Pharmacology & Pharmacy, 2012, 3, 433-438 http://dx.doi.org/10.4236/pp.2012.34058 Published Online October 2012 (http://www.SciRP.org/journal/pp) 1 Antibacterial Activity of Bougainvillea glabra, Eucalyptus globulus, Gnaphalium attenuatum, and Propolis Collected in Mexico Oswaldo Javier Enciso-Díaz1, Alfonso Méndez-Gutiérrez2, Lourdes Hernández De Jesús3, Ashutosh Sharma1, María Luisa Villarreal1, Alexandre Cardoso Taketa1* 1Biotechnology Research Center (CEIB), Autonomous University of Morelos State (UAEM), Morelos, Mexico; 2Cente r fo r Research and Teaching in Humanities of the State of Morelos (CIDHEM), Morelos, Mexico; 3Department of Pharmacy, National School of Biological Sciences, National Polytechnic Institute (IPN), Mexico City, Mexico. Email: *ataketa@uaem.mx Received July 4th, 2012; revised August 12th, 2012; accepted September 14th, 2012 ABSTRACT Bougainvillea g labra C., Eucalyptus globulus Labill., and Gnaphalium attenuatum DC., as well as propolis, have been used in Mexican trad itional medicine as a remedy to treat respiratory illnesses. There are few biological reports of such material collected in Mexico, despite the high demand for them as raw material for popular and industrial uses. The antibacterial activity of the plants and propolis studied here were evaluated against a panel of bacteria using three dif- ferent methodologies: agar disc diffusion, macro and micro dilution methods. E. globulus and G. attenuatum extracts showed the strongest active values (P > 0.05) in the agar disc diffusion method with a range of 8 - 22 mm inhibition zone, MIC values ranging from 25 - 250 g/mL, and MBC values of 25 - 500 g/mL obtained by macro and micro di- lution methods. B. glabra extracts were active against E. coli, S. typhi, K. pneumoniae, S. aureus and S. agalactiae, with 8 - 13 mm inhibition zone, MICs ranging from 500 - 3000 g/mL, and MBCs of 1000 - 3000 g/mL. The propolis ex- tract turned out to be active against E. coli, K. pneumoniae, S. aureus and S. a g alactiae, with values of 7 - 12 mm, MICs of 1000 - 2000 g/mL, and MBCs of 2000 - 2500 g/mL. E. globulus and G. attenuatum extracts were the most active in the three methodologies assayed. This is also the first time that the antibacterial activity of G. attenuatum has been experimentally demonstrated. The microdilution method showed to be more sensitive, less expensive and minor time-consuming technique compared with the other two. Keywords: Antibacterial Activity; Bougainvillea glabra; Eucalyptus globulus; Gnaphalium attenuatum; Mexican Ethnomedicine; Propolis 1. Introduction Acute respiratory infections represent a serious health problem worldwide, and in Mexico, in the 2008 national statistics list, these infections occupied the first position in the top 20 illness affecting more than 24 million people, especially young children between 1 and 4 years [1]. Mexico has an ancient tradition in the use of medicinal plants as first aid remedies, which actually remains in use now. An important number of products derived from plants are used to manufacture a broad range of herbal formulas for the treatment of respiratory diseases, which are commercialized in such different forms, as infusions, plasters, syrups, pills, and candies. These products are usually registered as supplements or dietary aids in order to avoid the more strict governmental health policies [2]. The local market is flooded with a variety of herbal products lacking any kind of quality control guarantying efficacy and safety. The most im- portant missing issue is a scientific approach to the speci- fication of the concentration levels of the crude extracts, as well as the pharmacological activity and safety. Some medici nal plants such as Bouga invillea glabra C. (Nyctagi- naceae), Eucalyptus globulus Labill. (Myrtaceae) and Gnaphalium attenuatum DC. (Compositae), as well as extracts from propolis, are popularly employed alone or in combination. Neglected is the importance of cha r a c t eri z- ing a plant extract in terms of the origin of the raw material: local, seasonality and ontogenetic variations depending on its site of collection for. It has been demonstrated that the metabolic profiling of a plant species can change dramatically affecting the pharmacological response in relation to different environments [3,4]. In the same way, *Corresponding a uthor. Copyright © 2012 SciRes. PP
