Mayra Herrera-Martíneza, Marco Vinicio Ramírez-Maresa,*, Eleuterio Burgueño-Tapiac, Ernestina Cepillo-Portugalb, Coral Mirón-Enríqueza, Beatriz Hernández-Carlosa,*
a Instituto de Recursos, Universidad del Mar, Puerto Ángel, Oaxaca, 70900 México. *Correspondence to: Beatriz Hernández-Carlos, Marco Vinicio Ramírez-Mares, Instituto de Recursos, Universidad del Mar, Puerto Ángel, Oaxaca, 70900 México. Phone: +52 95 85 84 30 49; Fax: +52 95 85 84 30 78. E-mail: bhcarlos@angel.umar.mx, marcoram@angel.umar.mx.
b Preparatoria Agrícola, Universidad Autónoma de Chapingo, Chapingo, Estado de México, 56230 México.
c Departamento de Química Orgánica, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala. Col. Santo Tomás, México D.F. 11340, México.
Received August 2012.
Accepted December 2012.
ABSTRACT
A baccharane-type triterpene (1), four oleanane-type triterpenes (2-5) and twelve oleanane-type bidesmodic saponins (6-17) were subjected to antioxidant, antimicrobial and antitopoisomerase evaluation. Moderate antimicrobial activity was observed (zone of inhibition in millimeters); triterpenes 2-4 were toxic to Staphylococcus epidermidis (13.20 ± 0.00; 11.75 ± 0.07; 9.85 ± 0.21, respectively), while baccharis oxide (1) inhibited the growth of S. epidermidis (14.75 ± 0.35), Candida albicans (20.55 ± 0.92), Escherichia coli (14.80 ± 1.13) and Klebsiella pneumoniae (16.35 ± 0.49). Polygalacic acid (4), 6, 7, 15 and 16 showed growth inhibition of C. albicans (13.50 ± 0.71; 11.00 ± 00; 8.50 ± 0.71; 11.70 ± 0.14; 10.50 ± 0.71, respectively). The growth inhibition of S. epidermidis was achieved by 8 (9.00 ± 1.41) and 15 (9.00 ± 0.00); while the last compound inhibited the growth of K. pneumoniae (9.50 ± 0.71). The tested compound concentration for antitopoisomerase activity was between 1.8-13.8 times of those used as positive controls (CPT= 0.1435 μM, ETP = 0.0849 μM), showing that 1 (1.1726 μM), is topoisomerase I inhibitor. Methylated oleanolic acid (3a, 0.7128 μM), together with 9 (0.3650 μM), 13 (0.3294 μM) and 14 (0.3691 μM) showed antitopoisomerase II activity. None of all tested compounds showed significant antioxidant activity using the DPPH method.
Keywords: Topoisomerase inhibitor, antimicrobial activity, baccharis oxide, bayogenin, polygalacic acid, saponins.
RESUMEN
Se evaluó la actividad antioxidante, antimicrobiana y antitopoisomerasa de un triterpeno tipo bacharano (1 ), cuatro triterpenos tipo oleanano (2-5) y doce saponinas bidesmódicas tipo oleanano (6-17). Los triterpenos 2-4 mostraron actividad antimicrobiana (zona de inhibición en milímetros) en contra de Staphylococcus epidermidis (13.20 ± 0.00; 11.75 ± 0.07; 9.85 ± 0.21, respectivamente), en tanto que el óxido de baccharis (1 ) inhibió el crecimiento de S. epidermidis (14.75 ± 0.35), Candida albicans (20.55 ± 0.92), Escherichia coli (14.80 ± 1.13) y Klebsiella pneumoniae (16.35 ± 0.49). El ácido poligalácico (4), 7, 6, 15 y 16 inhibieron el crecimiento de C. albicans (13.50 ± 0.71; 11.00 ± 00; 8.50 ± 0.71; 11.70 ± 0.14; 10.50 ± 0.71, respectivamente), el crecimiento de S. epidermidis fue inhibido por 8 (9.00 ± 1.41) y 15 (9.00 ± 0.00). Este último compuesto inhibió además el crecimiento de K. pneumoniae (9.50 ± 0.71). En la evaluación antitopoisomerasa de 1-17 usando concentraciones entre 1.8 y 13.8 veces mayores que las correspondientes a los controles positivos (CPT= 0.1435 μM, ETP = 0.0849 μM) se demostró que 1 (1.1726 μM) es inhibidor tipo I de la enzima. El derivado metilado del ácido oleanólico (3a, 0.7128 μM) y las saponinas 9 (0.3650 μM), 13 (0.3294 μM) y 14 (0.3691 μM) mostraron actividad antitopoisomerasa II. En la evaluación antioxidante por el método del radical libre DPPH ningún compuesto mostró actividad significativa.
