37Rv and Mycobacterium smegmatis mc2155 were grown in Middlebrook 7H9 broth supplemented with 0.2% of glycerol and 10% of OADC (oleic acid, albumin, dextrose, and catalase) The inoculum was adjusted according to the turbidity of tube No. 1 of the McFarland nephelometer, then 1: 10 was diluted with the same medium to obtain 6 x 106 colony-forming units (UFC)/mL. Appropriate positive and negative controls were included with each test. Following addition of the mycobacterial inoculum the plates were incubated for 5 days in case of M. tuberculosis and 2 days in case of M. smegmatis. Plates were developed by adding alamar blue, and visual and fluorometric readings were recorded in a plate fluorometer (Fluoroskan Ascent-Thermo). The minimal inhibitory concentration (MIC) was defined as the lowest concentration of the compound that prevented change of color from blue to pink in relation to positive and negative controls. 2.5 In Vivo assay
For animal studies, ICR mice of 25+5 g of body weight of both male and female sex were used (PROPECUA, S.A. de C.V., Mexico City). The animals were kept for 7 days in plastic boxes, according to the conditions of animal housing (controlled temperature 24+2 °C, relative humidity 60-65%, light/dark cycle 12 x 12 h) for adaptation. The animals were fed with a standard diet (Lab Rodent Diet 5001, PMI Nutrition International, Inc., Bienwood, MO, USA) and water on demand. After the conditioning period, the mice were marked and weighed, and distributed randomly. The animal experiments and study design were approved by the Laboratory Animal Care Committee of Instituto Politécnico Nacional (IPN) and conducted in compliance with the Official Mexican standard NOM-062-ZOO-1999 regarding technical specifications for production, care, and use of laboratory animals (Norma Oficial Mexicana 062-ZOO-1999). 2.5.1 Assessment of Acute Toxicity Evaluation of the acute toxicity of the ethanolic extract of the aerial parts of B. odorata was carried out following the protocol 423 described by Organization for Economic Co-operation and Development and Test Guideline (OECD TG 423, 2008) (Supplementary material). 2.5.2 Evaluation of Hypolipidemic Activity 2.5.2.1 Induction of hyperlipidemia by Hypercholesterolemic Diet Five groups of eight male ICR mice each were established. The mice in the hyperlipidemic groups were fed for 6 days with a hyperlipidemic diet (1% cholesterol, 0.5% sodium cholate, 5% butter, 30% sucrose, 10% casein; food standard Rodent Lab 5001, 53.5% (Hisashi, 1986; Pahua-Ramos et al., 2012) and water ad libitum). Simultaneosly, mice used as negative controls were fed standard food, and positive controls were fed with the same hyperlipidemic diet but no treatment. The lipid-lowering activity of the ethanolic extract of B. odorata was determined according to the methodology described by Pahua-Ramos et al., 2012 (with modifications, Supplementary material). 2.5.2.2 Induction of hyperlipidemia with Triton WR 1339 (Tyloxapol) Five groups of eight male ICR mice were established, and hyperlipidemia was induced by a single intraperitoneal dose of Triton WR 1339 (Tyloxapol 400 mg/kg) (Mendieta, et al., 2014). The mice were treated with ethanolic extract or phenolic compound (1) 1 hour before and 22 and 48 hours after the injection of Tyloxapol. Group I (negative control) did not receive Tyloxapol; group II (positive control) was only administered with Tyloxapol; groups III, IV, and