Antibacterial Activity of Bougainvillea glabra, Eucalyptus globulus, Gnaphalium attenuatum, and Propolis Collected in Mexico 434 propolis, a resin with a highly complex composition, can present different antimicrobial effectiveness depending on the solvent used in the extraction process, as well as the origin of the resin [5]. Although there are guidelines, the reality is that researchers adopt a wide range of criteria which depends on individual situations and conditions to define such parameters as plant material, techniques employed, growth medium and tested microorganisms in the antibacterial studies of medicinal plants [6]. This complex issue alerts us to the need to define the pharmacological properties of a plant material in time and space. Even though there are a number of antibacterial studies of Bougainvillea glabra [7,8], Eucalyptus globulus [9-11], Gnaphalium sp . [12, 13], and propolis [5,14 ], each one was based on only one methodology. On the contrary, we are reporting the acti- vity of these same plants, collected in the state of More- los, as well as propolis fro m Oaxaca State, Mexico, which we subjected to three different methodologies: agar disc diffusion, macro and micro dilution methods. The panel of Gram positive bacteria included Staphylococcus aureus, Streptococcus agalactiae, Listeria monocytogenes and Bacillus subtilis; and the Gram negative bacteria were Escherichia coli, Klebisiella p neumoniae and Salmonella typhi. 2. Materials and Methods 2.1. Plant Material Plants were collected in Morelos State, Mexico, in the year 2007, and propolis samples were collected from beehives in March, 2007, in Oaxaca State, Mexico. Voucher specimens were authenticated by Juan Carlos Juárez and then deposited at the HUMO Herbarium, CEAMISH (Centro de Educación Ambiental e Investi- gación Sierra de Huautla) UAEM, under the numbers 26348 (B. glabra), 26347 (E. globulus), and 26346 (G. attenuatum). 2.2. Preparation of Plant Extracts Dried aerial part of the plants (200 g) were powdered and put into a container with 300 mL of cold water, and then heated to boiling. After cooling down the mixture decoction to room temperature, 700 mL of EtOH was added, and then macerated for 2 weeks in a dark and cool place. This procedure has been used by some industries to prepare a mother tincture. In this study, these macerates were filtered and the solvent was evaporated under reduced pressure to obtain an ex tract that was used to prepare the dilutions employed for th e biological testing. Plant crude extracts and propolis resin were dissolved in ethanol or DMSO prior to use in the antibacterial assay. 2.3. Test for Antibacterial Activity The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) were established for the hydroalcoholic extracts for each species, and for the propolis extract, us ing both macro and micro dilution methods with tetrazolium chloride dyeing agent. Ad- ditionally, a disc diffusion method was applied to record the diameter of inhibitory zone promoted by the extracts. The antibacterial tests were adapted from the National Committee for Clinical Laboratory Standards (CLSI), formerly NCCLS, gu idelines f or document M26-A ( 1999) [15] and M38-A (2002) [16]. 2.4. Microorganisms The b acteria u sed in the pr esent study were Gram positive: Staphylococcus aureus (ATCC 25923), Streptococcus agalactiae (ATCC 25924), Listeria monocytogenes (ATCC 244), and Bacillus subtilis (ATCC 6633); and Gram negative: Escherichia coli (ATCC 25922), Klebi- siella pneumoniae (ATCC 10031) and Salmonella typhi (ATCC 6539). The b acteria were provided from th e Micro- biology Laboratory Culture Collection, National School of Biological Sciences, National Polytechnic Institute, Mexico. The microorganisms wer e first inc ubated at 37˚C ± 0.1˚C for 24 h in the nutrient broth (Mueller-Hinton). The bacterial suspensions were prepared and adjusted by comparison against 0.5 of the MacFarland turbidity standard (5 × 107 cells/mL) tubes, and further diluted to obtain a final concentration of 5 × 106 cells/mL that was standardized in our laboratory by a turbidity measure at 420 nm wavel ength prior to use. 2.5. Agar-Disc Diffusion Method Sterilized filter papers discs with 6 mm diameter were impregnated with 2 mg extracts, and then placed into Petri dishes (9 cm) with Mueller-Hinton agar (Oxoid), which were previously streaked with a swab containing the bacteria. The plates were incubated at 37˚C ± 0.1˚C for 24 h. Diameter of inhibition zones appearing around the discs were measured and recorded in mm. Chloram- phenicol (2 g/disc) was used as a positive control, and negative control discs were prepared with ethanol only. 2.6. Microdilution Method (MiD) Dilutions of the extracts solubilized in DMSO were prepared and dispensed into 96 microdilution wells to obtain a final volume of 200 L at a concentration range of 25 - 3000 g/mL of inoculum suspension. Microdilution trays were incubated at 37˚C ± 0.1˚C for 24 h to determine the MIC and the MBC. To establish the MIC values, 20 l of tetrazolium chloride at 20 mg/mL in Copyright © 2012 SciRes. PP