Palabras clave: Inhibidor de la topoisomerasa, actividad antimicrobiana, óxido de baccharis, bayogenina, ácido poligalácico, saponinas.
INTRODUCTION
]]> Several assays have been developed to evaluate the compounds ability to modulate biochemical events presumed to be mechanistically linked to carcinogenesis (Shureiqi et al., 2000). Examples of such assays include: a) topoisomerase inhibitors, which constitute a class of agents that inhibit carcinogenesis via their antiproliferative or cell-differentiating action and chemotherapy and chemoprevention (Cho et al. , 2000); b) antimicrobial activity, allows the identification of novel agents capable of interfering with a specific molecular target, that may avoid the shortcomings of conventional chemotherapy because certain antimicrobials exhibit selective cytotoxicity against a broad spectrum of human cancer cells (Schweizer, 2009); c) antioxidant activity, as potent scavengers of Reactive Oxygen Species (ROS) may serve as a possible preventive intervention for free radical-mediated diseases such as cancer (Ralph et al., 2010).Up to day, scarce studies are available on antitopoisomerase properties of oleanane-type triterpenes and its glycosides in spite of that they have shown potential in the research of anticancer drugs (Shanmugam et al., 2012; Sparg et al., 2004). It has been described that oleanolic acid and its derivates inhibit the activity of human DNA topoisomerase II (topo II) (Mizushina et al., 2003), while Soyasaponin I and several Aesculosides were described as topo II (Suzuki et al., 2003) and topo I (Wang et al., 2010) inhibitors, respectively. On the other hand, the antimicrobial activities of oleanane-type triterpenes (Katerere et al., 2003) and their glycosides (Bader et al., 2000) have been known. For example, it was demonstrated that oleanane type triterpenes inhibited the growth of Gram-positive and -negative bacteria (Moodley et al., 2011; Djoukeng et al. , 2005) and polygalacic acid glycosides have shown to be potent fungicides (Bader et al., 2000). Antioxidant activity of oleanane type-triterpenes has been described as moderate (Dini et al., 2009). Among tetracyclic triterpenes, those of baccharane-type are less common than oleanane-type ones. For this reason biological studies of baccharane triterpenes or their glycosides are limited, for example: ((-glucuronidase inhibitory activity exhibited by Baruol (Núñez et al., 2004) or trypanocidal and inmunomodulatory activities described for baccharis oxide (Da Silva Filho et al., 2004; Missima et al., 2007).
It is known that pentacyclic triterpenes and their glycosides displayed antimicrobial and cytotoxic activities (Cipak et al., 2006; Castro et al., 1997). The aim of our work was to carry out the screening of antimicrobial, antioxidant and antitopoisomerase activities for one baccharane-type triterpene (1), four oleanane-type triterpenes (2-5) and saponins of bayogenin, polygalacic acid and 16-hydroxyprotobasic (6-17); herein we show the potential of baccharis oxide (1), polygalacic acid ester (4a), saponins of bayogenin (9) and polygalacic acid (12E, 13, 14) in the research of drugs with anticancer activity.
MATERIAL AND METHODS
General experimental procedures
Baccharis oxide (1): Ethyl acetate extract (4.5832 g) of Baccharis conferta roots (226.6 g) was dissolved in 400 mL of ethyl acetate and 571.4 mg of 1 was formed as a white solid, which was re-crystallized from methanol and identified by comparison of its spectroscopic data with those described (Nurnberg et al., 1998).
