Ethnopharmacological versus random plant selection methods for the evaluation of the antimycobacterial activity

Ethnopharmacological versus random plant selection methods for the evaluation of the antimycobacterial activity

Danilo R. Oliveira^I,*; Gilda G. Leitão^I; Tatiane S. Coelho^II; Pedro E. Almeida da Silva^II; Maria Cristina S. Lourenço^III; ARQMO^IV; Suzana G. Leitão^V

^INúcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco H, Brazil

^IILaboratório de Micobactérias e Biologia Molecular, Faculdade de Medicina, Fundação Universidade Federal do Rio Grande, Brazil

^IIIInstituto de Pesquisa Clínica Evandro Chagas, Serviço de Bacteriologia, Setor de Testagem de Drogas, Fundação Oswaldo Cruz, Brazil

^IVAssociação de Comunidades Remanescentes de Quilombos do Município de Oriximiná, Brazil

^VFaculdade de Farmácia, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco A 2ºandar, Brazil

ABSTRACT

The municipality of Oriximiná, Brazil, has 33 quilombola communities in remote areas, endowed with wide experience in the use of medicinal plants. An ethnobotanical survey was carried out in five of these communities. A free-listing method directed for the survey of species locally indicated against Tuberculosis and lung problems was also applied. Data were analyzed by quantitative techniques: saliency index and major use agreement. Thirty four informants related 254 ethnospecies. Among these, 43 were surveyed for possible antimycobacterial activity. As a result of those informations, ten species obtained from the ethnodirected approach (ETHNO) and eighteen species obtained from the random approach (RANDOM) were assayed against Mycobacterium tuberculosis by the microdilution method, using resazurin as an indicator of cell viability. The best results for antimycobacterial activity were obtained of some plants selected by the ethnopharmacological approach (50% ETHNO x 16,7% RANDOM). These results can be even more significant if we consider that the therapeutic success obtained among the quilombola practice is complex, being the use of some plants acting as fortifying agents, depurative, vomitory, purgative and bitter remedy, especially to infectious diseases, of great importance to the communities in the curing or recovering of health as a whole.

Keywords: bioprospecting, quilombola communities, traditional knowledge, ethnopharmacological, random approach

Introduction

Tuberculosis (TB) is the main cause of death by infectious illnesses in the world. It is estimated that approximately 1/3 of the world population may be infected with the TB bacillus, Mycobacterium tuberculosis (Singh, 2004; Maher & Raviglione, 2005). According to data of the Brazilian Ministry of Health (MS/SVS, 2005), Brazil has an average incidence rate of 43,78 cases of TB per 100,000 inhabitants; and in the Pará state the average is of 50,15 cases per 100,000 inhabitants. In 2005, the municipality of Oriximiná presented an alarming number of TB cases, 95,98 cases per 100,000 inhabitants (MS/UFPA, 2009). This heterogeneous geospatial distribution of TB cases in different regions of the country keeps a close relationship to their socioeconomic conditions. In the Amazon region, major part of the population suffers from chronic problems of malnutrition, precarious habitation and sanitation. In addition, the forest people who live in remote areas, far from civilization, have great difficulty in accessing primary health care (Basta et al., 2004). Another factor that has favored the higher incidence of TB in the North is the fact that there live about 60% of the indigenous population and that they have suffered real epidemics of the disease with an incidence 10 times higher in relation to TB average of the Brazilian population (Coimbra Jr. & Basta, 2007).

The progression of TB is mainly governed by the integrity of the host immune response, which may be effective in the microbial control shown by the action of macrophages, CD4 lymphocytes, interferon gamma and tumor necrosis factor. But in many cases, the bacillus cannot be destroyed and can remain in dormant state for years. Between 90 to 95% of those infected people fail to express the active form of the disease. However, others manifest the disease, usually in a condition of immunosuppression (Ducati et al., 2006; Barker, 2008). The inflammatory and pathologic processes produce symptoms typical of TB such as fever, weakness, weight loss, night sweats, chest pain, dyspnea, and cough. The pathology in advanced stages can also lead to the rupture of blood vessels causing hemoptysis (Baker, 2008).