V were administered with Tyloxapol intraperitoneally and treated by intragastric administration of 10, 100, 1000 mg/kg of the ethanolic extract or compound 1. Two hours after the last administration of the extract, blood samples were obtained. All blood samples were allowed to clot and centrifuged at 13000 rpm for 15 minutes. Subsequently, the recovered serum was analyzed for total cholesterol (TC), triglycerides (TG) and high-density lipoprotein (HDL) in the Selectra II Vita Lab autoanalyzer using commercial kits. With the values obtained from this analysis the concentration of low-density lipoprotein (LDL) was calculated using the Friedewald formula [LDL= (CT-HDL) - (TG x .45) (Friedewald, 1972). 2.6 Statistical Analysis The results from the hypolipidemic evaluation of the ethanolic extract of the aerial parts of B. odorata and of compound 1 were analyzed by the one-way ANOVA test, with subsequent comparative analysis by Tukey's method; the data obtained underwent normality tests by the Shapiro-Wilk, Anderson-Darling, and Jarque-Bera methods. Statistically significant difference was considered to be p<0.05. All data were analyzed and graphed with the software Past version 3.09
3. Results and Discussion 3.1 Phytochemical Study of Bidens odorata The aerial parts of B. odorata collected in Calpulalpan, Tlaxcala, were dried, fragmented, and extracted consecutively with solvents of different polarities, starting with hexane (10g/0.57% yield) and CH2Cl2 (9.8g/0.56% yield), followed by EtOAc (5.6g/0.32% yield), ethanol (30.9g/1.76% yield), and finally aqueous (15g/4.28% yield). An EtOH extract of the aerial part was subjected to solvent partitioning to obtain three fractions: dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and water fractions. In the phytochemical study, compound 1 was isolated from the ethyl acetate fraction (Fig.1). This is first time this compound has been isolated. On the other hand, maceration of vegetative matter (350 g) yielded 15g of aqueous extract, of which 10g was separated by selective extraction with EtOAc. The fraction was partitioned with EtOH and CH2Cl2. The latter fraction was chromatographed from which was isolated 3,5-Dihydroxybenzoic acid (2) (Figure 2). Structural examination of both compounds was performed mainly by 1D and 2D NMR spectroscopy, mass spectrometry (ESI-MS) and Infrared Spectroscopy (IR). NMR, Mass and IR spectra data are detailed in the Supplementary material- Figure A2A14.
Figure 1. Glycosidic compound (1) Figure 2. 3,5-Dihydroxybenzoic acid (2)
3.2 Characterization of the isolated compound (Spectral Data) Glycosidic compound (1) ((2R, 3R, 4S, 5S, 6R)-3,4,5-Tryhidroxy-6-(((E)-3-(4-hydroxyphenyl) acryloyl) oxy) tetrahydro-2H-pyran-2-yl) methyl-4hydroxybenzoate, was obtained (200 mg/28.5% yield) as a white solid (mp=200 °C). 1H NMR (500 MHz, CD3OD) (ppm): spectrum showed downfield signals of AA’BB’-aromatic ring protons at δ 7.95 (d, 2H, J= 8.9 Hz, H-21,25), 7.46 (d, 2H, J=8.6 Hz, H-13,17), 7.13 (d, 2H, J= 8.9 Hz, H-22,24), 6.82 (d, 2H, J= 8.6 Hz, H-14,16), along with vinylic protons signals at δ 7.63 (d, 1H, J=15.9 Hz, H-11), 6.34 (d, 1H, J=15.9, H-10), the spectrum also exhibit signals of anomeric proton at δ 5.04 (d, 1H, J= 7.6Hz, H-1), and other signals belonging for a β-D-glucopyranoside at δ 4.57 (dd, 1H, J=11.9, 2.1Hz, H-6a), 4.34 (dd, 1H, J= 11.9, 6.9 Hz, H-6b), 3.79 (d, 1H, J= 9.4, 2.1Hz, H-5), 3.52 (m, 2H, H-2,3), 3.44 (m, 1H, H4).