Compounds 2, 4, 6-16 were obtained from Sicyos bulbosus and Microsechium helleri according to Hernández-Carlos et al. (2011), and 17 was obtained from Sechium mexicanum (Hernández-Carlos et al., 2009).
HPLC analysis of 6-17: Solutions of saponins (1 mg/0.1 mL) in methanol/water (65:35) were injected (20 |lL) to C18 column (250 x 4.7 mm, 5 mm) with methanol/water (65:35) as mobile phase at 0.6 mL/min and IR detector. Retention times observed were 34.55 min (6), 13.17 min (7), 12.75 min (8), 30.66 min (9), 12.78 min (10), 11.80 min (11), 11.25 min (12), 11.47 min (13), 12.36 min (14), 27.77 min (15), 31.33 min (16) and 11.04 min (17).
Triterpenes 3 and 5: The chloroform extract (1.0 g) of aerial parts of B. conferta was subjected to column chromatography (300 mm x 20 mm I.D.) on SiO2 and eluted with ethyl acetate-hexane mixtures. From fractions 12 and 14, 5 (16.6 mg) and 3 (18.0 mg) were obtained, which were identified by comparison of their physical and spectroscopic data with those described (Liang et al., 1988).
]]> Saponins 10E and 12E: Compounds 10 (20 mg) and 12 (20 mg) were incubated with ( β-glucosidase (60 mg) in a buffer solution pH 5.5 at 37°C. After 16 h, each reaction mixture was extracted with n-butanol (10 mL x 3), the organic phase was removed under reduced pressure and subjected to separation by HPLC (Perkin Elmer series 200) using normal phase column (CNH2, 250 x 10 mm, 5 mm, Alltech) with acetonitrile-water (5:4) as mobile phase, the flow rate of the mobile phase was 2.5 mL/min and the peaks were identified using an IR detector. Compounds 10E (tR = 9.2 min, 9.0 mg) and 12E (tR = 9.02 min, 9.8 mg) were obtained from 10 and 12, respectively, and identified by HPLC (Perkin Elmer series 200) analysis of the mixture of each reaction (glucose tR = 16.00 min, 10: tR = 10.70 min and 12: tR = 10.03 min).
Biological test
Chemicals: Peptone bacto, yeast extract, agar bacto, Mueller Hinton (MH) agar, MH broth, trypticase soy agar, trypticase soy broth, sabouraud dextrose agar, sabouraud dextrose broth and dextrose were purchase from Difco (Sparks, MD). Methanol (spectrophotometric grade), hexane (HPLC grade), camptothecin (CPT), etoposide (ETP), dimethyl sulfoxide (DMSO-Hybri-Max), adenine hemisulfate salt, chloranphenicol, nystatin, ascorbic acid and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were obtained from Sigma Chemical (St. Louis, MO).
Antioxidant activity: The antioxidant activities were measured through the 2,2-diphenyl-1-picrylhydrazyl (DPPH·) radical scavenging activity by Brand-Williams et al. , method (1995). A methanolic solution of sample (50 μL) at four concentrations (9.5, 19, 38, 76 μM) was added to 1.95 mL DPPH radical solution (7.6 x 10 -5 M/ methanol). The decrease in the absorbance at 515 nm was followed using a uv/ vis spectrophotometer (Beckman DU-530) until the reaction reached the steady state in the dark (Siddhuraju and Becker, 2003). The DPPH radical concentration in the reaction medium was calculated from the following calibration curve, determinated by lineal regression:
A515nm=0.009[DPPH·]T - 0.007
Where [DPPH·]T was expressed as μM, r2 = 0.998
The percentage of remaining DPPH· (% DPPH· REM) was calculated as follows:
Where [DPPH·]T was the concentration of DPPH· at the steady state time and [DPPH·]T=0 was the concentration of DPPH· at zero time. The % DPPH·REM against the standard concentration was plotted to obtain the amount of antioxidant necessary to decrease by 50% the initial DPPH· concentration (EC50). The time needed to reach the steady state to EC50 concentration (TEC50) was determined. Ascorbic acid was used as reference standard. All experiments were carried out in triplicate. The activity of each sample was expressed as percentage of that achieved for the reference standard.