TB has been considered a neglected disease, with little investment in research to develop new drugs. This is changing recently, according to the gravity and danger that TB represents, with its high rate of morbidity and mortality and the unavailability of new drugs to control and cure patients with resistant strains to multiple drugs (MDR) and extensively resistant to multiple drugs (XDR) (Rivers & Mancera, 2008).

Medicinal plants have attracted great interest as a source for finding new efficient drugs or prototypes to TB treatment, and the literature is covered with many examples of plant selection methods based on random or ethnopharmacological approaches (Cantrell et al., 1998; Newton et al., 2002; Leitão et al., 2006; Gautam et al., 2007; McGaw et al., 2008; Ramos et al., 2008; Mohamad et al., 2011).

The vast Amazonian biodiversity together with the traditional knowledge of the forest people could represent a potential source for the discovery of new therapeutic agents, including against TB. The city of Oriximiná, Pará State, Brazil, has 33 quilombola communities (Oliveira et al., 2010). These communities are ethnic groups with historical background, specific territorial relations, and presumption of black ancestry related to resistance to oppression suffered historically. In the late 18^th and early 19^th centuries the slaves that were used on cocoa, coffee, and cotton plantations as a labor force, fled to remote areas, especially to regions of lakes and waterfalls that were difficult to access. Many of these communities are still in full contact with the natural biodiversity of regions far from the urban area of Oriximiná. Their close contact with nature over centuries, the knowledge formed from an Indian-Black-Portuguese complex, and their geographic isolation, have brought to the members of these communities a vast knowledge of medicinal plants (Oliveira et al., 2011a).

In December 2007, the Federal University of Rio de Janeiro obtained the first approval in Brazil to access the traditional knowledge for bioprospecting purposes in quilombola communities from Oriximiná (Oliveira et al., 2010). This work reports part of the vast knowledge of the quilombola people from Oriximiná (Oliveira, 2009) and aimed at the bioprospection of the medicinal plants used by them for TB-related diseases by ethnopharcological approach and a comparison between random plant selection methods for the evaluation of the antimycobacterial activity.

Material and Methods

Characterization of the search area

The municipality of Oriximiná is located in northern Brazil, in the State of Pará, and has an area of 107,603 km², being the second largest municipality in the Brazilian territory. It borders Suriname, Guyana, and French Guiana to the north, the cities of Faro, Juruti, and Óbidos to the South and East, and the States of Amazonas and Roraima to the West. According to data from the 2010 census, Oriximiná has 62,963inhabitants, being 40,182 in urban areas and 22,781 in rural areas (IBGE, 2010).

Currently, there are 33 known "quilombola" communities in the municipality of Oriximiná, which are divided into eight territories (Água Fria, Boa Vista, Trombetas, Erepecuru, Alto Trombetas, Jamari/Último Quilombo, Moura, and Ariramba) that, together, encompass more than 600,000 hectares (Figure 1). The "quilombolas" were represented by their association called Associação de Comunidades Remanescentes de Quilombos do Município de Oriximiná - ARQMO (Association of the Remaining of "Quilombo" Communities from the Municipality of Oriximiná). In this work, five communities, representing two "quilombola" areas were chosen: Bacabal and Arancuã-de-Cima from the Trombetas region, as well as Serrinha, Jauari, and Pancada, from the Erepecuru region (Figure 1).

Ethnopharmacological data collection

This work has received authorization for access to the traditional knowledge associated with bioprospecting purposes by the Directing Council of Genetic Heritage (Conselho de Gestão do Patrimônio Genético), through the Resolution no. 213 (6.12.2007), published in the Federal Official Gazette of Brazil on 27 December 2007 (Oliveira et al., 2010).

The selection of the interviewees began with the search for key informants who were respected people in the community, such as the community coordinator, matriarch or patriarch, and/or community health agent. Eventually, they led to the local specialists, who were "quilombolas" with wide experience in the use of medicinal plants, such as extractivists, woodsmen, healers, faith healers, prayer ladies, midwives, and "puxadores" or "puxadoras", who are like traditional chiropractors. For data acquisition four ethnobotanical field trips were performed between the period of June 2006 (after signing the Prior Informed Consent between UFRJ and ARQMO), until the month of September 2008. Each field work had a residence period of 30 to 60 days in the communities studied.