Antibacterial Activity of Bougainvillea glabra, Eucalyptus globulus, Gnaphalium attenuatum, and Propolis Collected in Mexico Copyright © 2012 SciRes. PP 435 water was added to each well. After incubation at 37˚C ± 0.1˚C for 30 min, the presence of live bacteria was recorded by visual inspection using the red formazan derivative. Before the addition of tetrazolium chloride, an aliquot was taken from the wells, where the turbidity was absent, and then inoculated in a tube with broth medium. The concentration point in which the bacterial growth was not observed was defined as the MBC value. 2.7. Macrodilution Method (MaD) Extracts were assayed at the same concentration and inoculation conditions as those used in the MiD method; however, a volume of 2 mL inoculums per tube was employed. The determination of MIC and MBC were carried out in the same way explained above for the MiD method. 3. Results Extraction procedures yielded 2.89%, 6.11%, and 2.60% w/w of t he hy dr oalc ohol ic extra cts of B. glabr a, E. globulus and G. attenuatum, respectively. The results of bacterial susceptibility testing for these extracts, as well as for propolis, are reported in Table 1. T he cr ude ex trac ts of E. globulus showed to be the most active one, with MIC values lower than 25 g/mL and up to 50 g/mL (MiD), 50 - 100 g/mL (MaD), and MBC values of <25 - 100 g/mL (MiD), <25 - 250 g/mL (MaD), and diameter of inhibitory zone at 8 - 22.5 mm, against all bacteria assayed (P > 0.05). G. attenuatum turned out to be the second most active extract, with a broad spectral action against all tested strains, and with MIC values of 50 - 200 g/mL, MBC of 100 - 250 g/mL, and diameter of inhibitory zone of 8 - 21.5 mm. Table 1. Antibacterial activity of crude extracts of B. glabra, E. globulus, G. attenuatum, and propolis using different method- ologies. Extacts Tests S. aur. S. aga. L. mon. B. sub. E. coli K. pneu. S. typ . DD 13(**) 13(***) 0 (***) 0 (**) 8 (*) 0 (****) 8 (**) MaDMIC 1000(C) 1000(C) n.a. n.a. 1500(B) n.a. 1000(B) MaDMBC 2000(c) 2000(a) n.a. n.a. 1500(c) n.a. 2000(a) MiDMIC 2500(A) 3000(A) n.a. n.a. 500(C) 2500(A) 1500(A) Bougainvillea glabra MiDMBC 3000(a) n.a. n.a. n.a. 1000(d) 3000(a) 2000(a) DD 22.5(*) 16.5(**) 15(*) 15(*) 8 (*) 15(*) 14.5(*) MaDMIC 100(D) 50(E) 50(B) 100(A) 100(F) 50(E) 100(D) MaDMBC 250(d) 100(c) 100(b) 250(a) 250(f) 100(e) 250(c) MiDMIC 50(E) <25(F) 50(B) <25(C) 50(G) 50(E) 50(E) Eucalyptus globulus MiDMBC 100(f) <25(d) 100(b) 25(c) 100(g) 100(e) 100(d) DD 20(*) 21.5(*) 12(**) 16 (*) 8 (*) 12(**) 8 (**) MaDMIC 100(D) 250(D) 100(A) 100(A) 250(D) 100(D) 250(C) MaDMBC 250(d) 250(b) 250(a) 250(a) 500(e) 250(d) 500(b) MiDMIC 100(D) <25(F) 50(B) 50(B) 200(E) 50(E) 50(E) Gnaphalium attenuatum MiDMBC 150(e) <25(d) 100(b) 100(b) 250(f) 100(e) 100(d) DD 10(***) 12*** 0 (***) 0 (**) 7 (**) 7 (***) 0 (***) MaDMIC n.a. n.a. n.a. n.a. 2000(A) 1000(C) n.a. MaDMBC n.a. n.a. n.a. n.a. 2000(b) 1000(c) n.a. MiDMIC 2000(B) 1500(B) n.a. n.a. 2000(A) 2000(B) n.a. Propolis MiDMBC 2500(b) 2000(a) n.a. n.a. 2500(a) 2500(b) n.a. DD 12 16 15 11 16 19 13 MaDMIC 16 8 12 12 16 12 16 Chloramphenicol MiDMIC 8 4 4 4 8 12 4 DD: disc diffusion method (mm); MaD: macrodilution method (g/mL); MiD: microdilution method with a 96-well microtiter plate (g/mL); S. aur. (Staphy- lococcus aureus), S. aga. (Streptococcus agalactiae), L. mon. (Listeria mono cytogenes), B. sub. (Bacillus subtilis), E. coli (Escher ichia coli), K. pneu. (Kleb- siella pn eumoni a), and S. typ. (Salmonella typhi); n.a.: no active at a concentration of 3000 g/mL. Values pres ented are av erages of t hree r epl icates. V alue s in parentheses in the same column following different asterisk, capital, and lower case letters designate a significant difference, in the DD, MICs, and MBCs values, respectively, by Duncan’s multiple range test, P < 0.05.
