Cuban flora as a source of bioactive compounds

Summary

Tropical flora constitutes a rich source of substances that can be applied directly, or as compounds, to the development of new pest control agents and pharmaceuticals. Partly due to its great diversity, Cuban flora has not yet been closely studied as a potential source of chemical pesticides. Mass screening of a variety of plants is needed to determine the presence of bioactive compounds which could be used instead of commonly adopted agrochemicals and drugs. This study from the Centre for Plant Protection in Havana has focused on a selection of plants as potential sources of new natural products for botanical pesticides and medicines. For the first time in Cuba, this research has been carried out using a refined bio-assay. Extracts from plants belonging to the Anacardiaceae, Asteraceae, Clusiaceae, Myrtaceae, Piperaceae, Anonaceae and Poaceae families were evaluated in terms of their effectiveness against bacteria, fungi, insects, mites and Artemia (brine shrimp). Plants from four of these families displayed activity and were then selected for further fractionation and purification. From a social and economic perspective, the identification of local candidates for developing phytosanitary products offers greater safety and efficiency for Cuban agriculture. This research could eventually assist other developing countries in developing national policies for agricultural management.

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Introduction

Natural products have been utilised by humankind for their own advantage for thousands of years. They have been used for food, clothing and cosmetics; the construction of shelters and traps; tools and weapons; poisons for game and fish; and finally as medicines and crop protection agents (see Copping and Duke, 2007).

There is a substantial scientific and ethno-botanical literature that lists plants with known pest control properties (see, for example, Isman, 2006). Over two thousand plant species are known to have these properties, and many of the plants are used by farmers in developing countries (Grainge and Ahmed, 1988). However, there is a need to identify the most active plant extracts and constituents for further development and utilisation (Hostettmann et al 2000).

Tropical plants that grow under climatic conditions favouring microbial or insect attack have developed a vast array of defence molecules. Therefore, they constitute a particularly rich source of substances which can find an application - either directly or as lead compounds for the development of new drugs or pest control agents (Hostettmann and Potterat, 1997).

A wide range of abundant and diverse terrestrial species are found to live in the tropical climates of Central America. These characteristics reflect the complex, unique quality of unspoiled natural eco-systems (Caledecott et al, 1994). Cuba's flora is particularly rich, with an estimated 6,500 vascular plant species of which 50% are endemic. Despite this, its huge potential as a source of bioactive products of natural origin has not been extensively exploited (Zambrana and Fernández, 2003). To date, only a small fraction of the plant species has undergone systematic phytochemical or biochemical research, leaving valuable sources for commercial products undiscovered.

There is anecdotal evidence regarding the biological activity of some of these plants and their popular use as natural pharmaceuticals and pesticides. However, in the majority of cases the active compounds have not been studied. There are also added complications concerning the available information that predominantly relates only to botanical data and traditional use, as well as being collected over 50 years ago (Roig 1988). In 1991 a national program led by the Cuban government to develop and unify traditional and natural medicine informed and encouraged the use and study of various medicinal plants (Del Toro García and Trapero Quintana, 2007).

Work in the area of discovering and developing substances with pesticidal activity has been more limited. Cuban farmers use their familiarity with plant properties empirically to protect their crops from pest attacks. However there is no systematic research to support this traditional knowledge nor to study in detail a broad range of plants as potential candidates for pesticides. Work addressing the isolation and characterisation of compounds responsible for pesticidal activity is scarce. The insecticidal, mollusquicidal, antimicrobial activity of approximately 40 species belonging to 25 families have been evaluated by various authors (see Pérez and Vázquez, 2001). Also several products from the neem tree have been successfully developed and four of them have undergone registration in Cuba (Estrada, 2002).

For the past 10 years, the Natural Pesticides Laboratory based at the Plant Protection Unit at the National Centre for Animal and Plant Health (CENSA) has been working towards the discovery and development of natural pesticides from vegetal species. The research has been aimed at the study of selected Cuban plants as sources of new bioactive natural compounds and focusing on the potential to develop them as botanical pesticides.