Ethnobotanical data was collected through semi-structured interviews, participating observation and walk in the woods (Albuquerque & Lucena 2004). The formularies applied contain socio-economic data (sex, age, professional, level of schooling, monthly family income, number of residents) and the medicinal plants information (common name, therapeutic indications, doses, preparation methods, counterindications and the local to obtain it). Thirty five quilombolas from the five communities studied, twenty women and fifteen men, were interviewed. Participants included four women and two men from Arancuã-do-meio, nine women and two men from Bacabal, three women and five men from Jauari, one women and four men from Pancada and three women and two men from Serrinha. They were between 19 and 87 years old. They survive mainly by fishing, hunting, and subsistence farming, and their only source of income is the extraction of the Pará nut ("Brazil nut"), which is available for only a few months of the year.

Quantitative data analysis techniques, such as the free-list salience index (S), and corrected major use agreement (MUAc), were also applied.

Free-List and Salience Index (S)

The free-list technique can identify items within an emic category or a cultural domain, and it offers a direct method to obtain data easily and simply. It is also used as an exploratory technique (Oliveira et al., 2011a,b). Using this technique, a direct list of the medicinal plants known and used by the informers was obtained, with the aim of searching for specific information on this cultural domain within the communities and its diffusion. Additionally, an ethnodirected inquiry regarding the plants used against TB and lung problems was conducted employing a glossary with the emic illnesses or symptoms like cough, dry cough, "whooping cough" ("tosse braba"), pneumonia, "weakness", tuberculosis, lung problem or illness, and infectious diseases of chest These therapeutic terms used locally were obtained in a previous study carried out in Oriximiná city (Oliveira, 2004) and through participant observation and ethnographic techniques. Taken together, these approaches suggest the most important cultural elements and the order of their importance (Albuquerque & Lucena, 2004). The S was calculated using the program ANTHROPAC 4.0 (Analytic Technologies, USA).

Major Use Agreement (MUA):

The Major Use Agreement (MUA) is a quantitative technique used to evaluate the agreement between the main uses cited by various informants (Oliveira et al., 2006). It is determined by the ratio between the number of informers who independently cited the species for a major use (MU) and the total number of informers who mentioned that species for any use (total uses, TU_s).

A correction factor (CF) was applied to calculate the corrected MUA (MUA_c), given by the formula: MUA_c = MUA x CF. The correction factor is the ratio between the number of informers citing each species for any use (TU_s) and the highest number of informers citing the most-cited species. In this study, Dipteryx odorata was the most-cited species and was cited by 24 informers (TU_s+) (Oliveira et al., 2011a).

Collection and identification of plant material

Plants were collected by one of the authors (DRO). with informants and woodsmen of the "quilombola" communities. Specimens were deposited at the Herbarium of the Amazonia National Institute of Research (INPA), where the parataxonomist Mr. José Ferreira Ramos, and specialists in specific botanical families, such as Dr. Fátima Salimena (Verbenaceae), Dr. Washington Marcondes-Ferreira (Apocynaceae), and Dr. Kátia Calago (Asteraceae), provided botanical identifications. Voucher numbers of plants collected by ethnopharmacological approach are listed in Table 1. Voucher number for plants collected by random approach: Acrocomia aculeata (Jacq.) Lodd. ex Mart. (INPA 224624), Ampelozizyphus amazonicus Ducke (INPA 224161), Anacardium giganteum W.Hanc. exEngl. (INPA 224700), Aspidosperma excelsum Benth. (INPA 224704), Aspidosperma rigidum Rusby (INPA 224692), Bauhinia platycalyx Benth. (INPA 223277), Brosimum potabile Ducke (INPA 224693), Brosimum sp. (without voucher number), Couepia paraensis (Mart.&Zucc.) Benth. (INPA 224627), Cucurbita moschata (Duch.) Duch.e xPoir. (INPA 223273), Endopleura uchi (Huber) Cuatrec. (INPA 224690), Machaerium ferox Mart. (INPA 233440), Operculina alata (Ham.) Urb. (INPA 223281), Simaba cedron (Rol) Ward (INPA 223283), Spondias mombin L. (INPA 224699), Trichilia quadrijuga Kunth (INPA 224620), Uncaria guianensis (Aubl.) Wild. (INPA 224608), Virola surinamensis (Rol) Ward. (INPA 224143).

Selection the plant species and the preparation of the extracts

The ETHNO plants (ten species) were selected from the free-listing for TB; and related diseases and the RANDOM pants (eighteen species) were selected by the other important medicinal uses in the communities, but not used for lung disturbances.