]]> Antibacterial and antifungal activities: Culture collection (ATCC, Manassas, VA): Gram negative bacteria: Klebsiella pneumoniae (ATCC 13883), Escherichia coli (ATCC 35218), Gram positive bacteria: Staphylococcus epidermidis (ATCC 12228) and the fungus Candida albicans (ATCC 14053). The antibacterial and antifungal activities were determined using the agar diffusion method. The bacterial strains were placed in plates of trypticase soy agar and the fungus in plates of sabouraud dextrose agar. After 24h incubation at 37°C (bacteria) and 30°C (fungus), four of five colonies were inoculated in 4 mL of Mueller-Hinton broth or sabouraud dextrose broth and incubated for 2 h at 37°C and 30°C, respectively. These inocula were adjusted to the 0.5 MacFarland standard (0.048 M BaCl2 0.5 mL + 0.18 M H2SO4 99.5 mL). For susceptibility testing, each 150 μL of adjusted bacterial or fungal suspension was spread on the sterile medium (trypticase soy agar or sabouraud dextrose agar) using sterile cotton swabs. The positive controls employed were chloramphenicol (30 mg) and nystatin (100 units) in the antibacterial and antifungal assays, respectively. Application of the samples (1-16) and controls (25 μL) was done directly in the solid medium. The application point was marked on the lower surface of the Petri dish. The preparations were left to diffuse. Subsequently the plates were incubated at 37°C for 24 h in the case of the bacteria; while the fungus was cultured at 30°C for 48 h. Plates prepared using the same procedures without samples or antibiotic, but with DMSO (25μL) were equally set as negative control. After incubation, the growth inhibition rings were quantified by measuring the diameter for the zone of inhibition in millimeters from the lower surface of the plates. All assays were carried out in triplicate.Yeast antitopoisomerase assay: Theyeast strains used in this study were Saccharomyces cerevisiae mutant cells JN362a, JN394, JN394 t-1 and JN394t2-5, containing recombinant forms of topoisomerase I and II enzymes and were kindly provided by Dr. John Nitiss of St. Jude Children's Research Hospital, Memphis, Tennessee. The requirement of topoisomerase II (Topo II) for the completion of mitosis makes this enzyme essential for cell division and cell proliferation. Differentiated cells express very low levels of Topo II, while highly proliferative and tumor cells often express 25-300 times the levels of quiescent cells (Heck and Earnshaw, 1986). A yeast S. cerevisiae strain clone forming assay was used as a model to measure topoisomerase I and II inhibition (Nitiss and Nitiss, 2001). Briefly, yeast cells were grown in YPD medium (yeast extract, peptone and dextrose) for 18 h in a shaking incubator. The logarithmically growing cells were then counted using a hematocytometer and adjusted to a concentration of 2 x 106 cells/ mL media. In all cases, cells were pre-grown at the same temperature that was used to measure drug sensitivity. Yeast cells (6 x 106 cells) were incubated at the optimal temperature for 24 h in the shaking incubator, in the presence of the 1-17. Compounds 1-17 solutions in DMSO (50 μL) were prepared in concentrations given in the Table 1. The concentrations of the compounds used in this assay were based on the solubility factor in DMSO (enough DMSO to dissolve 5 - 10 mg of each sample).
The same concentrations were used with the JN362a, JN394t-1 and JN394t2-5 strains. DMSO (1.66%) was used as negative control, while CTP (50 mg/μL), a topoisomerase I inhibitor, and ETP (100 mg/μL), a topoisomerase II poison, were the positive controls. Viable counts were determined by duplicate plating to YPDA medium solidified with 1.75% agar Bacto. Plates were incubated at the optimal temperature for growth of the cells to determine viable titer (25°C for temperature sensitive top2 mutants, 30°C otherwise). The percent survival was determined by comparison of the number of colonies counted in the no-drug control culture with those in the drug-treated culture. All experiments were repeated at least three times, and the means and population standard deviations were calculated for each of the data.
