Abstract
Extracts from Pittocaulon. spp. (Asteraceae) have been used in folk medicine to treat many ailments, particularly as anti-inflammatory and antimicrobial. Neither the bioactive components responsible nor the mechanisms involved have been evaluated. Here we report the antibacterial activities of their methanol and CH2Cl2 extracts and the effects on some fungal strains treated with CH2Cl2 extracts from root, stems, and flowers of P. praecox. (Cav.) H. Rob. & Brett., P. hintonii. H. Rob. & Brett., P. filare. (McVaugh) H. Rob. & Brett, P. velatum. (Greenm) H. Rob. & Brett., and P. bombycophole. (Bullock) H. Rob. & Brett. Thirteen extracts were obtained with MeOH and CH2Cl2 solvents from aerial parts and roots of five Pittocaulon. spp. for the first time in this species, and their antimicrobial activities were determined. The antimicrobial activities of these extracts were evaluated against the bacteria Staphylococcus aureus., Staphylococcus epidermidis., Bacillus subtilis., and Vibrio cholerae. (one El Tor strain, and a clinic case CDC-V12). Additionally, the CH2Cl2 extract was evaluated against Aspergillus niger., Fusarium moniliforme., Fusarium sporotrichum., Rizoctonia solanii., and Trichophyton mentagrophytes.. The most sensitive bacteria against these extracts were B. subtilis., S. epidermidis., and V. cholerae., a key bacterium in contaminated water. However, CH2Cl2 extracts from P. praecox., P. bombycophole., and P. hintonii. exhibited a significant antifungal activity against the fungal strains F. sporotrichum., R. solanii., and T. mentagrophytes.: at 1000 μg per disk, these extracts completely inhibited the mycelial growth of these fungi.
Introduction
Pittocaulon. (ex Senecio). form together with several related genera a monophyletic group within the subtribe Tussilagininae of the Senecioneae (Asteraceae). The genus Pittocaulon. has been segregated from the huge cosmopolitan genus Senecio.. This segregation is supported by means of classic methods (Robinson & Brettell, Citation1973) and by molecular phylogenetic techniques (Bain & Golden, Citation2000). There are five Pittocaulon. species endemic to México with one also occurring in Guatemala. They are found in tropical dry habitats often on rocky outcrops or steep cliffs. They are shrubs or small trees with broom-like succulent branches with water-storing pith and bark (Olson, Citation2005). The branches bear small clusters of leaves during the rainy season and are leafless during the drought the rest of the year. The stems of P. praecox,. the most abundant species, are frequently infested by a scale insect, Ceroplastes albolineatus. (Coccidae, Homoptera). We have previously reported the chemical-ecological relationship and the occurrence of pyrrolizidine alkaloids (PAs) from this host plant (Marín Loaiza et al., Citation2007a, Citationb).
The occurrence of PAs in most species of the genus Senecio. is well established (Hartmann & Witte, Citation1995). Preliminary studies with Pittocaulon. revealed the presence of furanoeremophilanes (Ortega et al., Citation1975; Bohlman & Zdero, Citation1976) and two structural types of PAs (i.e., macrocyclic 1,2-unsaturated alkaloids of the otonecine type and simple monoesters). 1,2-Unsaturated PAs are protoxic, whereas 1,2-saturated PAs are nontoxic, as far as liver toxicity is concerned (Marín et al., Citation2007b). The co-occurrence of macrocyclic otonecine derivatives and simple 1,2-saturated monoesters is rarely observed among Senecio. species. Preliminary studies indicated the presence of different stereoisomers of the 1,2-saturated necine base 1-hydroxymethyl-7-hydroxypyrrolizidine depending on the Pittocaulon. species analyzed (Marín et al., Citation2007b).
Our studies on the chemical structure of secondary metabolites from flowers, stems, and root extracts of Asteraceae and other botany families with biological activities show the presence of a series of compounds including alkaloids and other secondary metabolites (Calderon et al., 2001; Cespedes et al., 2000, 2004; Torres et al., Citation2003; Marín et al., Citation2007a, Citationb).
