Data and Genetic Diversity

Karla C.B. Santana,

¹

Diego S.B. Pinangé,

¹

Karina P. Randau,

¹

Marccus Alves

¹

and Ana M. Benko-Iseppon

^2,

*

Introduction

For many populations around the world, phytomedicines fi gure as the main form of treatment for a wide range of health problems, mostly being the fi rst line of defense against potential health hazards (Rahman 2012). Although access to the modern medicine is available in most countries, the use of medicinal herbs has retained its popularity due to historical and cultural reasons (Agra et al. 2008). Based on the historical evolution of the use of medicinal plants, in 1978 the World Health Organization (WHO) started to recognize medicinal herbs as an alternative therapy for human diseases. Additionally, the use of natural compounds from biological sources, as well as ethnobotanical knowledge, has emerged as an important source for discovering new products (Albuquerque and Hanazaki 2006, Li and Vederas 2009, Desmachelier 2010, Newman and Cragg 2012). Furthermore, in recent years, bioinformatic approaches, associated to the advanced techniques of separation, structure elucidation, screening and combinatorial synthesis have led to the consolidation of the usage of plants as a source of new drugs (Saklani and Kutty 2008, Sharma and Sarkar 2013).

Brazil is recognized by high and well-documented biodiversity mainly based on abundant records of use of its fl ora for traditional uses and medicinal purposes (Desmachelier 2010, Nogueira et al. 2010, Brandão et al. 2012). Additionally, in the northeastern region of Brazil, natural products have been largely used for medicinal purposes. Further, in the poorest regions and even in big cities, medicinal plants are still commercialized in popular street markets, as well as cultivated in residential backyards (de Almeida 1993, Agra et al. 2008, Veiga Junior 2008, Pasa et al. 2011, Benko-Iseppon and Crovella 2010, Benko-Iseppon et al. 2012). In a recent survey conducted by Benko-Iseppon et al. (2012) regarding the potential use of

1 Federal University of Pernambuco, Genetics Dept., Recife - PE, CEP 50.670-420 Brazil.

2 Department of Genetics, Federal University of Pernambuco, Av. Prof. Moraes Rego s/n, CEP 50732-970 Recife, PE, Brazil

* Corresponding author: ana.benko.iseppon@pq.cnpq.br

medicinal plants in the Brazilian northeastern region, most species and even genera used for therapeutic and medicinal purposes were recognized as endemic. Thereafter, more multidisciplinary studies such as taxonomy, physiology, genetics and phytochemistry are mandatory in order to shed light on the identifi cation and assignment of new compounds, their uses and applications. In this sense, population studies have proven to be essential, especially considering diverging genetic background together with environmental forces acting on plant phytochemical content.

Among several plant groups used in Brazilian folk medicine, Euphorbiaceae is one of the most recorded families for medicinal use (Agra et al. 2008, Benko-Iseppon and Crovella 2010, Forzza 2010, Benko-Iseppon et al. 2012). According to APG III (APG 2009) the so-called Euphorbiaceae sensu stricto consists of four subfamilies—Acalyphoideae, Crotoneideae, Euphorbioideae and Cheilosoideae. Within Euphorbioideae the genus Euphorbia L. fi gures as the largest Euphorbiaceae genus and one of the most diverse groups of the plant kingdom, with approximately 2,160 species (Webster 1994a, Steinmann and Porter 2002, Bruyns et al. 2006). Euphorbia encompasses a large morphological and adaptive diversity, refl ected sometimes also in the types of metabolisms found as, for example, E. tirucalli, a species that displays the C3 or C4 photosynthetic systems depending on the plant part analyzed (Hans 1973, Van Damme 2001, Sage et al. 2011).

Regarding taxonomic data, Euphorbia has been largely discussed as a natural genus (Webster 1987, Webster 1994b, Park et al. 2000). Some studies such as those performed by Aqueveque et al. (1999) and Domsalla et al. (2010) have proposed a taxonomic delimitation based on chemotaxonomy of fl avonoid and latex proteinase profi les. With respect to the current classifi cation, some authors consider that the group support is weak and presents many inconsistencies (Steinmann and Porter 2002, Bruyns et al. 2006, Park and Jansen 2007, Tokuoka 2007). Nevertheless, Zimmermann et al. (2010) proposed the current most accepted division of Euphorbia in four subgenera: Chamaesyce, Euphorbia, Esula and Rhizanthium.

With regard to ecological and economic importance, Euphorbia species have often high invasive potential, with consequent damage to agriculture, including the reported E. hirta L. and E. heterophylla L.

