Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2018, Article ID 3610364, 11 pages https://doi.org/10.1155/2018/3610364

Research Article In Vitro Bioactivity of Methanolic Extracts from Amphipterygium adstringens (Schltdl.) Schiede ex Standl., Chenopodium ambrosioides L., mexicanum DC., Eryngium carlinae F. Delaroche, and Pithecellobium dulce (Roxb.) Benth. Used in Traditional Medicine in

Peter Knauth ,1 Gustavo J. Acevedo-Hernández,2 M. Eduardo Cano,3 Melesio Gutiérrez-Lomelí,4 and Zaira López 1

1 Cell Biology Laboratory, Centro Universitario de la Cienega,´ Universidad de Guadalajara, Av. Universidad 1115, 47810 Ocotlan,´ JAL, Mexico 2Laboratorio de Biolog´ıa Molecular Vegetal, Centro Universitario de la Cienega,´ Universidad de Guadalajara, Av.Universidad1115,47810Ocotlan,´ JAL, Mexico 3Laboratorio de Biof´ısica, Centro Universitario de la Cienega,´ Universidad de Guadalajara, Av. Universidad 1115, 47810 Ocotlan,´ JAL, Mexico 4Laboratorio de Alimentos, Centro Universitario de la Cienega,´ Universidad de Guadalajara, Av. Universidad 1115, 47810 Ocotlan,´ JAL, Mexico

Correspondence should be addressed to Zaira Lopez;´ [email protected]

Received 29 November 2017; Accepted 29 January 2018; Published 28 February 2018

Academic Editor: Gloria Brusotti

Copyright © 2018 Peter Knauth et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Seven out of eight methanolic extracts from fve native to Mexico were inactive against ten bacterial strains of clinical interest. Te fruit extract of Chenopodium ambrosioides inhibited the bacteria Enterococcus faecalis (MIC = 4375 �g/ml), Escherichia coli (MIC = 1094 �g/ml), and Salmonella typhimurium (MIC = 137 �g/ml). Te fruit extract of C. ambrosioides was with CC50 =45�g/ml most cytotoxic against the cell-line Caco-2, followed by the leaf extract from Pithecellobium dulce (CC50 =126�g/ml); interestingly, leaves of C. ambrosioides (CC50 =563�g/ml) and bark of P. du l ce (CC50 =347�g/ml) extracts were much less cytotoxic. We describe for the frst time the cytotoxic efect from extracts of the aerial parts and the fowers of Cirsium mexicanum (CC50 =323�g/ml and CC50 =250�g/ml, resp.). Phytochemical analysis demonstrated for both extracts high tannin and saponin and low favonoid content, while terpenoids were found in the fowers. For the frst time we report a cytotoxicological study on an extract of Eryngium carlinae (CC50 =356�g/ml) and likewise the bark extract from Amphipterygium adstringens (CC50 =342�g/ml). In conclusion the fruit extract of C. ambrosioides is a potential candidate for further biological studies.

1. Introduction medicinal plants have been studied in order to discover pure compounds that show any of the aforementioned biological Plants are well known as a rich source of bioactive com- activities. pounds with a wide range of pharmaceutical applications, for Most of the antibiotics used in clinical and veterinary instance, neuro- or psychoactive compounds (e.g., atropine, medicine are from microbial origin (bacteria, fungi) and only cafeine, cocaine, and morphine), substances with anti- a few of them are synthetic. However, since the end of the infammatory (e.g., acetylsalicylate) or cardiovascular efects “GoldenAgeofAntibioticDiscovery”inthe80softhelast (e.g., digoxin), or anticancer activity (e.g., taxol, vinblas- centuryonlyasmallnumberofnewantibioticshaveemerged, tine). Hence, over the last few decades raw extracts from while on the other hand more and more antibiotics have lost 2 Evidence-Based Complementary and Alternative Medicine

Table 1: Fractions of the plants employed and concentration of the methanolic extracts. Previously reported biological activity of extract and their reference. Plant sample Plant fraction Action Reference Voucher Amphipterygium adstringens Bark Antibacterial [10, 24–26] (cuachalalate) CBM Cytotoxic [24, 27] CBL201701 80 mg/ml Proliferation [2] Antiprotozoal [25, 28–30] Chenopodium ambrosioides Leaf Fruit Antifungal [31] (epazote) ELM ESM Antibacterial [32] CBL201702 20 mg/ml 20 mg/ml Cytotoxic [33] Not cytotoxic [9] Cirsium mexicanum Flower Aerial parts Anticancer [14] (cardosanto) CsFM CsM Antidiabetic [14] CBL201703 48 mg/ml 40 mg/ml

Eryngium carlinae Aerial parts Antidiabetic [30] (hierba del sapo) HsM CBL201704 50 mg/ml