Plant materials used for the preparation of extracts (Table 2) were air-dried, powdered, and macerated with ethanol to make ethanolic extracts. Aqueous extracts were obtained by decoction prepared at 1% (w/v), approaching the traditional method of use. Saps and latex were obtained directly from incisions or perforations of the plants. Teas, saps, and latex were freeze-dried.

Isolates and strain preparation of Mycobacterium tuberculosis

Mycobacterium tuberculosis H₃₇Rv (ATCC27294) pan-susceptible strain was used for all experiments in both microbiology laboratories (FURG and Fiocruz). Additional assays with a rifampicin-resistant strain (ATCC 35338, His-526-Tir) were performed at the FURG laboratory. The isolates were maintained in Ogawa-Kudoh medium for ca. 14 days. The bacterial suspensions were prepared in sterile water containing 3-mm glass beads. The suspensions were homogenized by vortex agitation and turbidity was adjusted in agreement with tube one of the McFarland scale (3.2 x 10⁶ colony-forming units/mL). The inoculums were prepared by diluting the bacterial suspension 1:20 in Middlebrook 7H9 OADC medium (4.7 g Middlebrook 7H9 base; Difco, Becton Dickinson USA) enriched with 10% (v/v) oleic acid-dextrose-albumincatalase (BBL).

In vitro evaluation of antimycobacterial activity

Plant extracts selected by ethnodirected method (ETHNO) obtained with the free-list and by random method (RANDOM) were screened against M. tuberculosis H₃₇Rv strain by microdilution method using resazurin as indicator of cell viability. Initially, a fixed concentration of 200 µg/mL was used. Media plus bacteria, with and without rifampicin, were used as positive and negative controls, respectively. For the active samples and the positive control, the minimum inhibitory concentration (MIC) was determined.

Data analyses

The Chi-square Test and Fisher Exact Test were used to analyze differences between the positive and negative results obtained by the ETHNO and RANDOM, employing the statistical package SigmaStat 3.0 (Jandel Scientific, USA).

Results and Discussion

Thirty four individuals were interviewed, who reported 254 ethnospecies and a total of 2,508 use indications. Among these, 233 plant species were identified, belonging to 211 genera and 72 botanical families (Oliveira et al., 2011a). Another survey was conducted to assess the plants used by the "quilombolas" to treat TB and TB-related diseases and symptoms. In the communities, there isn't a clear perception and clinical diagnosis of TB. In this way, an ethnodirected method of free-list focusing on cough, dry cough, "whooping cough" ("tosse braba"), pneumonia, "weakness", tuberculosis, lung problem or illness, and infectious diseases of chest was employed, which led to the survey of 43 ethnospecies, displayed in Table 1, listed in order of their Salience (S). They are distributed into 29 botanical families, being Fabaceae the most well represented family (18.3%), principally for subfamily Caesalpinioideae.

Out of these 43 ethnospecies, only seven were cited by more than 20% of the interviewees (Frequency - FR>20%) for TB-related diseases; seven displayed a Salience value (S) above 0.1; and eight had a MUAc value higher than 25%. So, at least five ethnospecies - represented by seven identified plant species have potential for TB bioprospecting by ETHNO. They are: Dipteryx odorata (S=0,541; MUAc=79,2%; Fr=64,5%), Chenopodium ambrosioides (S=0,24; MUAc=45,8%; Fr=45,2%), Hymenaea courbaril, H. intermedia, H. oblongifolia (S=0,179; MUAc=41,7%; FR=29%), Plectranthus amboinicus (S=0,163; MUAc=37,5%; Fr=25,8%) and Cereus sp (S=0,12; MUAc=41,7%; Fr=22,6%).

The symptoms of TB appear confused with other bronchial and respiratory diseases as well as malnutrition and weakness. Moreover, older people in these communities refer to TB as the "weakening". The body weakness as a factor for the development of TB, in the view of traditional communities, is reported by other authors (Maués, 1990; Santos & Muaze, 2002), and is extremely well discussed globally that TB is a disease that manifests itself often in cases of disability or low immune system, especially in the case of individuals with Acquired Immune Deficiency Syndrome (AIDS) and those who suffer from chronic malnutrition. Therefore, many times, the indicated remedies are the same one to treat respiratory problems in general and to treat anemia and to fortify the body.

