Minimum inhibitory concentrations of medicinal plants used in Northern Peru as antibacterial remedies

Aim

The plant species reported here are traditionally used in Northern Peru to treat bacterial infections, often addressed by the local healers as “inflammation”. The aim of this study was to evaluate the Minimum Inhibitory Concentration (MIC) of their antibacterial properties against Gram-positive and Gram-negative bacteria.

Materials and methods

The antimicrobial activity of ethanolic and water extracts of 141 plant species was determined using a deep-well broth microdilution method on commercially available bacterial strains.

Results

The ethanolic extracts of 51 species inhibited Escherichia coli, and 114 ethanolic extracts inhibited Staphylococcus aureus. In contrast, only 30 aqueous extracts showed activity against E. coli and 38 extracts against S. aureus. The MIC concentrations were mostly very high and ranged from 0.008 to 256mg/ml, with only 36 species showing inhibitory concentrations of <4mg/ml. The ethanolic extracts exhibited stronger activity and a much broader spectrum of action than the aqueous extracts. Hypericum laricifolium, Hura crepitans, Caesalpinia paipai, Cassia fistula, Hyptis sidifolia, Salvia sp., Banisteriopsis caapi, Miconia salicifolia and Polygonum hydropiperoides showed the lowest MIC values and would be interesting candidates for future research.

Conclusions

The presence of antibacterial activity could be confirmed in most species used in traditional medicine in Peru which were assayed in this study. However, the MIC for the species employed showed a very large range, and were mostly very high. Nevertheless, traditional knowledge might provide some leads to elucidate potential candidates for future development of new antibiotic agents.

1. Introduction

In developing countries, Traditional Medicine (TM) is often the only accessible and affordable treatment available. In Latin America, the World Health Organization (WHO) Regional Office for the Americas (AMRO/PAHO) reports that 71% of the population in Chile and 40% of the population in Colombia have used TM. In many Asian countries TM is widely used, even though Western Medicine is often readily available. In the US the number of visits to providers of Complementary Alternative Medicine (CAM) now exceeds by far the number of visits to all primary care physicians (WHO 1999a,b; 2002), and CAM is becoming increasingly popular in many developed countries (WHO 1998), and a US survey reported the use of at least one of 16 alternative therapies increased from 34% in 1990 to 42% in 1997 (UNCCD 2000).

The expense for the use of TM and CAM is growing exponentially in many parts of the world. The 1997 out-of-pocket CAM expenditure was estimated at $ 2.7 billion in the USA. The world market for herbal medicines based on traditional knowledge is now estimated at US$ 60 billion (Breevort 1998).

Peru is a country rich in biodiversity. For millennia, traditional healers have used the rich flora to cure ailments. The same plants are still being used today. TM continues to be very popular since a large part of the population has either no access to, or no resources to afford Western Medicine. Bacterial infections and inflammation are among the ailments treated by traditional healers. Northern Peru is believed to be the center of the Central Andean Health Axis (Camino 1992), and TM practices in this region remain an important component of everyday life (Bussmann and Sharon 2006; De Feo 1992; Joralemon and Sharon 1993; Polia 1988; Revene et al. 2008; Sharon 1978, 1980, 1994, 2000; Sharon and Bussmann 2006). TM is also gaining more and more respect by national governments and health providers. Peru’s National Program in Complementary Medicine and the Pan American Health Organization recently compared Complementary Medicine to allopathic medicine in clinics and hospitals operating within the Peruvian Social Security System (EsSalud 2000). The WHO has expressed high interest in TM. It is important to demonstrate scientifically that the remedies employed in folk medicine are indeed therapeutically active (Baker et al. 1995; Cox and Balick 1994; Elisabetsky and Castilhos 1990; Farnsworth et al. 1985; Muñoz and Sauvain 2002).