Pithecellobium dulce Bark Leaf Antibacterial [34] (guamuchil)´ GBM GLM Cytotoxic [1] CBL201705 20 mg/ml 20 mg/ml Not cytotoxic [1]

activity due to the rise of acquired resistance against them [1]. in Mexico as an infusion for its antiparasitic activity and In 2005, the World Health Organization (WHO) developed a other positive efects on the gastrointestinal tract [11]: among list of Critically Important Antimicrobials (CIA) for human its components, especially ascaridole and hydroperoxide medicine and published a global action plan to address derivates from monoterpenes with two double bonds exhibit this problem [2]. Terefore, now other niches (e.g., marine antiprotozoal activity (Table 1). Moreover, its essential oils environment) and sources (e.g., animals or plants) are being have antifungal activity; and methanol or petroleum ether investigated to discover new classes of antibiotics [3]. Mexico extracts of the bark or fowers have antibacterial efects possesses a very high plant biodiversity with about 26,000 (Table 1). Tere are, however, contradictory reports on cyto- species already identifed [4], and an estimated 4,500 plants toxic efects of its extracts (Table 1). Additionally, moder- are used for medicinal purposes [5]; but in vitro antibacterial ate antioxidant and anti-infammatory properties could be properties have been reported for only 343 plant species attributed to the chenopodiumamines A to C isolated from [6]. Although many phytochemicals (e.g., eugenol, menthol, ethanolic extracts [12]. Using an animal model, ethanolic thymol, and linalool) or plant peptides with antimicrobial extracts of leaves or stems exhibited anti-infammatory, activity are known, those molecules are normally not used antinociceptive, and healing efects, which validate its poten- as antibiotics. Moreover, among those medicinal plants from tial therapeutical use [13]. Mexico, only 300 of them have documented use in anticancer Cirsium mexicanum DC., known as “Mexican thistle” treatment: 181 are supported by scientifc evidence and 119 or “cardosanto,” is empirically employed by the Mexican have been used empirically [7]. population to treat cancer or diabetes [14], but there is no Amphipterygium adstringens (Schltdl.) Schiede ex Standl., scientifc contribution about the biological activity of this commonlyknowninMexicoascuachalalate,iswidelyusedas plant. a medicinal plant, especially to treat ulcers, cancer, gastritis, Eryngium carlinae F. Delaroche, known in Mexico as and other gastrointestinal disorders as well as promote “hierba del sapo,” is ofen used in traditional medicine to treat wound healing [8]. Extracts from the bark of this plant have diabetes. Although an ethanolic extract of the whole plant of antibacterial activity, which is attributed to a mixture of E. carlinae didnotlowerbloodglucoselevelsindiabeticrats, anacardic acids (Table 1). At a concentration of 10–40 �M a signifcant decrease of creatinine, urate, total cholesterol, those anacardic acids were cytotoxic to several cell lines, and triglycerides could be demonstrated, which may lower while other authors reported proliferative efects (Table 1). theriskofrenalandcardiovascularcomplicationscausedby Moreover, extracts containing masticadienonic acid and its diabetes mellitus [15]. Additionally, a methanolic extract from 3�-hydroxy-derivative exhibited in rats antiulcer activity [9] E. carlinae inhibits growth of Helicobacter pylori [16]. and anti-infammatory efects [10]. Finally, Pithecellobium dulce (Roxb.) Benth. is autoch- Chenopodium ambrosioides L., also known as “worm- thonous to Mexico and , where it is known seed,” “Jesuit’s tea,” or “epazote,” has been empirically used as “guamuchil”´ or “huamuchil,”´ but nowadays it can also be Evidence-Based Complementary and Alternative Medicine 3 found in Southeast Asia and India, where it is known as sequences. Tis method has been tested in numerous plant “Madras thorn.” In Mexico, the plant is traditionally prepared species and has been proposed as an alternative for the as aqueous infusions or ethanolic extracts from diferent parts authentication of medicinal plants [35, 36]. First, plant pow- of the tree: from leaves to alleviate pain and convulsions and der was obtained as described above, and 100 mg from each combined with salt to cure indigestion and from bark for sample was used for DNA isolation using the PureLink Plant treatment of dysentery or fever [17]. In rats, an ethanolic Total DNA Purifcation Kit (Invitrogen, Carlsbad, USA), seed extract has protective efects against ulceration [18] following the protocol suggested by the manufacturer. To test � andantidiabeticactivity[19],whileanaqueousleafextract the quality of isolated DNA, a 5 lsamplewasrunona0.8% shows antihyperlipidemic activity [20]. A hydroethanolic agarose gel stained with EvaGreen (Jena Bioscience, Jena, extract of the bark exhibited antimicrobial activity (Table 1). Germany). Quantifcation of DNA was achieved by UV spec- Recent cytotoxicological studies revealed cytotoxic efects of trophotometry using a NanoDrop 2000 Spectrophotometer