Beyond the medicinal plants, a curious question is the use of the "queixada" tooth for treatment of pneumonia or TB in the quilombola communities of Oriximiná. This use has also been reported also in Amazonian communities of the valley of the rivers Purus and Acre (Santos & Muaze, 2002). "Queixada" (Tayassu pecari Link.) it is an Amazonian wild boar frequently used in the feeding of the communities studied. They can be very aggressive when confronted. When angry, it snaps its teeth in order to warn the intruder that it is about to attack. The use of teeth in the treatment of "weakening" probably gives the strength and stiffness of the teeth of the species, as well as the dry and shrill sound emitted by queixada when it is angry or threatened. The tooth should be dried (or toasted) and ground to then be used alone or in combination with some of the plants listed in Table 1.

As a result of the plant use information for the lung disturbances and related diseases, ten species were tested by ethnodirected method (ETHNO) and eighteen by random method (RANDOM) for antimycobacterial activity. In the screening eight species gave positive results with MIC varying between 12,5-200 µg/mL, being five selected by ETHNO, and three by RANDOM (Table 2). The best result was obtained by ETHNO, because 50% of the species tested had some activity, while by RANDOM only 16.7% of species had some activity (Table 2). Most of the natural products from plant sources do not display strikingly potent activities (Newton et al., 2000), and for most of them, published MIC ranges of substances considered to show modest activity lay between 50 to 216 µg/ml (Okunade et al., 2004). Among all positive samples Dipteryx odorata (ETHNO) and Aspidosperma spp. (RANDOM) showed MIC below 25 µg/mL, which can be considered a good activity for ethanolic crude extracts when compared with the antimycobacterial literature for medicinal plants. Five plant extracts (Dipteryx odorata, Campsiandra comosa, Machaerium ferox, Aspidosperma excelsum and A. rigidium) were active against both strains of M. tuberculosis indicating the absence of a cross-resistance to RMP when the molecular basis of resistance is rpoB His-526-Tir mutation (Table 2). Importantly, this mutation is one of the most common in RMP resistan strains. In this study the target and possible antimicrobial mechanism of action these extracts was not identified, although it is possible that the antimicrobial activity is due to a single molecule or could also be the result of a synergic action among various compounds.

The ethnopharmacological data show the importance of Dipteryx odorata (cumarú = tonka been) in the treatment of pulmonary illnesses, by the highest salience index (S=0.541), corrected major use agreement (MUAc=79.2%) and frequency of citation (RF=64.5%). These data are consistent with the good antimycobacterial activity obtained for the crude ethanol extract from their barks (MIC 12.5 µg/mL). However, the seeds did not show positive results at the concentrations tested.

From the barks of Dipteryx odorata were isolated the triterpene lupeol, a mixture of fatty acid methyl esthers, the flavonoid luteolin, plus several isoflavonoids (Nakano & Suárez, 1969; Hayashi & Thompson, 1974; Nakano et al., 1979; Nakano & Yoshimura, 1979; Socorro et al., 2003). The MIC of 12.5 µg/mL obtained for cumarú barks, against both strains of M. tuberculosis, may be related to the presence of diterpenes, triterpenes, fatty acids and isoflavones, since these natural product classes have several representatives with known antimycobacterial activity described in the literature (Cantrell et al., 2001; Copp, 2003; Okunade et al., 2004; Carballeira, 2008). Considering that the cumarú positive sample was a crude ethanolic extract, this result becomes very promising for the identification of bioactive substances. Aspidosperma rigidum and Aspidosperma excelsum, known as carapanaúba, are rich in indole alkaloids (Vieira et al. 2010) and antimycobacterial activity has already been recently demonstrated for several substances of this class (Copp, 2003; Okunade et al., 2004), as well as for other species of the Apocynaceae family (Case et al., 2006; Gautam et al., 2007; Ramos et al., 2008; Mohamad et al., 2011).

Some species such as Allium cepa, Anacardium occidentale, Cynnamomum zeylanicum, Jatropha curcas, Psidium guajava and Zingiber officinale in Malaysian (Mohamad et al., 2011), besides Chenopodium ambrosioides, Ruta graveolens, Ocimum americanum, Allium sativum and Mangifera indica in Índia (Gautam et al., 2007) are also indicated as a possible source of new antimycobacterial agents, and in South Africa, Ruta graveolens has also highlighted its potential (McGaw et al., 2008).