Statistical Analysis: Results are expressed as the mean ± SD of values obtained in at least duplicate measurements from three different experiments. A one-way ANOVA, with Dunnett and linear trend post test were used for statistical analysis. A probability (P) value of <0.05 indicated a significant difference.
RESULTS AND DISCUSSION
Compounds 2-4 and 6-17 (Figure 1) were obtained from Sycios bulbosus (2-4, 6-12), Microsechium helleri (13-16) and Sechium mexicanum (17), as described in Hernández-Carlos et al. (2009; 2011) while saponins 10E and 12E were obtained by enzymatic hydrolysis of 10 and 12, respectively, with β-glucosidase and purified by HPLC. Triterpenes 1 , 3 and 5 isolated from B. conferta were identified by comparing their physical and spectroscopic data with those of published values. Methyl esters ( 3a, 4a) of 3 and 4 were obtained according to Hernández-Carlos et al. (2009). The pure substances and known 1 , baccharis oxide (Anthonsen et al., 1970); 2, bayogenin, (Eade et al., 1963); 3, oleanolic acid (Saad et al. , 1988); 4, polygalacic acid (Seiligmann-Rodest and Polonsky, 1963); 5, maniladiol (Quijano et al., 1998); 6, tacacoside C (Castro et al. , 1997); 7, durantin III (Hiradate et al. , 1999); 8, heterpappussaponin 5 (Bader et al., 1994); 9, tacacoside B3 (Castro et al., 1997); 10 (Hernández-Carlos et al., 2009); 11, heterpappussaponin 7 (Bader et al., 1994); 12, (Hernández-Carlos et al., 2009); 13 (Hernández-Carlos et al., 2011), 14 (Hernández-Carlos et al. , 2011), 15, amole F (León et al., 1998); 16, amole G (León et al., 1998) and 17 (Eskander et al., 2006) were subjected to antioxidant, antimicrobial and antitopoisomerase evaluation. Purity of each compound was examined by TLC (1-5) and HPLC (6-17) analysis, while the used concentration for all assays was based on the solubility factor of each compound in DMSO.
None of the studied compounds showed significant antioxidant activity. According to references (Wolska et al., 2010), CH2OH and COOH groups at C-24 and C-28 of oleanane triterpenoids are critical for bactericidal activity; in the antimicrobial assays of oleanane-type triterpenes 2-5, maniladiol (5) showed no activity in all evaluated strains (Table 1), while bayogenin (2) showed activity against S. epidermidis (I.D. 13.20 mm) and the polygalacic acid ( 4) showed activity against S. epidermidis (i.D. 9.85 mm) and C. albicans (I.D. 13.5 mm). No difference in the extent of activity between Gram-positive (S. epidermidis) and -negative (E. coli and K. pneumoniae) bacteria was observed with baccharis oxide (1 ).
Antimicrobial activity of bayogenin saponins: Antimicrobial activity (Table 1) of bayogenin was improved with its glycoside amole F (15), which displayed the best antimicrobial activity in S. epidermidis (I.D. 9 mm), C. albicans (I.D. 11.7 mm), and K. pneumonia (I.D. 9.5 mm). The related saponin, amole G (16) with one glucose unit less than 15 (at C3 of aglycone), only showed activity against C. albicans (I.D. 10.5 mm) together with tacacoside C (6), which is equivalent to 16 without the xylose unit at C3 rhamnose inner (Figure 1). However, tacacoside B3 (9) did not show any antimicrobial activity in spite of its difference with 6 and 15, which are an additional glucose unit at C3 of aglycone and a xylose unit at C3 rhamnose inner, respectively.