13
C NMR (125 MHz, CD3OD) (ppm): spectrum showed 17 signals assigned for 22 carbon atoms in the molecule,
which were assigned to two carbonyls, one ester and one α, β-unsaturated ester at δ 168.95 (C-19,9), four quaternary aromatic carbons at δ 162.58 (C-23), 161.35 (C-15), 146.89 (C-20), 127. 15 (C-12) and four CH aromatic carbon signals were observed at δ 132.66 (C-21,25), 131.26 (C-13,17), 117.20 (C-22,24), 116.93 (C-14,16). Signals at δ 146.89 (C-11), 114.95 (C-10) belong to vinylic carbons. This spectrum also showed six carbons for a β-D-glucopyranoside moiety at δ 101.54 (C-1) (signal of anomeric carbon), 77.90 (C-3), 75.69 (C-5), 74.82 (C-2), 71.80 (C-4), 64.65 (C-6). IR spectrum exhibited absorption bands due to hydroxyl (3370 cm-1), α, β-unsaturated carbonyl (1689 cm-1) and aromatic rings (1606 cm-1). Molecular formula was determined as C22H22O10 by ESI-QTOF-MS [M-H]- m/z 445.1183, C22H22O10, calcd: 445.1213). The unequivocal assignment of 1H and
13
C was established using 2D NMR experiments, such as COSY,
TOCSY, ROESY, HSQC and HMBC, which helped to assign the glucose and ester carbonyl signals and differentiate the signals from the aromatic protons. 3.3 Toxicity Test 3.3.1 Acute Toxicity LD50 for EtOH extract of B. odorata was reached to 2000 mg/kg in both sexes of ICR mice. It is considered a Category 5 substance according to OECD TG 423. None of the four doses of ethanolic extract analyzed produced toxicity during the 14 days of the study. In mice of both sexes, there were no alterations in body weight or change in water or food consumption. There were no changes in the behavior of the animals, nor were there any differences at the macroscopic level in the organs (heart, lungs, stomach, intestines, gallbladder, liver, and kidneys). 3.4 Evaluation of Hypolipidemic Activity 3.4.1 Induction of hyperlipidemia by hypercholesterolemic diet The mice showed no signs of toxicity during the experiment. Table 1 shows the results obtained after administration of ethanolic extract of the aerial parts of B. odorata. The hypercholesterolemic diet induced significant increases (p <0.05) in total cholesterol (TC), low-density lipoprotein (LDL-C), and triglycerides (TG) compared to the group of normocholesterolemic mice. On the other hand, the groups treated with different doses (10, 100, 100 mg/kg) of the ethanolic extract of the aerial part of B. odorata showed significant reduction in the concentrations of TC, LDL-C, and TG. It is important to mention that the doses of 100 and 1000 mg/kg were found to be most effective, because they rendered concentrations of HDL-C and TG similar to those of the normolipid group. Therefore, it was found that there is a doseeffect relationship for the ethanolic extract. (Supplementary material-Figure A15-A18). Table 1. Effect of the administration of ethanolic extract (e.e.) of the aerial parts of B. odorata on the concentration of TC, C-LDL, C-HDL and TG in male ICR mice on hypercholesterolemic diet. The data is represented by means ± se ANOVA p <0.05 * significant difference vs the hyperlipidemic control, × significant difference vs the normal control, post hoc Tukey's. Treatment
Normocholesterolemic Hypercholesterolemic (Diet) Hypercholesterolemic
B. odorata e.e (mg/kg) -----------
TC (mmol/L)
C-LDL (mmol/L)
C-HDL (mmol/L)
TG (mmol/L)
3 ± 0.05*
1.10 ± 0.09*
1.46 ± 0.06*
1.34 ± 0.10*
-----------
7.44 ± 0.12
6.46 ± 0.12
0.80 ± 0.03
2.52 ± 0.15
10
3.48 ± 0.11*
2.54 ± 0.12*
1.08 ± 0.07*
1.40 ± 0.08*
(Diet) + ethanolic extract (e.e)
100
3.02 ± 0.12*
1.42 ± 0.07*
1.60 ± 0.07*
0.96 ± 0.02*×
1000
3.10 ± 0.10*
1.26 ± 0.11*
1.62 ± 0.08*
1.24 ± 0.07*
3.4.2 Induction of hyperlipidemia with Triton WR 1339 In Table 2 shows the lipid-lowering effect of the ethanolic extract of parts B. odorata in Triton WR 1339-induced hyperlipidemia mouse model. The extract at a dose of 100 mg/kg reduced total cholesterol levels significantly (p<0.05) compared to the hyperlipidemic group. For the LDL-C parameter, all doses were effective compared to the hyperlipidemic control, but it is important to mention that the 100 mg/kg dose also exhibited a significant decrease in the concentration of LDL-C with respect to the normal control. HDL-C showed a significant increase for the 100, 1000 mg/kg dose, which was different from the hypercholesterolemic control and normocholesterolemic groups. Mice treated with the ethanolic extract at all three doses showed a decrease in triglyceride concentration; again, the 100, 1000 mg/kg dose showed greater effectiveness with respect to TG concentration in the hyperlipidemic control and normocholesterolemic groups. (Supplementary material-Figure A19-A22). Table 2. Effect of the administration of ethanolic extract (e.e) of aerial parts of Bidens odorata on the concentration of TC, LDL-C, HDLC and TG in male ICR mice treated with Tyloxapol. The data is represented by means ± se. ANOVA p <0.05 * significant difference vs the hyperlipidemic control, × significant difference vs the normal control, post hoc Tukey's.