It is expected that the traditional knowledge about medicinal plants indicates the presence of biologically active substances. The collection of plants for biological testing from its traditional use can be a great advantage, or a shortcut, increasing the chances of discovering new drugs (Elisabetsky & Shanley, 1994; Soejarto, 1996). Table 3 shows the enormous potential of the ethnopharmacological approach found in several studies, compared with the randomized.

In this study, the best results for antimycobacterial activity were also obtained with the ethnopharmacological approach -50% ETHNO x 16,7% RANDOM (Table 2), however, because of the limited number of the samples, it was not found significant difference (p>0,05) between these approaches by the use of the Chi square and Fisher exact tests. Dipteryx odorata is the species with the highest salience index and showed a good antimycobaterial activity (MIC 12.5 µg/mL). This result points out a preference towards the ethnopharmacological information. In recent work published by our group (Oliveira et al., 2011a), the ethnodirected approach using the salience index and the major use agreement also improved the probability of finding activity species for acetylcholinesterase inhibition. In contrast, the study of Case et al. (2006) that used the informant consensus model (informant agreement ratio) to select plants used in traditional medicine for persistent respiratory symptoms among the Manus (Papua New Guinea), was inaccurate in predicting antimycobacterial activity plants. However, these authors related that "Due to the complexity of the human body, it cannot be assumed that in vitro bioassay results translate to human systems, for either a positive or negative result. Consequently, the species identified in the survey may be beneficial in the treatment of TB for many reasons apart from direct antimycobacterial activity, e.g., they may provide symptomatic relief from cough or have immunostimulatory effects. It must also be noted that the in vitro test may not be predictive of activity in vivo. With infinite resources, samples could be submitted to other relevant assays such as immune-modulating assays to better understand and assess the biological activity of traditional medicine" (Case et al., 2006). Oliveira et al. (2011a,b) discuss that some plants could have an adaptogen effect that contribute to quilombola health by an unspecific way. Some plants have the role of panacea, being used to cure all ills.

In the "quilombola" communities of Oriximiná TB is often called the "weakening". So, the ETHNO's good results can be even more significant considering that the therapeutic practices among the "quilombolas" from Oriximiná are complex. The use of fortifying agents, depuratives, vomitory agents, purgatives, bitter remedies, as well as curing infectious diseases, weakness, and memory loss, play an important role in the processes of curing diseases and/or health recovery, acting as order to restore overall health (Oliveira et al., 2011a,b). Similar data were surveyed by Rodrigues & Carlini (2004; 2006) in the Sesmaria quilombola community in the State of Mato-Grosso, in a transition area between Cerrado and Pantanal biomes, where certain species are characteristic for their versatility, or nonspecific therapy, is also employed for rejuvenation, to energize, to muscle building and to fortify the brain. Another study at the Pará State also showed a substantial number of general "cure alls" or panaceas, fortifiers, tonics, nerve tonics and aphrodisiacs, as a reflex of the caboclo culture (Branch & Silva, 1983; Berg, 1984; Amoroso & Gély, 1988).

A broad literature review about the 43 cited ethnospecies, used against TB and TB-related diseases and symptoms, revealed that 93% of them have been described as useful for the treatment of respiratory diseases and 86% were indicated as tonic and stimulant, corroborating with the ETHNO information (Table 4).

Acknowledgements

This work was supported by CNPq and FAPERJ. Authors wish to thanks Lucia Andrade from Comissão Pró-Índio de São Paulo for providing original files of the map of the quilombola region, upon which a new one was built by Paula S. de O. Barbosa. Carlos Bêta and Mira Carvalho, directors of Unidade Avançada José Veríssimo, of the Universidade Federal Fluminense, located in Oriximiná, contributed with infrastructure used for this project. We are especially thankful to the quilombolas who provided housing for the researchers involved in this ethnoknowledge study.

Received 23 Dec 2010

Accepted 8 Mar 2011

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Publication Dates

• Publication in this collection
  20 May 2011
• Date of issue
  Oct 2011

History

• Received
  23 Dec 2010
• Accepted
  08 Mar 2011