amethanolicleafextractontheprostatecancercelllineCaP, (Termo Fisher Scientifc, Waltham, USA). Primers and PCR conditions were as those reported by the Consortium for whilenoefectonthebreastcancercelllineMCF-7was Barcode of Life (CBOL) Plant Working Group [35]. Primers observed [21]. � used for amplifcation of matK gene were 3F-KIM-F (5 - When plants are selected on the basis of their use in � CGT ACA GTA CTT TTG TGT TTA CGA G-3 )and1R- traditional medicine, the frst extraction step to obtain a raw � KIM-R (5 -ACC CAG TCC ATC TGG AAA TCT TGG extract should mimic the extraction process as described � TTC-3 ), and for amplifcation of rbcL gene primers rbcLa- by the traditional healer [22]. In an initial screening for � F(5-ATG TCA CCA CAA ACA GAG ACT AAA GC- biological activity, the concentration ranges usually from 10 � � 3 )andrbcLa-R (5 -GTAAAATCAAGTCCACCRCG- to 10,000 �g/ml, but only raw extracts with a half maximal � ≤ � 3 ) were used. Amplifcation reactions were performed in a efective concentration EC50 100 g/ml are considered fnal volume of 20 �l containing 20 mM Tris-HCl (pH 8.4), active [23] and subjected to further analysis. 50 mM KCl, 2 mM MgCl2, 0.2 mM dNTPs mix, 1 �M each Of the fve plant species included in the present study, two primer, 10 ng of genomic DNA and 2 units of Taq DNA have not been extensively studied scientifcally and for the polymerase (Jena Bioscience, Jena, Germany). Amplifcations remaining three plants the published literature shows con- were performed in a Multigene Termocycler (Labnet Inter- ∘ tradictory results concerning their cytotoxicity. Terefore, we national, Edison, USA) using the program: 1 step at 94 Cfor ∘ ∘ considered these fve plants, which are all endemic to Mexico, 5 min, 35 cycles consisting of 30 s at 94 C, 1 min at 52 C, and ∘ ∘ as good candidates for assessment of their in vitro cytotoxicity 1minat72 C and a fnal extension step of 5 min at 72 C. using the human epithelial colorectal adenocarcinoma cell Amplifcation products were separated by electrophoresis line Caco-2, a standard model to discover novel anticancer in 0.8% agarose gels and stained with EvaGreen. Te PCR agents in colon cancer. Additionally, their antimicrobial productswerepurifedusingtheGenElutePCRClean-UpKit activity, useful to discover novel agents to prevent and treat (Sigma-Aldrich, St. Louis, USA) following the manufacturer’s nosocomial infections, was evaluated. instructions. Te DNA concentration of the samples was again estimated using a NanoDrop Spectrophotometer and 2. Methodology PCR products were sent for sequencing to the Genomic Ser- vices Laboratory at Langebio-Cinvestav (Irapuato, Mexico). 2.1. Plant Material and Extraction. Dried bark of A. adstrin- DNA sequences were used for plant identifcation through gens wasboughtatalocalmarketinOcotlan´ (, the Identifcation Engine at the Barcode of Life Data System Mexico), while the samples of C. ambrosioides, C. mexicanum, (http://www.boldsystems.org) and were submitted to the E. carlinae, and P. du l ce were collected in “La Cienega”´ region public sequence repository GenBank. (Jalisco, Mexico) between June and July. All fresh material was delivered to the laboratory and dried at room temper- 2.3. Phytochemical Characterization. Only plants or parts ature (RT) by ventilation for 10 d. Aferwards the collected of them, which have not been analysed previously, were material was separated according to plant parts (Table 1). phythochemically characterized [37]: fne plant powder was Each part was ground (electrical grinder) and passed through suspended at 100 mg/ml in distilled water with shaking a 30 mesh sieve to get a fne powder. Ten 10 g of plant powder (80 rpm) overnight at RT in the dark and subsequently cen- from each sample was macerated with 100 ml methanol by trifuged (2 min, 10,000 ×g). shaking at 80 rpm for 24 h at RT. Next, the macerates were � fltered, frst through Whatman flter paper #4 and then Flavonoids.1mlsupernatant,dilutedwith900 ldistilled through a Nylon membrane 0.45 �mporesize.Finally,4ml water, was mixed with 5 ml 1% NH4Clandthenfltered. of the fltrates was distributed into amber vials, the solvent Aferwards, 5 drops of concentrated H2SO4 were added. was evaporated using a nitrogen evaporator (Mini-Vap, CRS, An unstable yellow colour is formed in the presence of fa- Louisville, USA), the vial was weighed, and the precipi- vonoids. tate was resuspended in 1 ml methanol. All samples were ∘ Tannins.1mlsupernatantwasboiledfor2minandthen5 stored at 4 Cuntiluse. drops of 0.1% FeCl3 were added. A brownish green or blue black colour indicates the presence of tannins. 2.2. Molecular Identifcation through DNA Barcoding. DNA barcoding is a molecular method which allows the rapid, Terpenoids.100�l methanolic plant extract, diluted with 5 ml accurate, and automatic identifcation of plants using DNA methanol, was mixed with 2 ml dichloromethane and then 4 Evidence-Based Complementary and Alternative Medicine

Table 2: Qualitative phytochemical analysis of 4 studied extracts: ++, present; +, slightly present; and −, not present.