In some cases, alkaloids are responsible for the natural resistance to the action of insects, fungi, and bacteria (Seigler, Citation1997). Thus, these compounds could have applications as pesticides (Reina et al., Citation1997), allelochemicals (Schmeller et al., Citation1997; Pasteels et al., Citation2003), insecticides (Hartmann & Witte, Citation1995; Pasteels & Hartmann, Citation2004), antimicrobials (Jain & Sharma, Citation1987; Singh et al., Citation2002), and deterrents (Van Dam et al., Citation1995), among other activities such as carcinogenic, mutagenic, and toxic (Mattocks, Citation1968, Citation1986; McLean Citation1970; Mattocks & Cabral, Citation1982; Mattocks et al., Citation1991; Mattocks & Jukes, Citation1992; Rubiolo et al., Citation1992).
No species of the Pittocaulon. genus have been studied until now with respect to bioactive extracts and compounds. As a contribution to the knowledge base and continuing the work with native Asteraceae, the aim of this work was to evaluate the antibacterial and antifungal activity of the MeOH and CH2Cl2 extracts of Pittocaulon. spp.
Materials and Methods
Plant materials
Pittocaulon velatum. (Greenm) H. Rob. & Brett. and P. hintonii. H. Rob. & Brett. were collected in the state of Michoacán, México, along the road from Ciudad Hidalgo to Zitácuaro (2235 m) and Municipio of Coalcomán (Rancho la Parota) (1230 m), respectively. P. filare. was collected about 5 km south of the intersection to Los Asmoles on the non–toll highway Colima-Manzanillo (464 m) in the state of Colima, México. P. bombycophole. was collected along the road between Buenavista and Coaxcaclán, in the state of Guerrero, México, at 1728 m. The botanical identification of the plants was done by Dr. Mark Olson (Institute of Biology, UNAM). Voucher specimens are deposited at the herbarium of the Institute of Biology, UNAM (MEXU).
Pittocaulon praecox. (Cav.) H. Rob. & Brett. was collected within the campus of the Universidad Nacional Autónoma de Mexico (UNAM) (2277 m), México D.F, Mexico. Flowers, roots, and shoots were collected during spring (March 2004). Flowers, roots, and shoots of P. bombycophole. (Bullock) H. Rob. & Brett, P. velatum. (Greenm) H. Rob. & Brett., P. filare. (McVaugh) H. Rob. & Brett, and P. hintonii. H. Rob. & Brett. were collected during March 2005. All the plant materials were air-dried, ground, powdered, and kept dry until extraction.
Isolation and general procedures
The stems, roots, and flowers were dried and separated mechanically and finely chopped. Each of the samples (bark, roots, stems, and flowers) were separated in two batches and extracted three times, using CH2CI2 at r.t. for 48 h and after that methanol at r.t. for 72 h. The crude extracts were then evaporated to dryness under vacuum conditions.
Evaluation of biological activity (microorganisms and growth medium)
The antibacterial and antifungal activities of the extracts () were determined.
Table 1 Extracts tested
Due to a very low or null activity, in a preliminary trial, those extracts with letters a. to q. were not tested. Testing paper disks (6 mm, Whatman) were impregnated with 10 μ L solution containing 100 μ g of each extract to perform the test against the Gram-positive bacteria Bacillus subtilis. (ATCC6633), Staphylococcus aureus. (ATCC12398), and Staphylococcus epidermidis. (wild type 1), and the Gram-negative Vibrio cholerae. (all strains correspond with biotype El-tor., two collections: CDC-V12 and CC (a wild type 1, isolated from a clinic case). The fungal strains Aspergillus niger. (ATCC64958), Fusarium moniliforme. (ATCC96574), Fusarium sporotrichum. (Wild Type 2), Rhizoctonia solani. (wild type 2*), and Trichophyton mentagrophytes. (ATCC9972) were assayed against all extracts.
Wild type 1: Strain cultured and donated by Laboratorio de microbiolog´a of FES-Cuautitlán, UNAM, Mexico D.F. Wild type 2: Strain cultured and donated by Laboratorio de Análisis Cl´nicos of FES-Iztacala, UNAM, Mexico D.F. Wild type 2*: strain isolated from infected beans culture by Prof. Dr. Rodolfo de la Torre, Laboratorio de Microbiolog´a, FES-Iztacala, UNAM, Mexico D.F.