Treatment
Normocholesterolemic Hypercholesterolemic (Tyloxapol) Hypercholesterolemic (Tyloxapol) + Ethanolic extract (e.e)
B. odorata e.e (mg/kg) -----------
TC (mmol/L)
C-LDL (mmol/L)
C-HDL (mmol/L)
TG (mmol/L)
6.72 ± 0.13*
3.56 ± 0.06*
3.58 ± 0.14*
2.48 ± 0.09*
-----------
10.32 ± 0.46
8.54 ± 0.13
1.88 ± 0.06
3.06 ± 0.20
10
9.66 ± 0.20
6.44 ± 0.14*
2.54 ± 0.10*
2.14 ± 0.11*
100
7.38 ± .15*
2.68 ± 0.15*×
4 ± 0.17*
1.64 ± 0.09*×
1000
9 ± 0.15
5.48 ± 0.24*
3.6 ± 0.10*×
1.98 ± 0.11*
The ethanolic extract of the aerial parts of B. odorata showed high lipid-lowering activity, particularly at the 100 and 1000 mg/kg dose, with a significant effect on decreasing cholesterol, triglycerides, and low-density lipoproteins, and increasing HDL. These results show that the species B. odorata collected in Tlaxcala has lipid-lowering activity similar to that collected in Coahuila and evaluated by Moreno et al., 2017. Additionally, in the present study it was determined that metabolite 1 is one of those responsible for the lipid-lowering activity of this species. Therefore, the compounds extracted from this species behave as potent hypolipidemic agents, perhaps as antiatherosclerotics since the reduction of LDL cholesterol is essential in preventing the formation of atherosclerotic plaque. Additionally, the decrease in TG concentration is important, because it is also considered a risk factor for the development
of cardiovascular disease. Another interesting result was that B. odorata extracts induced an increase of high-density lipoprotein (HDL), which plays a major role in the transport of excess cholesterol in cells, through a reverse transport system. HDL is also involved in the protection of membranes against oxidative damage. In addition, clinical and epidemiological studies have shown that increasing the concentration of HDL potentially contributes to antiatherogenesis, possibly through an ability to inhibit LDL oxidation and protect endothelial cells from the cytotoxic effects of oxidized LDL (Assmann and Nofer, 2003; Shamala, et al., 2016). Monitoring of the ethanolic extract of the aerial part of B. odorata by TLC and NMR allowed us to find a greater proportion of glycosidic compound (1). It has been described that the metabolites structurally related to compound 1, are antioxidant and have protective function against cardiovascular and coronary diseases, since they have demonstrated ability to decrease LDL oxidation, inhibit lipoperoxidation and suppress the progressive increase of atherosclerotic plaque (Shamala, et al., 2016). It was interesting to know if compound 1 was responsible for lipid-lowering activity by the model induced with Triton WR 1339. Table 3 shows the results of the lipid-lowering effect of compound 1 (EtOH extract) (Supplementary material-Figure A23-A26). Table 3. Effect of the administration of compound 1 on the concentration of TC, C-LDL, HDL-C and TG in male ICR mice treated with tyloxapol for 3 days. The data is represented by means ± se. ANOVA p<0.05 * significant difference vs the hyperlipidemic control, post hoc Tukey's.