Extract Flavonoids Tannins Terpenoids Saponins ESM ++ ++ ++ − CsM + ++ + ++ CsFM + ++ ++ ++ HsM + ++ + ++

3mlconcentratedH2SO4 was added. Afer 20 min a stable 2.6. Cell Viability and Necrosis Induction. To determine the layer was formed and a reddish-brown colour in the interface metabolic activity the old medium was removed, the cells indicates positive results for terpenoids. were washed twice with phosphate bufered saline (PBS), and 100 �l medium containing 2 �l WST-1 was added. Afer Saponins.5mlsupernatantwasboiledfor2min,dilutedwith incubating for 4 h absorbance at 440 nm was measured with distilled water, and shaken to form foam; then 3 drops of olive an ELISA-reader (Multiskan FC). Neutral red uptake (NRU) oilwereaddedandagainshakenvigorously.Teformationof andtrypanblueassaysweremadein12-wellMTPand an emulsion indicates the presence of saponins. thecellsweregrownoncoverslides.Aferthelast24hof incubation 20 �l of 0.33% neutral red solution (Santa Cruz, 2.4. Antimicrobial Assays. Te following microorganisms Santa Cruz, USA) was added into each well and incubated were acquired from ATCC: Bacillus cereus #49064, Enterococ- for additional 4 h. Aferwards, the cover slides were washed cus faecalis #29212, Klebsiella pneumoniae #10031, Salmonella once with PBS and observed under the microscope (Axioskop typhimurium #29630, Streptococcus pyogenes #19615 and the 40FL, Zeiss, Oberkochen, Germany). To detect necrosis, following are obtained from the Microbiology Laboratory 0.05% (fnal concentration) trypan blue (Biowest) was added collection (CUCI, University of Guadalajara): Escherichia andincubatedfor5to10minandthecoverslideswere coli, Proteus mirabilis, Pseudomonas aeruginosa, Serratia washed with PBS and then observed under the microscope marcescens, and Staphylococcus aureus [38]. Agar plates with (Zeiss Axioskop 40FL). All tests were done in triplicate. Lysogeny Broth (LB; MP Biomedicals, Santa Ana, USA) or Muller-Hinton¨ (MH; MP Biomedicals, Santa Ana, USA) were ± 7 2.7. Statistical Analysis. Values are expressed as means 1.96 inoculated with 10 cfu of the model organism. Sterile sensi- ∗ standard error of mean (1.96 ∗ SEM). Diferences between discs (6 mm diameter) were impregnated with 2 �land10�l groups were determined by one-way analysis of variance extract (20–80 mg/ml), 2 �l chloramphenicol (Cm, 25 mg/ml; (ANOVA) and subjected post hoc to Tukey’s HSD multiple Sigma-Aldrich, St. Louis, USA) was used as positive control, comparison tests using the program Origin 5.0 (Microcal and 10 �l methanol was used as mock control. Afer their �< ∘ Sofware, Northampton, USA). A value of 0.05 was con- incubation at 37 C for 24 h the diameter of the inhibition sidered to indicate statistical signifcance. Te half maximal zone was measured (Kirby-Bauer method). For extracts with cytotoxic concentration (CC50) was calculated by nonlinear positive results the minimal inhibitory concentration (MIC) curve ft with dose-response function using the program was determined using 96-well microtiter plates (MTP): all Origin 5.0. wells contained 50 �lmediumandwereseriallydiluted � starting with 50 l extract (or control) in the frst row; fnally 3. Results all wells were inoculated with 50 �lmediumadjustedto0.5 8 McFarland (approx. 10 cfu/ml). Te MTPs were incubated at ∘ 3.1. Phytochemical Analysis. Methanolic extracts from difer- 37 Cfor24handthegrowthwasmeasuredbyabsorptionat ent parts of A. adstringens, C. ambrosioides, C. mexicanum, E. 625 nm with an ELISA-reader (Multiskan FC, Termo Fisher carlinae, and P. du l ce were prepared at concentrations ranging Scientifc, Waltham, USA). All tests were done six times. from 20 to 80 mg/ml and designated by the abbreviations shown in Table 1. 2.5. Cell Line and Culturing Conditions. Te cell line Caco- As many reports on the composition of A. adstringens, 2 (ATCC, Manassas, USA) was grown in DMEM/F12 (Cais- C. ambrosioides, and P. du l ce arealreadypublishedelsewhere, son, Smithfeld, USA) supplemented with 10% FBS (Biow- those were not analysed here. Tus, the phytochemical com- ∘ est, Kansas City, USA) at 37 C, 4% CO2,and95%RH. position of four extracts that have not yet been characterized Experiments were carried out in 96-well MTP containing was determined qualitatively (Table 2). Tis analysis revealed 4 100 �l medium inoculated with 10 cells. Afer 24 h incuba- that the fruits of C. ambrosioides do not contain saponins. tion for cell attachment 100 �l of methanolic plant extract (40–60 mg/ml) or control (methanol or H2O2) was added 3.2. Molecular Identifcation. All plants collected in the and serially diluted 1 : 2. Aferwards, the cells were incubated wild were identifed through PCR amplifcation and DNA for additional 24 h before the analysis. For qualitative assays sequencing of regions of rbcL and matK plastid genes, which 5 1.5 ∗ 10 cellsweregrownin1mlmediumin12-wellMTP havebeenproposedasthecoreplantbarcode[35].For on cover slides and otherwise using the same culturing rbcL fragments, the DNA sequence length was between 527 conditions. and 529 bases, while the sequence length of matK was Evidence-Based Complementary and Alternative Medicine 5