(Willard and Griffi n 1993, Aarestrup et al. 2008). On the other hand, Bellini et al. (2008) reported the signifi cance of some Euphorbia invasive species as pioneer plants and in the biological control of pest mites in plantations, hindering the spread of pathogens. Some Euphorbia taxa have commercial signifi cance, as the crown-of-thorns (E. milii) and the Poinsettia (E. pulcherrima), a species largely used especially for Christmas decorations. Also the Baseball-plant (E. obesa) deserves mentioning as an ornamental plant (Lee 2000, Mwine and Van Damme 2011); besides, some species with abundant latex as E. tirucalli and E. lathyris are known for their potential for biodiesel production (Duke 1983, Van Damme 2001).

Biochemical diversity and medicinal use of Euphorbia species

Most Euphorbia species are useful in popular medicine, although often the dosages, effi cacy and possible toxic effects remain unclear. Several species have been used in the treatment of various diseases, such as skin diseases, gonorrhea, migraines, intestinal parasites and warts (Singla and Pathak 1990, Appendino and Szallasi 1997, Shi et al. 2008). Some researchers have demonstrated that Euphorbia species also feature antitumor (de Melo et al. 2011, Zhang et al. 2011), antimicrobial (Sudhakar et al. 2006, Murugan et al.

2007, Akinrinmade and Oyeleye 2010, Benko-Iseppon and Crovella 2010) and antiviral activities (Hezareh 2005, Tian et al. 2010). Further effects as analgesic and antipyretic (Ma et al. 1997), anti-anaphylactic (Youssouf et al. 2007) and antioxidant (Barla et al. 2007) have been also reported. Records indicate activity in intestinal motility (Hore et al. 2006) and aid in the treatment of diabetes mellitus (Widharna et al. 2010).

Such observations led to the development of several patented drugs by compounds from various Euphorbia species, as listed by Mwine and Van Damme (2011).

These medicinal properties are considered to be derived from the rich content of secondary metabolites in species of Euphorbia. A large number of studies have revealed the presence of alkaloids, diterpenes, glucosinolates, tannins, triterpenes, steroids and large amounts of phenolic compounds (Seigler 1994, Shi et al. 2008), even considering the still scarce amount of data available regarding the chemical diversity of these compounds among populations. In 1999, Aqueveque et al., in a chemotaxonomic study with 12 Chilean species of Euphorbia including E. hirta, isolated 22 fl avonoids (13 fl avonols, eight fl avones and

one fl avanone) using thin layer chromatography. The fl avonolic profi le proved to be an excellent marker on Euphorbia, due to the high number of compounds observed, allowing an effective comparison among species, also uncovering a closer relationship among species of subgenus Chamaesyce when compared to the other Euphorbia members analyzed.

On the other hand, addressing the triterpenoid profi les from latex of 56 accessions of European leafy spurges, Holden and Mahlberg (1992) observed similar qualitative and quantitative profi les for the species E. amygdaloides, E. agraria, E. cyparissias, E. lucida, and E. seguierana, while 37 accessions of the E. esula complex were separated into 15 groups based on variability of the components, suggesting that different profi les exist within populations of Europe and North America. Further, the composition of triterpenes in E. esula presented a high level of qualitative and quantitative stability under diverse environmental and physiological conditions, indicating a genetic basis for triterpenoid synthesis (Mahlberg et al.1987).

Genetic diversity in Euphorbia L.

Among several concepts adopted for biological diversity, perhaps the simplest one may be defi ned as the variation present in all species of plants and animals associated with their genetic material and the ecosystems in which they thrive. The relevance of biodiversity for humankind has been well recognized and reported in the recent decades and many consider that diversity is essential for allowing sustainable development of various human activities, in different forms and levels (Frankel and Bennet 1970, Shiva 1994, Swingland 2001, Rao and Hodgkin 2002). Thus, the importance of adopting a holistic view of biodiversity, including applied research, such as agricultural biodiversity, as well as linking conservation with sustainable use, has been the central theme in research activities conducted in the last decades (Dias and Kageyama 1991, Arora 1997, Frankham et al. 2002).

In the literature, the idea that different types of molecular markers have the ability to differentiate genomes of interest is already widespread, generating data on diversity levels and distribution, generally using genetic polymorphism as background. Information on genetic diversity, and therefore gene frequency, produce additional insights that can be combined with morphological, physiological and agronomic data, providing a complete analysis especially when combined with other research areas (Carvalho et al. 2000a, 2000b, Huang et al. 2002, Faleiro 2007), including ethnobotanical and phytochemical profi ling, as in the present chapter.