Plants with potential medicinal activity have recently come to the attention of Western scientists, and studies have reported that some are bioactive (e.g. Perumal Samy and Ignacimuthu 2000). Potentially active compounds have been isolated from a few of the plants tested (D’Agostino et al. 1995a,b; Okuyama et al. 1994; Rodriguez et al. 1994; Umana and Castro 1990). Plant species from the Cordillera Blanca in Peru, demonstrated antimicrobial, anti-cancer, and wound-healing activities (Bussmann et al. 2008, 2009a,b; Hammond et al. 1998;, Lee at al. 1999; Neto et al. 2002; Villegas et al. 1997). However, despite the fact that the center of healing traditions in Northern Peru is located in the Trujillo/Chiclayo coastal region, no in-depth studies had been undertaken.

In this communication we report on antibacterial assays for 141 plant species with a wide range of traditional uses.

2. Materials and Methods

2.1. Plant Material

Plants were collected in Northern Peru (Fig. 1) in the field, in markets, and at the homes of traditional healers (curanderos) during August-September 2001, July-August 2002, July-August 2003, June-August 2004, July-August 2005, July-August 2006, June-August 2007, June-August 2008, March-April 2009 and June-August 2009. The specimens are registered under the collection series “JULS,” “ISA,” “GER,” “EHCHL,” “RBU/PL,” “ACR,” “KMM,” and AKT,” depending on the year of fieldwork and collection location. Surveys were conducted in Spanish by fluent speakers. Surveyors would approach healers, collectors and market vendors and explain the premise for the study, including the goal of conservation of medicinal plants in the area. All were asked to participate, but due to expected resistance information could not be recorded from everybody. From those who gave their prior informed consent, information was collected regarding their knowledge and inventory of medicinal plants.

Figure 1

Study area: Peruvian Departments of Amazonas, Piura, Lambayeque, La Libertad, Cajamarca, San Martin, and the Ecuadorian Province of Loja.

Vouchers of all specimens were deposited at the Herbarium Truxillense (HUT, Universidad Nacional de Trujillo), and Herbario Antenor Orrego (HAO, Universidad Privada Antenor Orrego, Trujillo). In recognition of Peru’s rights under the Convention on Biological Diversity, most notably with regard to the conservation of genetic resources in the framework of a study treating medicinal plants, the identification of the plant material was conducted entirely in Peru. No plant material was exported in any form whatsoever.

2.1. Plant Material

Plants were collected in Northern Peru (Fig. 1) in the field, in markets, and at the homes of traditional healers (curanderos) during August-September 2001, July-August 2002, July-August 2003, June-August 2004, July-August 2005, July-August 2006, June-August 2007, June-August 2008, March-April 2009 and June-August 2009. The specimens are registered under the collection series “JULS,” “ISA,” “GER,” “EHCHL,” “RBU/PL,” “ACR,” “KMM,” and AKT,” depending on the year of fieldwork and collection location. Surveys were conducted in Spanish by fluent speakers. Surveyors would approach healers, collectors and market vendors and explain the premise for the study, including the goal of conservation of medicinal plants in the area. All were asked to participate, but due to expected resistance information could not be recorded from everybody. From those who gave their prior informed consent, information was collected regarding their knowledge and inventory of medicinal plants.

Figure 1

Study area: Peruvian Departments of Amazonas, Piura, Lambayeque, La Libertad, Cajamarca, San Martin, and the Ecuadorian Province of Loja.

Vouchers of all specimens were deposited at the Herbarium Truxillense (HUT, Universidad Nacional de Trujillo), and Herbario Antenor Orrego (HAO, Universidad Privada Antenor Orrego, Trujillo). In recognition of Peru’s rights under the Convention on Biological Diversity, most notably with regard to the conservation of genetic resources in the framework of a study treating medicinal plants, the identification of the plant material was conducted entirely in Peru. No plant material was exported in any form whatsoever.

2.2. Nomenclature

The nomenclature of plant families, genera, and species follows the Catalogue of the Flowering Plants and Gymnosperms of Peru (Brako and Zarucchi 1993) and the Catalogue of the Vascular Plants of Ecuador (Jørgensen and León-Yanez 1999). The nomenclature was compared to the TROPICOS database (Tropicos, 2010). Species were identified using the available volumes of the Flora of Peru (McBride 1936–1981), as well as Jørgensen and Ulloa Ulloa (1994), Pestalozzi (1998) and Ulloa Ulloa and Jørgensen (1993), and the available volumes of the Flora of Ecuador (Sparre and Harling 1978–2009), and reference material in the herbaria HUT, HAO, QCA, LOJA and QCNE.