Table 3: Molecular identifcation of the collected medicinal plants through DNA barcoding.

Plant sample DNA barcode GenBank ID Top scores % similarity D. ambrosioides 100.00 rbcL MG436873 D. multifda 100.00 Chenopodium ambrosioides C. album 99.62 (Dysphania ambrosioides) D. ambrosioides 100.00 matK MG572013 C. acuminatum 99.88 D. multifda 99.74 C. mexicanum 100.00 rbcL MG436872 C. undulatum 100.00 Cirsium mexicanum C. scariosum 100.00 C. discolor 100.00 matK MG572012 C. altissimum 100.00 C. horridulum 100.00 E. aristulatum 99.81 rbcL MG436874 E. pendletonense 99.81 Eryngium carlinae E. leavenworthii 99.62 matK No data No data No data P. du l ce 100.00 rbcL MG436875 P. lanceolatum 99.43 Pithecellobium dulce Acacia centralis 99.43 P. du l ce 100.00 matK MG572014 P. lanceolatum 100.00 Havardia mexicana 99.25

Table 4: Antibacterial activity of methanolic extract from Chenopodium ambrosioides fruits (ESM) compared to chloramphenicol (Cm). Values expressed as mean ± 1.96 ∗ SEM (� = 3) of the inhibition halo in mm.

Zone of growth inhibition [mm] Statistical Microorganism ESM (10 �l, 70 mg/ml) Cm (2 �l, 50 mg/ml) signifcance Enterococcus faecalis 13.3 ± 2.4 22.0 ± 1.1 � = 0.003 −4 Salmonella typhimurium 18.0 ± 1.1 37.3 ± 3.5 � = 4.8 ∗ 10 −4 Escherichia coli 12.0 ± 0.6 26.3 ± 2.4 � =3.2∗ 10 between 807 and 813 bases. For similarity searches, these Table 5: Minimal inhibitory concentration (MIC) for methanolic sequences were submitted to the Barcode of Life Data Systems extract from Chenopodium ambrosioides fruits (ESM) compared to (http://www.boldsystems.org/) using the Plant Identifcation chloramphenicol (Cm). tool and plant species showing that the top matching scores Minimal inhibitory concentration are depicted in Table 3 along with the corresponding Gen- Microorganisms (MIC) [�g/ml] Bank accession numbers for the DNA sequences obtained in ESM Cm this work. Despite several attempts, the matK primers failed to amplify the DNA from E. carlinae; thus identifcation of Enterococcus faecalis 4375 6 this plant could only be achieved using rbcL sequence. It must Salmonella typhimurium 137 6 be noticed, however, that at the moment neither the matK nor Escherichia coli 1094 6 the rbcL sequences for E. carlinae areavailableattheBarcode of Life Data System, and hence the best matches were closely related species. A. adstringens has been identifed earlier [38]. lower activity than a pure antibiotic (Table 4). Hence, only these combinations were subjected to a MIC determination 3.3. Antimicrobial Activity. In the screening for antimicrobial (Table 5). None of the other extracts showed any efect on the activity by the Kirby-Bauer method only the fruit extract tested bacterial strains. from C. ambrosioides caused a clear zone of inhibition against the three strains of E. faecalis, S. typhimurium, and 3.4. Cytotoxicity. Te cytotoxicity of the extracts on the E. coli; as expected, the raw extract ESM had a signifcant enterocyte cell line Caco-2 was estimated by the reduction 6 Evidence-Based Complementary and Alternative Medicine