Antimicrobial activity of polygalacic acid saponins: Durantin III (7) and heterpappussaponin 5 (8) displayed toxic activity against strains of C. albicans (I.D. 8.5 mm) and S. epidermidis (I.D. 9.0 mm) respectively, both compounds have one glucose unit at C3 aglycone. The rest of polygalacic saponins did not show antimicrobial activity, although all saponins tested here are structurally related, for example, 12 contains an additional glucose unit at C3 of aglycone and a rhamnose unit at C3 of xylose in comparison with 8, but did not show antimicrobial activity. It is noted that active saponins (7 and 8) contain five monosaccharide units while the rest of them contains six or seven monosaccharide units. The activity of polygalacic acid against several C. albicans strains is known (Bader et al. , 2000) and in this work we observed that the fungicide activity of polygalacic acid diminished when its glycosides were tested, this could be possible associated to the concentrations used of polygalacic acid and its glycosides (0.3202 μM and 0.1050-0.1374 μM, respectively). Then the effective concentration of polygalacic acid in the glycosides was diminished almost 50%.
]]>Antitopoisomerase activity: As shown in Table 2 and Figure 3, the JN394 strain was hypersensitive to CPT (100.0%) which is a Topo I poison. Growth inhibition of this strain was achieved by 1 (97.57%), 3a (28.60%), 6 (91.70%), 7 (73.90%), 8 (62.80%), 9 (64.10%), 10 (39.90%), 10E (68.27%), 11 (33.5%), 12 (35.65%), 13 (40.80%), 14 (75.64%), 15 (59.00%), 16 (96.68%), and 17 (58.81%). The strain JN394 is DNA repair-deficient and drug permeable (carry ise2 and rad52 mutations) (Nitiss and Wang, 1988). These mutations increase the sensitivity of these cells to drugs. The yeast JN362a is a DNA repair-proficient strain (Nitiss and Wang, 1988) and the compounds with antitopoisomerase activity do not inhibit the growth of this strain, but they do inhibit the JN394 strain growth. CPT as positive control, and a topoisomerase inhibitor, affects the JN394 growth but do not inhibit JN362a growth. The compounds with similar behavior to CPT were 1 , 3a, 9, 13 and 14 (Figure 3), which showed the following inhibition percent in the JN362a: -16.61, -14.25, 9.53, -9.46 and 2.12%, respectively. Compounds 6, 7, 8, 10, 10E, 11, 12, 15, 16 and 17, inhibited both strains and represent cytotoxic compounds with action mechanisms different to the inhibition of topoisomerases. The compounds 2, 3, 4, 4a, 5 and 12E did not inhibit any of the strains and therefore they are no considered cytotoxic. The strain JN394t-1 is isogenic to JN394 and contains a disrupted top 1 gene (Nitiss and Wang, 1988): the absence of this gene resulted in diminution of antitopoisomerase I drugs cytotoxicity. Positive control CPT (0.1435 μM) fails to reduce the growth of these mutant cells, 1 was incapable of affecting the growth of the cells with the top1 mutation, therefore topoisomerase I is the target of 1 (Table 2 and Figure 3) at 1.1726 μM (-5.95%). Cytotoxic activity against cancer cells is known for 8 and 11 (Bader et al. , 1996); however 8 and 11 did not show antitopoisomerase activity. In contrast to the result for 1 no resistance was observed when JN394t-1 cell (contains a disrupted top1 gene) were treated with 3a, 9, 13, 14 or the antitopoisomerase drug ETP, indicating that the observed effect is specific to antitopoisomerase II agents. The strain JN395t2-5 carries a top2 allele that is resistant to multiple classes of topoisomerase II poisons at its permissive temperature (25°C) (Jannatipour et al., 1993). Cells with the top2-5 mutation are able to grow in the presence of ETP (inhibition 30.03%), but not in the presence of CPT (99.82%). The compound 9 played as ETP. The strain has essentially the same sensitivity to CPT as JN394 (rad52 top 2+ cells), indicating that the observed resistance is specific to antitopoisomerase II poison agents that trap the enzyme-mediated DNA cleavage. The compounds 3a, 13 and 14 are inhibitors of the topoisomerase II enzyme, which have the ability to block the overall catalytic activity of the enzyme.