Treatment
Compound 1 (mg/kg)
TC (mmol/L)
C-LDL (mmol/L)
C-HDL (mmol/L)
TG (mmol/L)
Normocholesterolemic
-----------
2.88 ± 0.22*
0.96 ± 0.16*
1.48 ± 0.11*
1.24 ± 0.08*
Hypercholesterolemic (Tyloxapol)
-----------
6.12 ± 0.79
3.83 ± 0.76
0.58 ± 0.16
3.77 ± 0.60
25
4.72 ± 0.39
1.9 ± 0.18*
1.35 ± 0.26
3.47 ± 0.71
50
3.62 ± 0.57*
1.07 ± 0.29*
1.52 ± 0.28*
2.24 ± 0.44
100
2.56 ± 0.14*
0.64 ± 0.10*
1.43± 0.09*
1.31 ± 0.11*
Hypercholesterolemic Tyloxapol + Compound 1
3.5 Antimycobacterial Activity One of the common uses of B. odorata reported in traditional medicine is its use as an antitussive agent and to treat lung diseases. Since there are no previous reports in the literature related to the activity of B. odorata on these conditions, present work was undertaken to determine antimycobacterial activity of its different extracts. This study also describes for the first
time the antimycobacterial properties of compound 1 isolated from this species. According to Jimenez-Arellanes et al., 2013, in the ethnobotanical literature the definition of tuberculosis has not been described, even though numerous plant species have been described to treat symptoms related to the disease (such as cough with fever and lung pain, among others). Herein, the results of our antimycobacterial evaluation are shown in Table 4, revealing that hexane, CH2Cl2, EtOAc, and ethanolic extracts having antibacterial activity against Mycobacterium tuberculosis H37Rv; and only the extracts of hexane and dichloromethane had effect against Mycobacterium smegmatis mc2155. On the other hand, the ethanolic extract was subjected to fractionation by selective extractions, from which a purified compound of phenolic nature was obtained that presented an excellent activity with MIC of 3.125 g/mL. It should be mentioned that the aqueous crude extract did not display any antimycobacterial activity, but when it was subjected to fractionation it was possible to isolate 3,5hydroxybenzoic acid (compound 2), which unlike the aqueous extract, did show biological activity against M. tuberculosis H37Rv (Supplementary material-Figure A27). In this context, the antibacterial activity observed with the extracts of less polarity agrees with the results reported by other researchers (Jimenez-Arellanes, et al., 2010). This review includes 75 species of the medicinal flora of Mexico, which were evaluated in in vitro models against mycobacteria, being the extracts of less polarity that showed better bioactivity, mainly due to their lipophilic nature. However, the ethanolic extract and compounds 1 and 2, despite their polar nature, showed outstanding activity against M. tuberculosis H37Rv at a low concentration (Table 4). So far, few compounds with chemical structure similar to compound 1 have been studied for its antimycobacterial activity. For instance, Sashida in 1991 and Wang in 1997 reported the isolation of compound 1,6-di-O-pcoumaroyl--D-glycopyranoside from B. pilosa. This compound presented a mild antimicrobial activity against Klebsiella pneumoniae, Sthaphylococcus aureus and Mycobacterium aurum (125 μg/mL) and slightly better activity against Mycobacterium tuberculosis H37Ra (63 μg/mL). Table 4. a H37Rv: drug-sensitive Mycobacterium tuberculosis. b (1) Hexane extract; (2) CH2Cl2 extract; (3) EtOAc extract; (4) Ethanol extract; (5) Aqueous extract; (6) Compound 1; (7) Compound 2. MIC: minimum inhibitory concentration. The assays with a MIC >50, ˃ 200 µg/mL were considered inactive. MIC 2 (µg/mL) * Extract/Fraction
1b
2
3
4
5
6
7
100
12.5
12.5
12.5
˃ 200
3.125
50
50
100
˃ 200
˃ 200
˃ 200
˃ 50
˃ 50
Microorganism Mycobacterium tuberculosis H37Rv
a
Mycobacterium smegmatis 2
mc 155
4. Conclusions Bidens odorata is a species widely distributed and used in the traditional Mexican medicine, however there are few reports in this plant. In the present study, it was demonstrated that its ethanolic extract and the isolated glycoside compound (1) had an important effect on decreasing TC, LDL-C, and TG and increasing HDL-C, which could be potential lipid-lowering agents. The toxicological results demonstrated that ethanolic extract does not present toxic effects in mice. Extracts of different polarity of the aerial parts of this plant showed antimycobacterial properties, particularly the isolated compound 1, gave MIC of 3.125 μg/mL. These results show that B. odorata can be an excellent source of active molecules with antimycobacterial and hypolipidemic properties. The antimycobacterial activity found in this study contributes to finding
potential natural alternatives for the treatment of tuberculosis, thus alleviating the emerging drug-resistance problem shown by some tuberculosis strains. The above results found in this study supports the documented traditional use of B. odorata. Funding This work was supported by SIP-IPN (20181909 and 20171699), JLH, LGZV, LGS and MEVD are COFAA, EDI and SNI fellows. KMHS was CONACyT (335863) fellow. References Achika, J.I; Gerald, I.; Adedayo, A. 2014. A review on the phytoconstituents and related medicinal properties of plantas in the Asteraceae family. Journal of Applied Chemistry. 7(8),1-8. Argueta, A., Cano, L., Rodarte, M. 1994. Atlas de las plantas de la medicina tradicional mexicana I. Instituto Nacional Indigenísta, Ciudad de México, México. 1786 pp., http://www.medicinatradicionalmexicana.unam.mx/atlas.php (accesed 2017). Assmann, G., Nofer, J., 2003. Atheroprotective effects of high-density lipoproteins. Annu. Rev. Med. 54: 321–341. DOI: 10.1146/annurev.med.54.101601.152409 Beltrán, H. 2016. The Asteraceae (Compositae) from Laraos district (Yauyos, Lima, Peru). Revista Peruana de Biología. 23(2), 195-220. DOI: http://dx.doi.org/10.15381/rpb.v23i2.12439 Digital Library of Mexican Traditional MedicineIndigenous http://www.medicinatradicionalmexicana.unam.mx/index.php (accesed 2017).