140 140 ∗ 120 ∗ 120

100 100

80 ∗ 80 ∗ ∗ ∗ ∗ 60 ∗ 60 ∗ Relative activity Relative Relative activity Relative 40 40 ∗ ∗ ∗ ∗ ∗ ∗ 20 ∗ 20 ∗ ∗ ∗ ∗ ∗ ∗ 0 0 0.000 0.001 0.010 0.100 1.000 0.000 0.001 0.010 0.100 1.000 Concentration (mg/ml) Concentration (mg/ml) CBM CsFM ELM GLM HsM CsM ESM GBM Figure 1: Cytotoxicity of methanolic extracts on the cell line Caco- Figure 2: Cytotoxicity of methanolic extracts on the cell line Caco- 2: bark of A. adstringens (CBM, square), whole plant of E. carlinae 2: leaf (ELM, up triangle) and fruit (ESM, circle) of C. ambrosioides (HsM, down triangle), and fower (CsFM, circle) and whole plant as well as leaf (GLM, down triangle) and bark (GBM, square) of P. (CsM, up triangle) of C. mexicanum. Error bars indicate 1.96 ∗ SEM dulce. Error bars indicate 1.96 ∗ SEM (�=6). ∗ indicates statistically (�=6); ∗ indicates statistically signifcant diference to control and signifcant diference to control and to the next smaller value (�< to the next smaller value (� < 0.05). 0.05).

of CsFM confuence was clearly reduced and several cells of the tetrazolium salt WST by dehydrogenase activity. Most werestainedbluebyTBSwhilebyNRUthecellswerenot of the tested methanolic plant extracts had a cytotoxic stained or only slightly red (not shown). Te extract HsM ≥ � concentration of CC50 250 g/ml, which are considered at 500 �g/ml did not cause observable cellular alterations; low cytotoxicity values: the bark of A. adstringens (CBM) that is, the confuence did not decrease and the cells had with CC50 = 342 ± 38 �g/ml, fowers (CsFM) and whole plant functional membranes (not shown) and functional lysosomes (CsM) of C. mexicanum with CC50 = 250±55 �g/ml and with (red by NRU, Figure 3(d)), similar to the negative control CC50 = 323±93 �g/ml, respectively, and the whole plant of E. (Figure 3(e)). At 1,500 �g/ml of HsM the red staining of the carlinae (HsM) with CC50 = 356±114 �g/ml (Figure 1). While cells by NRU was fainter (not shown) but at 3,000 �g/ml of for P. du l ce the bark extract (GBM) was not cytotoxic either HsM the confuence was clearly reduced and the cells were with CC50 = 347 ± 78 �g/ml, its leaf extract (GLM) exhibited notstainedredbyNRUbutbluebyTBS(bothnotshown). frst signs of cytotoxicity with a CC50 = 126 ± 14 �g/ml. Tis Even though 500 �g/ml of ELM reduced the confuence contrast between diferent parts of a plant was even more of Caco-2 strongly, the cells were disintegrated and the pronounced for the C. ambrosioides extracts: while the leaf lysosomes were not stained by NRU (Figure 4(a)), while at extract (ELM) was the least cytotoxic with CC50 = 563 ± 250 �g/ml of ELM only the confuence was reduced but the 66 �g/ml, the fruit extract (ESM) was the most cytotoxic one lysosomes were still functional (not shown). In contrast, the with CC50 = 45 ± 7 �g/ml (Figure 2). extract from its fruits (ESM) at 20 �g/ml reduced slightly con- Tese results were confrmed qualitatively by neutral red fuence; at 50 �g/ml of ESM confuence was clearly reduced uptake (NRU), where functional lysosomes are stained red and the cells were deformed and barely stained by NRU and trypan blue staining (TBS), where necrotic cells with (Figure 4(b)) and many cells were stained blue by TBS (not disrupted membranes are stained blue. At 400 �g/ml of CBM, shown).Athigherconcentrations(≥100 �g/ml ESM) only Caco-2 cells started to show irregular shapes, although the cell debris could be observed (not shown; similar to the confuence was not reduced and most cells still had functional positive control Figure 3(f)). Tis cytotoxic efect was already lysosomes (i.e., red by NRU, Figure 3(a)) and membranes. At seen afer 4 h of exposure (not shown). GBM at 400 �g/ml 800 �g/ml of CBM the confuence was clearly reduced and diminished confuence and the cells were not stained red by the cells had only a faint red staining by NRU (not shown) NRU (Figure 4(c)); GLM at 100 �g/ml diminished lysosomal andmostwerebluebyTBS(notshown).CsMat400�g/ml functionality only slightly (Figure 4(d)) and cells were not causednoobservablechangesinconfuence,cellshape,mem- stained blue by TBS (not shown), but already at 200 �g/ml brane functionality (not shown), and lysosome functionality of GLM the cell membrane was disrupted (not shown) and (Figure 3(b)) and even at a much higher concentration of lysosomes were not functional anymore (not shown). 2,000 �g/ml of CsM most cells were still stained red by NRU, although the confuence was clearly reduced (not shown). Te 4. Discussion extract CsFM at 480 �g/ml caused no observable changes in confuence, cell shape, membrane functionality (not shown), Te methanolic extracts were evaluated against ten bacte- and lysosome functionality (Figure 3(c)); but at 1,200 �g/ml rial strains; three of them were Gram-positive and seven Evidence-Based Complementary and Alternative Medicine 7

(a) (b)

(c) (d)

(e) (f)

Figure 3: Neutral red uptake of Caco-2 afer 24 h exposure to diferent methanolic extracts: (a) bark of A. adstringens (400 �g/ml CBM); (b) whole plant of C. mexicanum (400 �g/ml CsM); (c) fowers of C. mexicanum (480 �g/ml CsFM); (d) whole plant of E. carlinae (500 �g/ml HsM). (e) Methanol as negative control and (f) H2O2 as positive control.