Although Euphorbia is the largest genus within Euphorbiaceae, surprisingly, there are few studies involving analysis of genetic diversity via molecular markers in this group. Most of them evaluated Euphorbia species from North America and Europe (Nissen et al. 1992, Park et al. 1997, Rowe et al.

1997, Morden and Gregoritza 2005), often focusing on phenetic and phylogenetic data, as well as indexes of population polymorphisms and differentiation, using RAPD (Random Amplifi ed Polymorphic DNA) markers (Rowe et al. 1997, Morden and Gregoritza 2005), Isoenzymes (Park et al. 1997), Alloenzymes (Park 2004), RFLPs (Restriction Fragment Length Polymorphism—Nissen et al. 1992, Rowe et al.

1997), sequencing of cpDNA (Chloroplast DNA—Nissen et al. 1992, Rowe et al. 1997) and ITS (Internal Transcribed Spacer—Morden and Gregoritza 2005), among others. Such evaluations allowed not only the identifi cation of signifi cant levels of polymorphism, but also some insights regarding the relationships between species and/or populations.

In Brazil, a single record is available regarding the analysis of diversity and structure of wild-poinsettia (E. heterophylla) populations performed by Frigo et al. (2009). In this evaluation, the esterase loci polymorphisms were analyzed revealing mostly high degree of differentiation in the sampled populations with subsequent defi cit of heterozygosity probably caused by inbreeding.

Euphorbia hirta L. and E. hyssopifolia L.

Belonging to the subgenus Chamaesyce, Section Anisophyllum (Yang and Berry 2011), E. hirta and E. hyssopifolia (see Fig. 8.1) are widely used as medicinal herbs in folk medicine around the world. Both are sub-spontaneous and ruderal species, native to the New World, tolerant to high temperatures and drought (Stehmann et al. 2009, ZCZ 2013a, 2013b). They are widely distributed in tropical and subtropical

regions, from the sea level up to 1500 m (Amorozo 2002, Schneider 2007, Euphorbia PBI 2013a, 2013b).

In Brazil, they occur in all regions and biomes, where they inhabit degraded areas, roadsides, cultivated fi elds and gardens (Steinmann et al. 2013a, 2013b).

The species E. hirta (synonym Chamaesyce hirta, among others) is a semi-prostate or erect herb with simple, opposite and ovate-elliptic or lanceolate leaves, usually asymmetric, serrated margins. The infl orescence is a cyathium with tiny orbicular glands, without petaloid appendages and pubescent fruits.

In English this species is called hairy-spurge, asthma plant, garden-spurge, pill-bearing-spurge, pill-pod broomspurge, pill-pod-sandmat, asthma weed, cat’s-hair, spurge or milkweed (Johnson et al. 1999, ZCZ 2013b), whereas in Portuguese is known as ‘erva-de-Santa-Luzia’, ‘erva-de-cobra’, ‘erva-de-sangue’,

‘erva-andorinha’, ‘burra-leiteira’, ‘tranca-cu’ and ‘quebra-pedra’ (Lorenzi 2000, Amorozo 2002, Guglieri- Caporal et al. 2011).

In turn E. hyssopifolia (E. brasiliensis and Chamaesyce hyssopifolia, among other synonyms) is an erect or semi-prostate herb with simple, opposite and oblong-elliptic to oblong-obovate leaves fi nely serrated margins. The infl orescence is a cyathium (terminal or pseudo-axillary) with oblong to reniform glands, petaloid appendages and glabrous fruits. It is recognized by the same common names in Portuguese—‘erva- de-andorinha’, ‘erva-de-Santa-Luzia’, ‘burra-leiteira’, ‘quebra-pedra’, ‘porca-parideira’, ‘sete-sangrias’

and ‘erva de cobra’ (Lorenzi 2000, Albuquerque et al. 2005, Agra et al. 2008, Pasa et al. 2011, Menezes et al. 2012) whereas in English it has been named hyssop-spurge, hyssop-leaf broomspurge, hyssop-leaf- sandmat and leafy-spurge (ZCZ 2013a).