Research into pesticides and pharmaceuticals of natural origin can be approached from various perspectives: active compounds characterisation, mode of action, synergetic effects, specificity and toxicology to name but a few. They initially involve establishing bioassay conditions, the isolation and characterisation of bioactive compounds and the determination of structural features related to biological activity. Based upon this protocol, Cuban pesticide research from natural products has evolved. Crude extracts are screened for biological activity using a general and simple bioassay (brine shrimp test); this system provides a platform for measuring a large number of samples so optimising in terms of time, cost and efficiency. Thus the active plant species are detected and then effectively measured. At the same time using directed bioassays systems, and the bioactive compounds identified, fractionates the active extracts.

The next steps are (1) structure activity relationships studies (SARs) for identifying structural features for improving efficacy, stability, selectivity and/or to decrease toxicity or effects on non-target organisms (2) the synthesis of analogues and toxicity and (3) ecotoxicity studies. The analysis of all these data allows the selection of promising natural products (lead molecules or botanical medicines/pesticides) that will go into a development stage as candidates. This may result in the development of commercial products.

General methodology

Selection of plants

Plants were selected using a chemotaxonomic approach, which was supported by all the information gathered from ethno-botanical and scientific research sources of data. They were chosen for both their potential applications as botanical pesticides and as a template for compounds. Very rare species that have not been studied before from a chemical or biological point of view (with greater possibilities of discovering novel compounds) were included as well as other species that are more abundant and have potential pesticide links. Plants belonging to Anacardiaceae (genus Mangifera), Annonaceae (genus Annona) Asteraceae (Lescaillea, Vernonia), Clusiaceae (Calophyllum, Clusia, Mammea, Rheedia), Myrtaceae (Psidium) and Poaceae (Arthrostilidium, Zea) families were collected.

Extraction and biological screening

Dry raw material from these plants was extracted by sequential maceration with solvents of increasing polarity. Crude extracts from more than 50 plants were evaluated for potency against Artemia salina Leach. All the crude extracts were tested following the brine shrimp lethality test protocol, using 96-well microplates, at final concentrations of 10, 100 and 1000 mg/ml. LC₅₀ values and 95% confidence intervals or limits were calculated using the Probit analysis, Polo computer program (LeOra Software, Berkeley, California). Samples with LC₅₀<1000µg/ml were considered active. In cases where the initial dose concentration range was too broad to permit satisfactory calculation of LC₅₀ values and the confidence intervals, the assay was repeated with dosage closer to the apparent LC₅₀ value.

On the basis of these screening results, the active crude extracts are chosen for further investigation. Therefore, evaluation against other biological targets (mites, insects, bacteria, and fungi), large-scale extraction and bio-guided fractionation of these species will be carried out at a later stage.

Results and discussion

Of the various procedures, the Artemia bioassay (brine shrimp test) was selected for screening the plants due to its general bioassay that is indicative of pesticidal, cytotoxic and other pharmalogical effects. Since its development, the brine shrimp bioassay, combined with specific anti-tumour bioassays, has led to the discovery of several bioactive plant materials. A positive correlation exists between brine shrimp lethality and 9KB (human nasopharyngeal carcinoma) cytotoxicity (p=0.036). Therefore, the brine shrimp is used as a pre-screen of plant extracts for anti-tumour compounds (McLaughlin et al, 1998). Consequently, extracts and compounds with high levels of activity against Artemia are identified as potential candidates for developing new pesticides and/or drugs.

A total of 150 crude extracts obtained from different plant tissues were studied. Table 1 below summarises the data where at least one extract met the activity criteria (LC₅₀<1000µg/ml). A mortality effect was found even after 6 hours of exposure to each active extract. The rest of the extracts did not show a concentration/response relationship and did not attain 50% mortality at the highest concentration tested. Some of these extracts decreased the motility of the nauplii but most of the tested organisms were still alive at 24 hours. A general tendency for the petrol crude to be the most active among the three extracts of different polarity obtained from the same plant material was observed. This information provides assessments to be made about the lipophilic nature of the active compounds and how this may influence the biological activity as a pesticide.

On the basis of the biological screening results indicating levels of activity, extracts from vegetal species belonging to two distinctive families were selected: Clusiaceae (Calophyllum pinetorum Bisse, Mammea Americana Lin) and Annonaceae (Annona muricata Lin, Annona reticulata Lin).