Antibacterial Activity of Bougainvillea glabra, Eucalyptus globulus, Gnaphalium attenuatum, and Propolis Collected in Mexico 436 B. glabra crude extract was mildly active against S. aureus, S. agalactiae, E. coli, and S. typhi, with MIC values of 500 - 3000 g/mL (MiD), 1000 - 1500 g/mL (MaD), and MBC of 1000 - 1500 g/mL (MiD) and 1500 - 2000 g/mL (MiD), and diameter of inhibitory zone at 8 - 13 mm (P > 0.05). This extract was not active against L. monocytogenes and B. subtilis at the maximal dosis assessed of 3000 g/mL. Propolis extract resulted to posses a weak activity against S. aureus, S. agalactiae, E. coli, and K. pneumoniae, with MIC values of 1500 - 2000 g/mL, MBC of 2000 - 2500 g/mL, and diameter of inhibi t ory zone of 7 - 1 2 m m. 4. Discussion The microplate and tube macro dilution methods em- ploying tetrazolium chloride dyeing agent, as well as the disc diffusion assay, are currently used methods applied to determine the antibacterial potency of crude extracts derived from natural products [17,18]. In the present work, the three methods were used to compare the activity of some important plants employed in Mexican folk medicine to treat respiratory diseases. These three methods covered a broad intrinsic variability of each technique, and extract complexity, and also permitted the comparison of the data obtained in this study with previously reported literature. In general, the MiD method was more sensitive and quick than the MaD assay, requiring a smaller sample amount. Additionally, we performed the disc diffusion method to compare our results with data from literature. This method is largely used due to it is a reliable, easy and inexpensive suscep- tibility test. In some occasions, the most active extract employing the disc diffusion assay did not agree with that determined by MiD test. The genus Bougainvillae comprises 18 species, and some of them have been used in traditional medicine to treat such disorders as diarrhea, stomach acidity, cough, sore throat, leucorrohea, hepatitis, and as an anti-inflam- matory, antiviral and antibacterial agent [7,8]. Leaves and inflorescence of Bougainvillaea g labra C. have been used in Mexican traditional medicine as a remedy for such respiratory illnesses as cough, cold, bronchitis and asthma [18,19]. Its antimicrobi al effect can be associated with the presence of betalains pigments [20], as well as steroidal compounds with anti-inflammator y activity [2 1]. Gupta et al. (2009) found that a 500 g/disc hydroalco- holic extract from leaves of B. glabra was active against S. aureus (10 mm), B. subtilis (15 mm), E. coli (12 mm), S. typhi (14 mm), and K. pneumoniae (16 mm). These results confirmed the level of activity reported in the present study ag ainst S. aureus (13 mm), E. coli (8 mm), and S. typhi (8 mm). However, the extracts were not active against B. subtilis and K. pneumonia, the most susceptible bacteria reported by Gupta when tested by disc diffusion assay. These differences could be attri buted to a chemical profiling v ariation associated with ch anges in the plant environment. The leaves extract and essen tial oil from Eucalyptus sp. have been used in the traditional medicine of different countries to treat patien ts with respiratory tract infections [10,11]. A study p erformed with E. globulus collected in Morelos, Mexico, using the methanolic extract from the leaves, reported MIC values of 5000 g/mL against S. aureus and 10,000 g/mL against E. coli [9]. These con- centrations are far higher than those MIC values we found in this investigation (S. aureus of 100 g/mL and to E. coli of 100 g/mL, both in the MaD method; and S. aureus of 50 g/mL and to E. coli of 50 g/mL, both in the MiD method). These huge differences can be e x p la i n e d due to intrinsic factors associated w i th th e collec ted plants, as seasonality and particular environments, that cause a change in the metabolic profiling, and consequently, in its activity. Gnaphalium sp. infusion is the most common herbal remedy used in Mexican ethnomedicine in the treatment of both adult and children’s cough