Medicinal
Flora
of
Mexico.
2012.
Dimo, T., Azay, J., Tan, P.V., Pellecuer, J., Cros, G., Bopelet, M., Jacques-Serrano, J. 2001.Effects of the aqueous and methylene chloride extracts of Bidens pilosa lea fon fructose-hypertensive rats. Journal of Ethnopharmacology. 76, 215-221. Gutierrez, J.P., Rivera-Dommarco, J., Shamah-Levy, T., Villalpando-Hernandez, S., Franco, A., Cuevas-Nasu, L., Romero-Martinez, M., Hernandez-Avila, M., Gomez-Acosta, L.M., Gaona-Pineda, E.B., Gomez-Humaran, I., Saturno, P., Avila, M.A., Mauricio-Lopez, E.R., Martinez, J., García Lopez, D.E. Encuesta Nacional de Salud y Nutricion 2016. Resultados Nacionales. Cuernavaca, Mexico: Instituto Nacional de Salud Publica (MX), 2016. Friedewald, W.T., Levy, R.I., Fredrickson, D.S. 1972. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifugate. Clinical Chemistry. 8(6), 499-502. García-Regalado, G. Plantas Medicinales de Aguascalientes. Universidad Autónoma de Aguascalientes. 2015, 498 pp., http://www.uaa.mx/direcciones/dgdv/editorial/docs/plantas_medicinales_aguascalientes.pdf (accesed 2017). http://www.medicinatradicionalmexicana.unam.mx/flora2.php?l=4&t=Aceitilla&po=tepehuan_del_sur&id=6126&clave_region=13 (accesed 2017). Garduño, L., Salazar, M., Salazar, S., Morelos, M.E., Labarrios, F., Tamariz, J., Chamorro, G.A. 1997. Hypolipidaemic activity of αasarone in mice. Journal of Ethnopharmacology. 55,161-163. DOI: https://doi.org/10.1016/S0378-8741(96)01492-4 González, E. M., López, E. I. L., González, E. M.S., Tena, F. J. A. 2004. Plantas medicinales del Estado de Durango y Zonas aledañas. CIIDIR-Durango, Instituto Politécnico Nacional. 144pp. Hisashi, M., Takeshi, C., Yasushi, K., Johji, Y., Tokunosuke, S., Hajime, F. Hitoshi, K. 1986. Effects of Crude Drugs on Experimental Hypercholesterolemia. I. Tea and Its Active Principles. Journal of Ethnopharmacology. 17, 213-224. DOI: https://doi.org/10.1016/03788741(86)90110-8 Jiménez, A. A., Cornejo, G.J., León, D. R. 2010. Las plantas mexicanas como fuente de compuestos antimicrobianos. Revista Mexicana de Ciencias Farmacéuticas. 41(1), 22-29. Jimenez-Arellanes, A., Meckes, M., Ramírez, R., Torres, J., Luna-Herrera, J. 2003. Activity against Multidrug-resistant Mycobacterium tuberculosis in Mexican Plants Used to Treat Respiratory Diseases. Phyther Resp. 17, 903-908. DOI: 10.1002/ptr.1377 Jiménez-Arellanes, M.A., Yépez-Mulia, L., Luna-Herrera, J., Gutiérrez-Rebolledo, G.A., García-Rodríguez, R.V. 2013. Actividad antimicobacteriana y antiprotozoaria de Moussonia deppeana (Schldl and Cham.) Hanst. Revista Mexicana de Ciencias Farmacéuticas. 42 (2), 31-35. Liang, Y.C., Yang, M.T., Lin, C.J., Chang, C.L.T., Yang, W.C. 2016. Bidens pilosa and its active compound inhibit adipogenesis and lipid accumulation via down-modulation of the C/EBP and PPARγ pathways. Sci. Rep. 6, 24285. DOI: 10.1038/srep24285. Martínez, M. 1996. Las plantas medicinales de México. 7ª Reedición, México. 656 pp. Meléndez- Camargo, M. E., B., Berdeja, and G., Miranda. 2004. Diuretic effect of the aqueous extract of Bidens odorata in the rat. Journal of Ethnopharmacology. 95 (2-3), 363–366. DOI: 10.1016/j.jep.2004.08.005