Gram-negative and all of them are of clinical interest. Te cell of the solvent, it is reasonable that those lipophilic anacardic line used was Caco-2 (human epithelial colorectal adenocar- acids are the cytotoxic principle for the cell line Caco-2 as cinoma) in order to elucidate possible antitumoral efects. well. Also Rodriguez-Garcia et al. (2015) found antiprolifer- ative efects by a methanolic bark extract (CBM) on human Amphipterygium adstringens. Te methanolic raw extract of cell lines like OVCAR-3, UACC-62, HT-29, PC-3, U251, NCI- the bark of A. adstringens (CBM) was cytotoxic for the cell H460, and 786-O ranging from 4.4 to 28.0 �g/ml [41]. On line Caco-2 at a concentration of CC50 = 342 ± 38 �g/ml, the other hand, Xiu et al. (2014) showed that anacardic which is in a similar range of observed cytotoxic efects of an acids promote proliferation of ovarian cancer cells, inhibit aqueous (CBW) and an ethanolic bark (CBE) extract on HeLa late apoptosis, and induce cell migration by lamellipodia with a CC50 =467�g/ml and CC50 =202�g/ml, respectively formation [42]. Surprisingly, our CBM extract did not inhibit [38]. It was previously shown that the bark contains anacardic bacterial growth, although we found that an ethanolic extract acids [39], and those compounds were cytotoxic to several (CBE) inhibited the growth of B. cereus (150 �g/ml), E. faecalis cell lines at concentrations of 10–40 �M(≈3.4–13.6 �g/ml) (660 �g/ml), and S. aureus (1,380 �g/ml) [38]. Canales et al. [27, 40]; as the cytotoxicity increased with decreasing polarity (2005) reported that an methanolic extract inhibited Sarcina 8 Evidence-Based Complementary and Alternative Medicine

(a) (b)

(c) (d)

Figure 4: Neutral red uptake of Caco-2 afer 24 h exposure to diferent methanolic extracts: (a) leaves from C. ambrosioides (500 �g/ml ELM); (b) fruits from C. ambrosioides (50 �g/ml ESM); (c) bark of P. du l ce (400 �g/ml GBM); (d) leaves from P. du l ce (100 �g/ml GLM).