and upper respiratory tract infections [12,13]. It has been reported that extracts from aerial parts of G. oxyphyllum, G. liebmannii and G. viscosum, exhibited a wider spectrum of activity against S. aureous, B. cereus, S. typhimurium, and E. coli. As far as we know, there are no antibacterial activities reported for G. attenuatum, a very important medicinal plant found from Mexico to Panama [22]. In our study, this plant was active against all tested bacteria, showing the wider spectrum of action, with high inhibition zones (8 - 21.5 mm) and low MICs ranging between <25 - 200 g/mL (MiD) and 100 - 250 g/mL (MaD); and MBC values of <25 - 250 g/mL (MiD) and 250 - 500 g/Ml (MaD). There are no biological or chemical studies for G. attenuatum, however, Fajardo-Ochoa (2004) reported th at Gnaphalium sp. may conta in sapo nin s with low n a rc o t iz i n g action responsible for lethargy and respiratory failure in newborns [13]. It has been demonstrated that the propolis extract from the state of Campeche, Mexico, was active against P. aeruginosa with a MBC of 3.33 mg/mL, followed by S. aureus ( MBC of 4.82 mg/mL) and Streptococcus pyo ge n es (MBC of 5.38 mg/mL). In that work, S. typhi turned out to be the most resistant bacteria (MBC of 9.82 mg/mL) [5]. Our study shows that propolis from Oaxaca was not active against B. subtilis, L. monocytogenes and S. typhi, with MIC and MBC values investigated at a maximal concentration of 3 mg/mL. However, this propolis extract exhibited MIC values of 1.50 - 2.00 mg/mL and MBC of 2.0 - 2.5 mg/mL against E. coli, K. pneumoniae, S. aureus and S. agalactiae, when tested by MiD method. Copyright © 2012 SciRes. PP
Antibacterial Activity of Bougainvillea glabra, Eucalyptus globulus, Gnaphalium attenuatum, and Propolis Collected in Mexico 437 In the same way that the antibacterial potency of honey varies very markedly, and depends on the flora source of the honey [23], the activity of pro polis resins can change depending on its content of flavonoids and aromatic acids and esters [14]. This variation in the secondary metabolite content can explain why propolis samples collected from various geographical regions of Turkey presented a marked antibacterial activity against the Gram positive strain (Listeria monocytogenes) and a low action against Gram negative bacteria (Salmonella en- teritidis) [24], contrasting with our results. Although the propolis extracts from Campe che and Oaxaca have shown to be less potent than the plants extracts investigated in this work, the use of propolis resin in combination with plant extracts can be supported by the fact that propolis have been demonstrated to potentiate the effect of certain antimicrobial drugs [25], as well as to prevent or reduce the development of tolerance to antibiotics [14]. The microplate method using color detection by formation of formazan derivative (with red color) showed to be better than the other two, displaying high active values. It was the most sensitive, less expensive and minor time-con- suming technique compared with the macrodilution as- say. 5. Conclusions Extracts prepared from aerial parts of Bougainvillea glabra, Eucalyptus globulus, and Gnaphalium attenuatum, as well as propolis are frequently commercialized in Mexico as ingredients of a variety of syrups, candies, and other remedies destined to relieve the symptoms of upper respiratory tract infections like cough and sore throat. Ethnobotanical stud ies strongly support their uses, which were confirmed by the present study, revealing that E. globulus and G. attenuatum extracts were the most active, showing broad spectrum activity, in contrast with B. glabra and propolis extracts which were less active and more selective in action. Here, we present the first report for the antibacterial activity of the aerial parts of G. attenuatum against a panel of Gram positive and negative bacteria. There was a good correlation between the macro and micro dilution methods, th e latter being faster, more sensitive and requiring less sample amount. 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