Bacteria were grown in brain heart infusion broth (Bioxon, Mexico City, Mexico) for stock cultures, and Mueller-Hinton broth (Bioxon) was used as a test medium due to its low interaction with the assayed compounds. The antibacterial activity of the extracts was assessed with the disk-diffusion method using Mueller-Hinton agar (Baron & Finegold, Citation1995) and determination of inhibition zones at different dilutions of compounds and extract. Filter paper disks (6 mm i.d., Whatman) with MeOH extract samples were impregnated with 10 μ L of a 10 μ g/μ L solution of each sample, and the filter paper disks of the CH2Cl2 extracts were impregnated with 10 μ L of a 40 μ g/μ L solution of sample and placed in Petri dishes containing the test organisms. Cultures were incubated at 37°C, and after 24 h, the diameter of inhibition zone was determined (millimeters). The mean value from at least three different experiments was used for statistical analysis, and each experiment was done in triplicate. The treatments were evaluated with a completely randomized design. The treatments were subjected to a one-way analysis of variance (ANOVA), and means were compared with the Student-Newman-Keuls (SNK) test (p = 0.05) under Microsoft MicroCal Origin 6.2 statistical program. Gentamicin was used as positive control.
The estimation of the minimal inhibitory concentration (MIC) was carried out by the broth dilution method (Vanden Berghe & Vlietinck, Citation1991). Dilutions of samples of the extracts from 3.0 to 0.01 mg/mL were used. Test bacteria culture was used at the concentration of 105 CFU/mL. MIC values were taken as the lowest extract concentration that prevents visible bacterial growth after 24 h of incubation at 37°C. Chloramphenicol was used as reference, and appropriate controls with no extract were used. Each experiment was performed three times.
The antifungal property of the extracts was tested by the agar-well diffusion method using Sabouraud dextrose agar. Standard reference antibiotics were used in order to control the sensitivity of the tested microorganisms, which were inoculated in Czapek-Dox broth medium; plates containing only the culture medium, with the addition or not of the solvents (methanol or water 10 μ L/sensidisk), were used as viability controls for each fungus studied. The fungi inocula (10 μ L of 3 × 106 spores/mL) were placed in a hole (0.4 mm2) made in the center of each Petri dish after solidification of the medium. The doses of the extract were 2 mg/sensidisk; positive control, ketoconazole 10 μ g/sensidisk; negative control: each one of the solvents used (water and methanol 10 μ L/sensidisk). The cultures were incubated at 28°C and controlled every 24 h for 14 days. Inhibition of radial mycelial growth diameters were measured daily and recorded as mean percentages (%) of growth (Wang & Ng, Citation2002).
Antifungal assays (IC50) and minimum fungicidal concentration (MFC)
These tests were carried out to analyze the fungicidal activity exhibited by each compound and extract while further studying their fungistatic activity. For quantitative assays of the extract, three doses were added to potato dextrose agar (4 mL) at 45°C, these being mixed rapidly and poured into three separate 6-cm Petri dishes. After the agar had cooled to room temperature, a small amount (1 × 1 mm) of mycelia, the same amount to each plate, was inoculated. Buffer only was employed for a negative control. After incubation at 23°C for 72 h, the area of the mycelial colony was measured and the inhibition of fungal growth and hence the IC50 was determined. For the extract and compounds, 14 days after the beginning of the assay, a circle of agar around the central hole was obtained, as well as the mycelium of fungi from these plates that exhibited negative growth. At the end of this period, the MFC values were recorded (Wang & Ng, Citation2002).
Statistical analysis
Data shown in figures and tables are average results obtained by means of three replicates and independent experiments and are presented as average ± standard errors of the mean (SEM). Data were subjected to analysis of variance (ANOVA) with significant differences between means identified by GLM procedures. Results are given in the text as probability values, with p < 0.05 adopted as the criterion of significance; differences between treatment means were established with a Student-Newman-Keuls (SNK) test. The I50 values for each activity were calculated by Probit analyses based on percentage of inhibition obtained at each concentration of the samples. I50 is the concentration producing 50% inhibition. The complete statistical analysis was performed by means of the MicroCal Origin 6.0 statistical and graphs PC program.
Results and Discussion
In our screening program looking for biological activities of succulent plants from semiarid regions, it was found that P. praecox, P. hintonii, P. bombycophole, and P. velatum. showed antibacterial and antifungal activity in a preliminary trial (the extracts from P. filare. did not show any activity). Based on this information and on the high resistance to insect pest and pathogen attack of this plant, we have carried out antimicrobial studies on the roots and aerial parts of these plants.