In traditional medicine, several parts of the plant have been used: leaves, fl owers, roots, latex, aerial parts and the whole plant. E. hirta has been studied more frequently, being widely used in different preparations for gastrointestinal disorders (gastritis, diarrhea, dysentery, colic, used as vermifuge, amoebicide, purgative, colagogue and to treat ulcers), bronchial and respiratory diseases (cough, coryza, hay asthma, asthma, bronchial affections), skin affections (acne, wart, rashes, furuncle, ringworm, measles, chickenpox, smallpox wounds, splinter removal), pinkeye, kidney stones, edema and hypertension, as galactagogue, and in snake bites (Watt and Breyer-Brandwijk 1962, Anjaria et al. 1997, Mhaskar et al. 2000, Agra et al. 2008, Kumar et al. 2010). In addition, it has been confi rmed that a wide range of properties, such as antibacterial, antiamoebic, anthelmintic, antifungal, antiplasmodial, antiviral, antineoplastic, spasmolytic, antidiarrhoeic, decreased the gastrointestinal motility, sedative, antidepressant, anxiolytic, analgesic, antipyretic, anti-infl ammatory, immunomodulatory, antiallergic, antihypertensive, as diuretic and as an adjunctive agent in diabetes mellitus treatment (Williams et al. 1997, Johnson et al. 1999, Tona et al. 2000, 2004, Hore et al. 2006, Ogbulie et al. 2007, Anuradha et al. 2008, Loh et al. 2009, Kumar et al.

2010, Ramesh and Padimavathi 2010, Shih et al. 2010, Widharna et al. 2010, Alisi and Abanobi 2012).

Figure 8.1. (A) Euphorbia hyssopifolia and (B) E. hirta (Credit to the authors).

The second species, E. hyssopifolia is used to treat warts, corns, conjunctivitis and in external ulcers, as a purgative, also as a diuretic and in dysuria, as emmenagogue, as well as to expel placenta, against fl u, coughs, fever, for eyes diseases and high blood pressure (Watt and Breyer-Brandwijk 1962, Arnason et al. 1980, Morton 1980, Agra et al. 2008, Brandão et al. 2012). It was also found that its metabolites have signifi cant inhibitory effects on HIV-1 reverse transcriptase (Matsuse et al. 1999, Lim et al. 1997).

The problem: two species one common name

It is well known that E. hirta and E. hyssopifolia are used in folk medicine with recognized variety of pharmacological actions, sometimes shared by both. It refl ects on the popular names they have in common: as Erva de Santa Luzia (related to the saint in charge of eye problems), quebra-pedra (‘stone breaker’ regarding its diuretic properties, a name also used for Phylanthus niruri L.), and erva de cobra (‘snake-herb’ related to its use after snake bites). However, most of the characteristic features of plant secondary metabolism regard its vast chemical diversity and intraspecifi c variation. Also, due to several reports regarding the toxicity related to species from this group, the quality of medicinal plants and their products should fulfi ll safety requirements, effi cacy and stability in order to be used by pharmaceutical industries (WHO 2002, Mwine and Van Damme 2011, Kheyrodin and Ghazvinian 2012). Then, the use of homogeneous genotypes with well-known and stable phytochemical and biological properties is an important requirement for use considering pharmaceutical purposes.

Unfortunately, for many plant species data on metabolites stability or toxicity is not available, demanding a better knowledge of their chemical features, besides some variations that they may present due to their occurrence in contrasting environmental and ecological conditions. Especially in the case of non-cultivated medicinal plants, plant parts collected from the fi eld may exhibit drastic differences in their compounds and therapeutic effects as well as in its toxicity. Therefore, it is critical to provide information about the phytochemical differences within species in different habitats as observed in Arnica Montana, considering different conditions of climate and altitude (Spitaler et al. 2006).

In the presented pilot study, two sources of polymorphisms were accessed: phytochemical (TLC—Thin Layer Chromatography) and genetic markers (ISSR—Inter Simple Sequence Region). For that we performed two experiments: (1) Chemical differentiation associated to color-associated characters (individuals of the same species with red versus green stem) and species distinction (leaves and stem of E. hirta versus the ones of E. hyssopifolia) and (2) Differences in secondary metabolism through methanolic screening, including fl avonolic population diversity together with evaluation of population’s genetic diversity. In the case of both species (E. hirta and E. hyssopifolia) the use of the same common name has induced herb sellers to provide parts of both species for different uses. Thus, the present study aimed to provide preliminary evidence on the diversity of the compounds contained in the species level and different areas of occurrence, and also an analysis of genetic diversity with ISSR.

Experimental design of an exemplary pilot test Specimen collection

Specimens of E. hirta (EHIR) and E. hyssopifolia (EHYS) were collected in August 2009, in the city of Recife in urban Atlantic coastal rainforest fragments, as well as, in a locality in the semi-arid region in the inland of state of Pernambuco, Northeast of Brazil (Fig. 8.2). The populations from the city were ca.

85 km distance from the inland population and about 4 km from each other, including periurban and rural areas, with different grades of antropization (Table 8.1).