Mendieta, A., Jiménez, F., Garduño-Siciliano, L., Mojica-Villegas, A., Rosales-Acosta, B., Villa-Tanaca, L., Chamorro-Cevalllos, G., Medina-Franco, J., Meurice, N., Gutiérrez, R., Montiel, L., Cruz, M.C., Tamariz, J. 2014. Synthesis and highly potent hypolipidemic activity of alpha-asarone- and fibrate -based 2-acyl and 2-alkyl phenols as HMG-CoA reductase inhibitors. 22, 5871-5882. DOI: http://dx.doi.org/10.1016/j.bmc.2014.09.022 Moreno-Pena, D.P., Cordero-Pérez, P., Leos-Rivas, C., Bucio, L., Viveros-Valdez, J.E., Muñoz-Espinosa, L.E., Galindo-Rodríguez, S.A., Rivas-Morales, C. 2017. Evaluation of hypocholesterolemic activity of extracts of Bidens odorata and Brickellia eupatprioides. Pak. J. Pharm. Sci. 2, 613- 617. Norma Oficial Mexicana 062-ZOO-1999. Technical specifications for the reproduction, care and use of laboratory animals. Revised in 2017. Organization for Economic Co-operation and Development (OECD). 2008. Guidelines for the Testing of Chemicals. Assay TG 423. Revised in 2017. Pahua-Ramos, M.E., Ortiz-Moreno, A., Chamorro-Cevallos, G., Hernández Navarro, M.D., Garduño-Siciliano, L., NecoecheaMondragón,H., Hernández-Ortega, M. 2012. Hypolipidaemic Effect of Avocado (Persea americana Mill) Seed in a Hypercholesterolemic Mouse Model. Plants Food Hum Nutr. 67, 10-16. DOI: 10.1007/s11130-012-0280-6 Rzedowski, G., C. de, J. Rzedowski. 2005. Flora fanerogámica del Valle de México. 2a. ed., 1a reimp., Instituto de Ecología, A.C. y Comisión Nacional para el Conocimiento y Uso de la Biodiversidad, Pátzcuaro (Michoacán). 1406 pp. Sashida, Y., Ogawa, K., Kitada, M., Karikome, H., Mimaki, Y., Shimomura, H. 1992. New aureone glucosides and new phenylpropanoid glucosides from Bidens pilosa. Chem. Pharm. Bull. 39, 709-711. DOI: https://doi.org/10.1248/cpb.39.709 Shamala, S., Baskaron, G., Mohd, Y.S., Zuki, A.B. 2016. Phytochemical investigation, hypocholesterolemic and anti-atherosclerotic effects of Amaranthus viridis leaf. RSC Adv. 6, 32685-32696. DOI: 10.1039/C6RA04827G Secretaría de Salud. 2014. Prevención y Control de la Tuberculosis: Programa Sectorial de Salud 2013-2018. México. Vibrans, H. 1995. Bidens pilosa L. y Bidens odorata Cav. (Asteraceae: Heliantheae) en la vegetación urbana de la Ciudad de México. Acta Botánica Mexicana. 32, 85-89. Wang, J., Yang, H., Lin, Z.W., Sun, H.D. 1997. Flavonoids from Bidens pilosa var. radiata. Phytochemistry. 46, 1275- 1278. DOI: https://doi.org/10.1016/S0031-9422(97)80026-X WHO. 2017. Tuberculosis [Internet]. Available from: http://www.who.int/mediacentre/factsheets/fs104/es/ (Accesed, 2017). Zavala-Mendoza, M., Alarcón-Aguilar, F.J, Pérez-Gutiérrez, S., Escobar-Villanueva, M.C., Zavala-Sánchez, A. 2013. Composition and Antidiarrheal Activity of Bidens odorata Cav. Evidence- Based Complementary and Alternative Medicine. 1, 1-7. DOI: 10.1155/2013/170290