The Clusiaceae family encompasses over 1000 species of wide distribution in the tropics as trees and shrubs. Previous phytochemical studies focused on obtaining biologically active compounds. This influenced a chemotaxonomic interest in the family and the presence of xanthones; coumarins, byphenyls, flavonoids and triterpenes have already been reported. In addition to their chemotaxonomic qualities, the coumarins also exhibit significant biological effects such as insecticidal, molluscicidal, piscicidal, cytotoxic, in vivo anti-tumour activity, antioxidant, tuberculostatic, anti-inflammatory, antileukaemic, antimicrobial and hypoglycaemic activities. Recent interest on these compounds has arisen as a result of their identification as potent inhibitors of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) (Hostettmann et al, 2000; Reyes-Chilpa et al 2004; Yang et al, 2006).

Annonaceae is a large family of tropical and subtropical trees and bushes. They have economic importance as fruits, edible oils and fragrances as well as ethnobotanical medicines. These plants contain bioactive compounds called annonaceous acetogenins which exhibit a broad range of biological activities: insecticidal, antitumoral, cytotoxic, anti parasitic, antimalarial, antimicrobial, immunosuppressant, antifeedant and pesticidal. The structure and levels of activity of these secondary metabolites and acetogenins vary greatly across the species studied. The unique nature of Cuban species and their potential as botanical pesticides was recently recognised when Annona bullata rich was found to be the most effective plant ever tested against lepidopteran pests (McLaughlin et al, 1998; Isman, 2006).

Table 1. Biological activity of crude extracts against Artemia salina

Conclusion

This study is the first one to be undertaken in Cuba in which the search for new sources of potential pesticides was carried out through a screening procedure for the presence of bioactive compounds from plant sources. The extended use of this general bioassay for studying Cuba's rich biodiversity as a source of bioactive substances will simplify and increase the capacities of evaluating natural products for commercial and biological evaluation. This in turn, will improve the general knowledge and understanding regarding our natural resources and consequently their sustainable use.

This work provides the practical base needed to introduce a sensitive bioassay into the work of determining and developing new pesticides and drugs from natural resources in Cuba using Artemia species and strains. The introduction of new bioassays systems into research at CENSA will enhance the development of more effective isolation and characterisation of natural products that may be candidates as pesticidal agents. Similarly some of these could be developed for pharmaceutical use. We have demonstrated that the new bioactive compounds can be rapidly detected and isolated through bioactivity-guided screening and fractionations of the plant extract. Once the active plant species have been detected, a series of more extensive and specialised bioassay can undertaken.

Natural products may play an important role in the future of medicine and Integrated Pest Management (IPM) either as new products directly; new chemical frameworks for production; or for identifying new modes of action. Currently there are exceptional opportunities for the development of phytochemical based products which avoid difficulties caused by pathogens or pest resistance, human toxicity, contamination of water courses, environmental persistence or the effects on non-target organisms in ecosystems. Another outcome from the study is that the structural of the bioactive ingredients could provide a scaffold for the development of semi-synthetic compounds that also have potent anti pesticide properties.

Finally the outcome of this study demonstrates that several species of Cuban flora (some of which were analysed for the first time) represents a new contribution to the knowledge of our botanical biodiversity. It also illustrates its potential as a source of compounds with qualities relating to, primarily, pesticidal but also anti-tumoral products. These new products would provide environmentally and therapeutically safe agents which would play a significant role in the pharmaceutical industry regarding the treatment of important diseases for which, at present, there is no effective medicine. From a social and economic perspective, the identification of candidates developing new phyto-sanitary products offer new alternatives that combine efficiency and safety for Cuban agriculture in the area of plant protection. This research and knowledge could then be transferred to assist other developing countries in developing national policies that relate to the management and enhancement of agriculture through crop protection.


Acknowledgements
We gratefully acknowledge the financial support of Rothamsted Research and the award of three Rothamsted International Fellowships.

Oriela Pino Pérez, Fanny Jorgé Lazo and Ondina Léon Díaz are based at la Dirección de Protección de Plantas, Centro Nacional de Sanidad Agropecuaria (CENSA), la Habana, Cuba. Email oriela@censa.edu.cu

B. P S. Khambay is a researcher in the Biological and Ecological Chemistry Department, Rothamsted, UK.

Christopher J. Branford-White is Director of the Institute for Health Research and Policy, London Metropolitan University, UK. Email cbranfordwhite@londonmet.ac.uk