lutea (125 �g/ml), S. aureus (250 �g/ml), and B. subtilis known that they contain mainly saponins, favonoids, and (1,500 �g/ml) [43], although Rodriguez-Garcia et al. (2015) essential oils [45]; here we report for the frst time a qualitative reported only an efective inhibition of Streptococcus mutans phytochemical analysis for the species E. carlinae:saponins (125 �g/ml CBM) while other bacteria are only inhibited and tannins were abundant; favonoids and terpenoids were at very high concentrations (>37 mg/ml CBM) [41]. Tis hardly detectable. While Castillo-Juarez´ et al. (2009) reported apparent contradiction may be explained by the fact that the that a methanolic extract inhibited H. pylori [16], we could degree of unsaturation of the fatty acid of anacardic acids has not fnd bacterial inhibition using the Kirby-Bauer method. a high impact on their antimicrobial activity [44]. A similar result was found for methanolic extracts from leaves of E. foetidum, which is also native to Mexico and Cirsium mexicanum. To the best of our knowledge, there are South America: one group reported inhibition of H. pylori, no scientifc reports on the biological activity of extracts from while another group found no inhibition of several other C. mexicanum,sowereporthereforthefrsttimethatextracts bacterialstrains(reviewedin[45]).Toourknowledge,here fromthewholeplantaswellasfromthefowersofthis we report for the frst time cytotoxicological results on E. species contain potentially active molecules, mainly tannins carlinae:wefoundwith356 ± 114 �g/ml HsM low cytotoxic and saponins, and, in contrast to the whole plant, the fowers efects on Caco-2. Until now, only for few triterpenoids contained also terpenoids; favonoids were barely detectable and triterpenoid-glycosides, isolated from diferent Eryngium in both extracts. Using the Kirby-Bauer test, no antibacterial species, there are reports about their moderate antiprolifera- activity could be detected and low cytotoxicity was observed tive activity on diferent human cell lines [45]. for both methanolic extracts with 323 ± 93 �g/ml CsM and 250 ± 55 �g/ml CsFM, respectively. Te latter results could Pithecellobium dulce. Kumar and Nehra (2014) found antibac- beconfrmedqualitativelybyNRUandTBS.Teseresults terial activity for a methanolic bark extract from P. du l ce do not indicate this plant as a good candidate for anticancer against E. faecalis, E. coli, K. pneumoniae, M. luteus, S. typhim- treatment as it is used empirically by the Mexican population urium,andS. epidermidis at MICs of 250–500 �g/ml [46] and [14]. the same group reported inhibition of bacterial growth for a methanolic leaf extract against B. subtilis, E. aerogenes, E. Eryngium carlinae. On the composition and biological activ- coli, Kpneumoniae, P. ae r ug ino s a , and S. epidermidis at MICs ity of extracts from E. carlinae few scientifc reports exist ranging from 375 to 1000 �g/ml[47].Wewerenotableto only: for the aerial parts of some Eryngium species it is detect bacterial inhibition using the Kirby-Bauer test, from Evidence-Based Complementary and Alternative Medicine 9 both extracts, but we found a cytotoxic efect on Caco-2 studies on biological activity. A preliminary qualitative phy- with CC50 = 347 ± 78 �g/ml for the bark extract (GBM) and tochemical analysis revealed the presence of favonoids, the methanolic leaf extract (GLM) was even more cytotoxic tannins, and terpenoids and the absence of saponins. with a CC50 = 126 ± 14 �g/ml. Tis is in a similar range of � CC50 =112 g/ml GLM for the cell line MCF-7 [48], a value 5. Conclusion also reported by Olmedo-Agudo et al. (2016) with CC50 > � 100 g/ml GLM [21]; the latter group determined cytotoxicity Tis ethnopharmacological evaluation helps to direct the � for the cell line CaP with CC50 = 3.7 g/ml, indicating that P. screening for plants with antitumoral properties. Te most dulce leaf extract has cytotoxic potential. promising candidate resulted to be the methanolic extract of fruits from C. ambrosioides (ESM), followed by the methano- Chenopodium ambrosioides.TeleavesofC. ambrosioides lic leaf extract from P. du l ce (GLM). In further studies the especially have been broadly studied, but the results vary cytotoxic and antibacterial principle, especially from ESM, strongly. While some authors fnd ascaridole and derivates will be elucidated. with about 50–80.0% as the main component [33, 49–51], othersfnditwithlessthan10%asaminorcomponent[52, 53]. It is supposed that, apart from the diferent geographic Abbreviations location, the part of the plant, or the processing method, MTP: Microtiter plate those diferences can be explained by the existence of diferent NRU: Neutral red uptake varieties of the plant: C. ambrosioides var. anthelminticum TBS: Trypan blue staining has a high ascaridole content while the other variety, var. CBM: Cuachalalate bark methanolic extract pubescens, does not [54]. Sousa et al. (2012) could not CsFM: Cardosanto fower methanolic extract detect antimicrobial activity from an ethanolic extract of the � CsM: Cardosanto aerial parts methanolic extract aerial parts up to 500 g/ml [55], as we did not observe it ELM: Epazote leaf methanolic extract either for a methanolic extract from the leaves. Jesus et al. ESM: Epazote fruit (seed) methanolic extract (2017) determined for an ethanolic crude extract from leaves GBM: Guamuchil´ bark methanolic extract only very high MICs against Mycobacterium tuberculosis GLM: Guamuchil´ leaf methanolic extract (1.25 mg/ml), E. faecalis (4.29 mg/ml), and P. ae r ug ino s a HsM: Hierba del sapo aerial parts methanolic extract. (68.75 mg/ml) [56]. Generally, the whole aerial part is not considered (very) cytotoxic: Rufa et al. (2002) did not detect cytotoxic efects up to 1,000 �g/ml for HepG2 [57], Koba et Conflicts of Interest al. (2009) found a CC50 =700�g/ml for HaCaT [50], and Te authors declare that there are no conficts of interest Barros et al. (2013) determined CC50 of 264 to 319 �g/ml for regarding the publication of this paper. HeLa, HepG2, and HCT-15 [58]; those values are in the range of CC50 = 563 ± 66 �g/ml ELM that we report for Caco-2. Although Degenhardt et al. (2016) reported cytotoxic efects References of an ethanolic leaf extract against blood cell lines with CC50 [1] L.-F. Nothias, R. Knight, and P. C. Dorrestein, “Antibiotic of 30–62 �g/ml [33] and Al-kaf et al. (2016) reported a CC50 ≈ 25 �g/ml for HT-29 for a hydrodistillate [51], both associated discovery is a walk in the park,” Proceedings of the National AcadamyofSciencesoftheUnitedStatesofAmerica,vol.113,no. the cytotoxic efect to the high ascaridole content of their 51, pp. 14477–14479, 2016. extracts, which coincide with a report on the cytotoxicity of [2] WHO, Global Action Plan on Antimicrobial Resistance,WHO, ascaridoles with CC50 = 4.2–23.7 �g/ml for diferent tumour Geneva, Switzerland, 2015. cell lines [59]. However, Koba et al. (2009) could demonstrate [3] R. I. 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