Search for polymorphisms

In the presented pilot study, two sources of polymorphisms were accessed: The phytochemical study was carried on into two experiments: the fi rst regarding the differentiation of species and color character differences in secondary metabolism through methanolic screening (Experiment 1) while the second

analyses focuses on population’s fl avonolic and genetic diversity using molecular markers (Experiment 2).

The workfl ow presented here considering the mentioned approaches (Fig. 8.3), may be applied to any near-related species and their populations (as in the case of the present work) or even to a single species with different populations.

Experiment 1

The assembly of the experiment was based on the previous observation of color difference (reddish and greenish), existing on the stems (stem) of both species. Therefore, this experiment aimed to verify if the separation of species from its phytochemical profi le, as well as assessing possible phytochemical variation associated with morphological differentiation of stem color.

Figure 8.2. Collected populations from the Pernambuco state (Brazilian northeastern region), including four areas from Atlantic forest (API = Apipucos; CDU = Cidade Universitária; LAR = Lagoa do Araçá and MEU = Mata do Engenho Uchoa) and one (SUR = Surubim) from Caatinga (semi-arid) environment.

Table 8.1. Studied populations with their provenances and bio-geographical characteristics.

Samples plot Cidade

Universitária

Apipucos Lagoa do Araçá

Mata do Engenho Uchoa

Surubim

Geographical coordinates

Latitude 8°3’4.09”S 8°1’10.34”S 8°5’32.41”S 8°5’53.91”S 7°50’35.82”S

Longitude 34°56’47.38”O 34°56’0.56”O 34°54’51.04”O 34°57’37.08”O 35°42’7.95”O

Altitude 17 m 10 m 6 m 23 m 317 m

Sample abbreviation

E. hirta EHIR-CDU EHIR-API EHIR-LAR EHIR-MEU EHIR-SUR

E. hyssopifolia EHYS-CDU EHYS-API EHYS-LAR EHYS-MEU EHYS-SUR

Plots characteristics

Anthropization

degree High High High Moderate Low

Biome Atlantic Forest Atlantic Forest Atlantic Forest Atlantic Forest Caatinga

Environment Urban Urban Urban Ruderal Rural

Climatic means in august 2009: (Source: INMET—Instituto Nacional de Meteorologia). Recife (CDU, API, LAR and SUR)—temperature 25ºC, humidity 80%, radiation 1500 kjm, precipitation 12 mm/day; Surubim—temperature 23ºC, humidity 70%, radiation 1700 kjm², precipitation 3 mm/day.

Atlantic Forest Caatinga Cerrado

One specimen of each species was collected at the same time in one of the plots of the ‘population’

experiment—CDU (details in Table 8.1). Three samples were selected from each species: leaves—L; green stem—GS and red stem—RS, resulting six samples—EHIR-L, EHIR-GS, EHIR-RS, EHYS-L, EHYS-GS and EHYS-RS. Methanol extracts were obtained by using 10 ml of methanol per 5 g of sample. Secondary metabolites were separated thought thin-layer chromatography, according to the methodology described for each metabolite, and compared with the available patterns (Table 8.2). Finally, the plates were analyzed for presence/absence of metabolites and the number of compounds obtained in each population.

Experiment 2

The leaves collected for this experiment were split for genetic and phytochemical analysis. The environment chosen had different levels of degradation and human disturbance. Each population included 10 individuals, for both analyses. The individuals were grouped in a bulk with 5 g of stem/leaf mass per individual, in a total of 10 bulks. The characteristics of the locations and samples analyzed are shown in Table 8.1. For the phytochemical data, a phenolic (fl avonoids and cinnamic derivatives) screening in chromatography was performed, where we used 10 ml of methanol per sample and the same method from Experiment 1.

In relation to the molecular analysis, the genomic DNA extraction from each bulk was performed according to the protocol described by Weising et al. (2004). The molecular marker employed was the dominant ISSR marker. The choice of ISSR marker was based on the premise that their target sequences are abundant throughout the eukaryotic genome, regard rapidly evolving regions and show high reproducibility (Fang and Roose 1997, Esselman et al. 1999, Pinangé 2009, Vasconcelos et al. 2012). Thus, the amplifi cation reactions followed the protocol described by Bornet and Branchard (2001), with modifi cations performed Figure 8.3. Workfl ow illustrating the main steps for phytochemical screening, chemical analysis and also genetic diversity with ISSR (Inter Simple Sequence Repeat) markers, also considering morphological and biogeographical peculiarities of the samples analyzed.

1. EHIR-L; 2. EHIR-GS; 3. EHIR-RS 4. EHYS-L; 5. EHYS-GS; 6. EHYS-RS

analytical thin-layer

chromatography Generation of phenogram and distance analysis