2.3. Preparation of Extracts

For each species tested, above ground material (in case of trees leaves or bark as indicated by the collaborating healers) was collected, and the entire material used for extract preparation. This corroborates with the traditional preparation (Bussmann and Sharon 2006). Plant material was dried at 35°C for three days. After drying, the material was ground with an industrial grinder, and 2 samples of 5g. of plant material each were weighted out. One sample was submerged in 100ml of 96% ethanol and left to macerate for 7 days, while another sample was submerged in 100ml of boiling distilled water and left to macerate for 24h. After maceration the plant material was filtered and 100 ml 96% ethanol was added to the water extracts to allow faster solvent removal. The solvent was then evaporated to complete dryness using a standard Buchi rotary-evaporator. The resulting dry extracts were re-suspended in 5 ml distilled water. In order to determine the real concentration of each extract, 1ml of previous homogenization of the respective extracts was removed and again completely oven-dried and then weighed to determine amount of extract per ml of final solution. The remaining extract was used for MIC assays.

2.4. Antimicrobial assays

2.4.1. Bacteria and culture media

Staphylococcus aureus ATCC 25923 (Gram-positive) and Escherichia coli ATCC 25922 (Gram-negative) were used for the current study. Bacterial cultures were grown on 5% sheep red blood agar (SBA). Following the initial incubation, organisms were suspended in 10ml of physiological saline solution and optical density readings were compared to a 0.5 McFarland standard. For the MIC determination bacterial solutions of 5×10 ^{colony-forming units (cfu) ml were employed.}

2.4.2. Minimal inhibitory concentration (MIC) determination

The antibacterial activity of the plant extracts was determined using sterile 2ml 96-well plates (Wiegand et al. 2008). The 12 wells of each row were filled with 0.5 ml sterilized Mueller Hinton agar. Sequentially, wells 2–11 received an additional 0.5 ml of a mixture of culture medium and plant extract serially diluted to create a concentration sequence from 0.512 ml to 0.008 ml. Well 1 served as growth control, well 12 as antibiotic control. Tetracycline Hydrochloride (0.1mg/ml) and Amoxicillin (0.1mg/ml) were used as controls for the S. aureus and E. coli assays respectively. The respective antibiotics were chosen because they are often employed as first line antibiotics in the respective bacterial infections. The MIC of Tetracycline Hydrochloride (for S. aureus assays) was 0.25 μg/ml and the MIC of Amoxicillin (for E. coli assays) was 8 μg/ml. The deep-wells were incubated for 24h at 37°C. The resulting turbidity was observed, and after 24h MIC was determined to be where growth was no longer visible by assessment of turbidity by optical density readings at 600nm with a Beckman DU-70 UV-Vis Spectrophotometer. At least three repetitions were run for each assay. Strong activity was defined as MIC < 5 mg/ml.

3. Results and Discussion

The selection of plant material for this study was based on ethnobotanical data on the traditional use of the plants in treatment of bacterial diseases, and conditions classified by the traditional healers as “infection” and “inflammation,” the latter characterized by reddening (e.g. in wounds), or internal afflictions causing gastric discomfort (Table 1). The plant species were initially tested in simple agar-bioassays, which included plants that are used or other purposes by the local population (Bussmann et al. 2007, 2008, 2009a,b). The initial testing yielded 141 species with antibacterial activities which were chosen for this study to establish their MIC values. Because many traditional preparations are prepared by maceration in ethanol or water, we tried to use both extraction methods to prepare the starting extracts for this study.