The antibacterial activity was carried out against Gram-positive Bacillus subtilis, Staphylococcus aureus., and Staphylococcus epidermidis. and the Gram-negative Vibrio cholerae.. All extracts assayed were active against all Gram-positive bacteria, and the most active extract was PBoRDc (CH2Cl2 extract of roots of P. bombycophole.) with a MIC of 0.25 mg/mL. shows the MIC and MBC of the assayed extracts. After the evaluation of the results, we found that the PBoTDc, PBoTMe, PBoFMe, PBoRDc, PPrTDc, PHnTMe, and PHnTDc extracts showed the highest inhibitory activity against Gram-negative tested bacteria and did not show the same activity against Gram-positive tested bacteria (), as the zone (mm) diameters were greater than shown by the other assayed extracts (data not show).
Table 2 MIC and MBC of antibacterial growth inhibition activity on bacterial inoculum by extracts of Pittocaulon. sppFootnotea..
All P. bombycophole. extracts showed a variable but consistent composition of 7-angeloylturneforcidine (16.0% to 76.0%), the most abundant/common alkaloid, followed by 9-angeloylturneforcidine (∼ 10.0%) and neopetasitenine (16.0% to 70.0%), together with other alkaloids such as senkirkine (Marín et al., Citation2007b). These alkaloids have also been reported for other species of the Senecio. spp. (Hartmann & Witte, Citation1995; Fu et al., Citation2004).
In view of the potent activity of P. bombycophole. root CH2Cl2 extract against bacteria, these extracts (PBoRDc), P. praecox. (PPrFDc: flowers-CH2Cl2), and P. hintonii. (PHnFDc, PHnRDc: flowers and roots CH2Cl2 extracts, respectively) were assayed against different fungal strains. These extracts were assayed against R. solanii., F. sporotrichum, F. moniliforme, A. niger., and T. mentagrophytes. (). The activity level shown by PBoRDc, PPrFDc, PHnFDc, and PHnRDc extracts against these fungi was relatively high compared with positive control, ketoconazole (), the most active being PBoRDc and PPrFDc with an inhibition of 45% at 1000 μ g/mL per disk against all strains assayed (). In addition, these extracts showed a total inhibition (100%) against these fungi at 4000 μ g/mL per disk (data not show). Similar effects were shown by different extracts regulating completely the mycelial growth of fungal strains at 300 μ g/mL per disk and the mycelial growth (Storey et al., Citation1991; Tinney et al., Citation1998; Hol & Van Veen, Citation2002; Loizzo et al., Citation2004).
Table 3 Fungal qualitative evaluation of P. praecox., P. bombycophole. and P. hintonii. extracts.
On the other hand, with PBoRDc, PPrFDc, PHnFDc, and PHnRDc extracts, MFC and IC50, values were determined. The concentration that completely inhibited mycelial growth (MFC) ranged from 2500 to 4000 μ g/mL, and the IC50 values were almost half of the concentrations (data not show).
Interestingly, in P. bombycophole. extracts, the high percentage of 7-angeloylturneforcidine, 9-angeloylturnefor-cidine, and neopetasitenine and in P. praecox. extracts, the high presence of 7-angeloyldihydroxyheliotridane, 9-angeloyldihydroxyheliotridane, is noteworthy, and senkirkine in aerial parts, and senecionine, integerrimine, platyphylline, neoplatyphylline, and senkirkine, together a very low percentage of 7-angeloyldihydroxyheliotridane, in roots may be noted (Marín et al., Citation2007b).
In summary, when antifungal activity was assayed with P. bombycophole. extracts, the IC50 was as low as that observed with the positive control, ketoconazole (data not shown). These results can be due to a synergistic effect caused by the components of these extracts, which was not observed until now in these species. The activity can be attributed to any of the mixture of compounds present in the samples (e.g. pyrrolizidine alkaloids, furanoeremophilanes or different kind of compounds that have not been studied on these species yet). The synergistic effect is one of the most important characteristics exhibited by natural extracts, increasing their efficacy in contrast to that which could be obtained with the equivalent amount of the active constituents alone.
These results reveal that these compounds found in Pittocaulon. spp. extracts act on phytopathogenic fungi and show antibacterial and antifungal activities. This could indicate that such metabolites can play an important role in the resistance and chemical ecology behavior of this genus.
Acknowledgments
We acknowledge the financial support of DGAPA-UNAM grants IN-243802 and IN-211105. J.C.M.L thanks a DGAPA-PAPIIT IN211105-3 project, and DGEP for a doctoral fellowship (01.01.2007). The authors thank Dr. Mark Olson (Biology Institute, UNAM) for the taxonomic identification of plants.
Notes
*Dedicated to Professor John Thor Arnason of the University of Ottawa, Department of Biology, on the occasion of his sixtieth birthday.
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