Baccharis oxide (1 ) resulted topoisomerase I inhibitor and it represents the first compound that is described of a baccharane-type triterpene with both antimicrobial and antitopoisomerase activities. Bayogenin (2) and polygalacic acid ( 4) showed not topoisomerase activity but their glycosides 9, 13 and 14 did. Oleanolic acid (3) is described as topoisomerase to evaluation using a yeast antitopoisomerase assay, being active only the derivative (antitopoisomerase II). The results suggested that 3 could be active but it is incapable of achieving the enzyme, contrary to the situation in the ester derivative (3a), where the polarity of 3 was diminished. This could facilitate the passing through the wall and membrane cell of the yeast. Considering these results, bayogenin (2), which contains one hydroxyl group less than polygalacic acid, probably could exhibit antitopoisomerase activity through its methyl ester. Biological tests of polygalacic acid and bayogenin are scarce due to several causes: a) they are not common in plants as oleanolic acid (Neto, 2011); b) they are obtained by acid hydrolysis of saponins and c) both compounds are poorly soluble in aqueous or organic solutions. These results are important examples of the biological activities of the oleanane type hydroxylated triterpenes, which are highly polar (Bader et al., 2000).
Cytotoxic activity against cancer cells by maniladiol (5) has been described (Ling et al. , 1982), in this work maniladiol did not show antimicrobial or antitopoisomerase activities due to the lack of a carboxyl group at C28, which is important for both activities (Wolska et al., 2010; Mizushina et al. , 2003), therefore maniladiol acts as cytotoxic in cancer cells through a different mechanism of topoisomerase inhibition.
Polygalacic acid glycosides that has antitopoisomerase II activity are 13 (0.3294 μM) and 14 (0.3691 μM), where the oligossacharide chains help to enhance the availability of 4, no matter 13 has one glucose unit more than 14 at C3 aglycone. However, 10 (0.3556 μM), 11 (0.3540 μM), and 12 (0.3294 μM) were cytotoxic to S. cerevisiae, even though these compounds are structurally related with the no cytotoxic saponin 12E. As it is observed in Figure 2, compound 12 lost its cytotoxic activity when it was hydrolyzed to 12E. All tested saponins are capable of crossing cell membranes due to their surfactant properties and their activities seem to be related to the sugar attached at C3 and C28 of aglycone. For example, the bayogenin saponin 9 possess one additional glucose unit at C3 aglycone in comparison with 6, therefore the antitopoisomerase II poison activity observed for 9 seems dependent on the glucose extra at C3 aglycone. Meanwhile, the antitopoisomerase activity of polygalacic acid saponins ( 13 and 14) was observed with two or one glucose unit at C3 aglycone.
Saponins tacacoside C (6) and tacacoside B3 (9) had been described as cytotoxic in cancer cells (Castro et al., 1997), according to our results this activity could be due to antitopoisomerase II activity of 9 and not yet identified the cytotoxic mechanism of 6.
CONCLUSIONS
]]> In this work it is shown that there is anti-topoisomerase I activity of baccharis oxide (1); the antitopoisomerase II activity was observed with methyl oleanolic acid (3a), polygalacic acid saponins (13, 14) and the bayogenin saponin (9). Moreover, the moderate antimicrobial activity (1-4, 6-8, 15, 16) and the good cytotoxicity against S. cerevisiae through topoisomerase inhibition was shown by 1 , 3a, 9, 13 and 14 establishing the potential of these compounds in the development of anticancer drugs. Also, in this work it was described for the first time the antimicrobial and antitopoisomerase activity of a baccharane-type triterpene.
ACKNOWLEDGMENTS
Stimulating supports of UMAR, SIP-IPN and CONACYT are acknowledged. We gratefully acknowledge J.R. Hanetho for language revision.
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This paper is dedicated to Professor Pedro Joseph-Nathan in recognition of his 50 years of outstanding scientific trajectory.
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