Table 1 shows the antibacterial activity of Northern Peruvian medicinal plants against Gram-positive and Gram-negative bacteria. The extracts were subjected to the determination of MIC values. The ethanolic extracts of 51 species inhibited Escherichia coli, and 114 ethanolic extracts inhibited Staphylococcus aureus. In contrast, only 30 water extracts showed activity against E. coli and 38 extracts against S. aureus. The MIC concentrations ranged from 0.008 to 256mg/ml. The very high values in many species indicate only a very limited antibacterial efficacy. The ethanolic extracts exhibited stronger activity and a much broader spectrum of action than the water extracts. The most interesting activity on E. coli was obtained from ethanolic extracts of Baccaris sp., Ochroma pyramidale, Croton lechleri, Banisteriopsis caapii, Miconia salicifolia, and Eugenia obtusifolia. Only the latter species also showed strong activity in the aqueous extract. A much wider range of species, including most species active against E. coli showed inhibition of S. aureus. Poropohyllum ruderale, Senecio sp., Corynaeae crassa, Dioscorea trifida, Senna monilifera, Spartium junceum, Pelargonium odoratissimum, Satureja pulchella, Cuphea sp., Malva parviflora, Brosmium rufescens, Syzygium aromaticum, Sanguisorba minor, Citrus limetta, Verbesine sp. and 2 unidentified species all showed MIC values between 1–4mg/ml. Most of them however did not portray any efficacy in aqueous extract. Hypericum laricifolium, Hura crepitans, Caesalpinia paipai, Cassia fistula, Hyptis sidifolia, Salvia sp., Banisteriopsis caapi, Miconia salicifolia and Polygonum hydropiperoides showed the lowest MIC values and would be interesting candidates for future research. Most MIC values reported in this work were largely higher than those obtained for South American species (Bastos et al. 2009; Jimenez et al. 2001; Meléndez et al. 2006; Zampini et al. 2009) and African studies (Kirira et al. 2006). However, they were in range or lower than concentrations reported by Kloucek et al. (2007), Nascimento et al. (2000) and Psewu et al. (2008).

Most species effective against S. aureus are traditionally used to treat wound infection, throat infections, serious inflammations, or are post partum infections. Interestingly many species used in cleansing baths also showed high activity against this bacterium. Many of these species are either employed topically, or in synergistic mixtures, so that possible toxicity seems not to be an issue. The species effective against E. coli were mostly employed in indications that traditional healers identified as “inflammation”.

Most of the plants used by the healers have antibacterial activity, but only 8 of the 141 plants (5.6%) examined in this study show any MIC values of 200 or less mg/ml of extract. Of these 8 plants 5 are used to treat diseases believed to be in bacterial origin by TM, one is a disease not believed to be caused by bacteria and one is used for undefined treatment purposes.

Nine out of 141 plants (6.3%) tested that were not used for diseases believed to be bacterial in origin by TM, 5 showed high antibacterial activity with MIC values below 16 mg/ml. Four of these were among the most potent plants tested with MIC values of 2 or less mg/ml including the hallucinogen and extracts used to treat diabetes and epilepsy. Diseases such as diabetes often compromise the health of the individual and antibacterial treatments can be warranted for secondary complications of the disease. In addition, TM does determine sometimes that diseases not originally believed to be bacterial in origin, such as ulcers, are actually caused by bacteria. Currently TM is seriously looking the role of inflammation (which can certainly be bacterial in origin) in heart disease.

The results presented in this paper demonstrate that most of the plants used by the healers in Peru to treat disease of bacterial origin do show limited antibacterial activity and that some treatments for diseases not currently believed to be of bacterial origin also show antibacterial activity. These facts support the medicinal value of traditional Peruvian remedies and suggest that those plants widely used by the curanderos could be new sources of antibacterial therapies.

4. Conclusions

The antibacterial activity of 141 ethanolic and aqueous extracts belonging to 140 plant species used in TM in Northern Peru was demonstrated. Most species tested showed limited antibacterial activity. It is important to note however, that most species are not employed as single plant extracts in the traditional context. The results indicate that the often very elaborate traditional knowledge can serve as guideline to provide leads for further testing of potentially interesting plants that can serve for further studies that would allow the clinical validation of the traditional uses and the application of the species in modern treatment forms. Further studies on the toxicity of the species employed, as well as their application in often complex traditional mixtures would allow to elucidate possible candidates for future development of antimicrobial agents.

Acknowledgments