Secondary Metabolites from Rubiaceae Species

This study describes some characteristics of the Rubiaceae family pertaining to the occurrence and distribution of secondary metabolites in the main genera of this family. It reports the review of phytochemical studies addressing all species of Rubiaceae, published between 1990 and 2014. Iridoids, anthraquinones, triterpenes, indole alkaloids as well as other varying alkaloid subclasses, have shown to be the most common. These compounds have been mostly isolated from the genera Uncaria, Psychotria, Hedyotis, Ophiorrhiza and Morinda. The occurrence and distribution of iridoids, alkaloids and anthraquinones point out their chemotaxonomic correlation among tribes and subfamilies. From an evolutionary point of view, Rubioideae is the most ancient subfamily, followed by Ixoroideae and finally Cinchonoideae. The chemical biosynthetic pathway, which is not so specific in Rubioideae, can explain this and large amounts of both iridoids and indole alkaloids are produced. In Ixoroideae, the most active biosysthetic pathway is the one that produces iridoids; while in Cinchonoideae, it produces indole alkaloids together with other alkaloids. The chemical biosynthetic pathway now supports this botanical conclusion.


Introduction
The Rubiaceae family is characterized by the production of bioactive metabolites with great pharmacological potential. These metabolites can be used as chemotaxonomic markers even for genera and subfamilies [1,2]. Usually, taxa are classified according to different botanical characteristics; classical taxonomic systems only consider the plant morphological characters, while modern systems correlate their various combinations, including the chemical composition. Studies correlating classical plant taxonomy to chemical data can be found as far back as 1699 [3].
Phytochemical compounds can be a useful tool for characterizing, describing and classifying plant species. The distribution of secondary metabolites in Rubiaceae follows patterns that may help characterize the botanical group (subfamily, tribe or genera). These patterns relative to chemotaxonomy are often used to establish the botanical origin [4].
In recent years, Rubiaceae species have been thoroughly studied from a phytochemical viewpoint. However, very few studies have used this knowledge as a tool in taxonomic studies. When conducting bioprospecting studies of a plant, all botanical and chemotaxonomic information is of great importance, since it increases the likelihood of finding bioactive compounds, which enables the discovery of new Nature-originated drugs [5]. Therefore, the present study aims to conduct a literature survey on phytochemical studies addressing species of Rubiaceae published from 1990 to 2014, and describe their secondary metabolites occurrence and distribution in the subfamilies, tribes and main genera of this family.

Taxonomic Classification of Rubiaceae
The Rubiaceae family has a cosmopolitan distribution, mostly concentrated in the tropics. Being one of the largest in the Magnoliopsida class, it ranks fourth in diversity of species among Angiosperms [4]. It includes approximately 637 genera and 13,000 species [5,6]. In Brazil, nearly 120 genera and 1400 species occur, representing one of the most important economic, ornamental and medicinal plant families in the Brazilian flora [7].
The Rubiaceae family taxonomic classification is complex and there are still some gaps which have to be filled. According to the classification of Robbrecht [8], the Rubiaceae family is divided into four subfamilies: Rubioideae, Cinchonoideae, Antirheoideae and Ixoroideae. However, more recent studies suggest this family to be divided into three subfamilies: Rubioideae, Cinchonoideae and Ixoroideae, as some authors do not recognize Antirheoideae as a subfamily, since molecular studies have shown it to be polyphyletic with no standardized occurrence of a chemical marker [9][10][11][12][13][14][15][16]. Due to the abundance of species, the subfamilies were divided into 43 tribes (an intermediate clade between genus and subfamily) [16], which are listed in Figure 1.
Due to the lack of studies that can complement the extant information on geographical distribution, morpho-anatomical characteristics and molecular data, there are still genera and species not allocated into any tribe [16]. The evaluation of the chemical profile of these species may indicate a more complete phylogenetic distribution, since the secondary metabolites are the results of adaptation and evolution of a specific taxon to environment [17]. Thus, the profile of secondary metabolites distribution can bring new information for the taxonomic classification of this family.

Chemical and Biological Aspects of Rubiaceae
The Rubiaceae family presents a large diversity of substances such as iridoids, indole alkaloids, anthraquinones, terpenoids (diterpenes and triterpenes), flavonoids and other phenolic derivatives, with emphasis on production of bioactive alkaloids [2]. Alkaloids are secondary metabolites that can generate various drugs with important pharmacological effects and used to find out physiological The plant evolution process, from a morphological point of view, occured by the successive appearance of small weeds, larger herbs, shrubs and, finally, trees achieving the climax with primitive angiosperms. Then, the evolutionary polarity became inverted, woody plant being gradually replaced by herbaceous plants [40,41]. As explained by Gottlieb: "The most conspicuous evolutionary trend in the gross morphology of land plants concerns the successive appearance of small weeds, larger herbs, shrubs and, finally, trees. This trend had attained or even had passed its climax with the primitive angiosperms and within this division the evolutionary polarity became inverted, woody plants being gradually replaced by herbaceous plants.  These successional phenomena are paralleled by micromolecular compositions. The ubiquitous flavonoids excepted, polyketides and terpenoids dominate the chemical compositions of bryophytes and pteridophytes. Shikimate-derived aromatics became numerically significant only in gymnosperms and attain predominance over other biosynthetic classes in primitive angiosperms. Concomitantly, here secondary metabolism reflects the trend from woody to herbaceous forms by inactivation of cinnamoyl/cinnamyl-derivatives through two phenomena: (i) extension of the shikimate pathway by reduction of cinnamyl alcohols to allylphenols and propenylphenols and (ii) gradual curtailment of the final steps of the shikimate pathway. The former alternative is most frequent in the primitive magnolialean block, where oxidative oligomerization of the precursors leads to neolignans. The first consequence of the latter alternative, the accumulation of phenylalanine and tyrosine, again very frequent in the magnolialean block, occurs also in the rosiflorean block. Oxidative elaboration of these amino acids leads to benzylisoquinolines. Further shortening of the shikimate pathway is restricted to the rosiflorean block. It leads to the accumulation of chorismic acid, the precursor of anthranilateand of tryptophane-derived alkaloids, and of shikimic acid, the precursor of gallic acid-and ellagic acid-derived tannins. With gallic acid, the possibilities of diversifying the production of micromolecules through gradual curtailment of the shikimate pathway seem to be exhausted. In the most highly advanced, mostly sympetalous, angiosperms, shikimate-derived secondary metabolites play a relatively minor role. In these lineages, the full potential of acetate utilization leads to polyacetylenes, while mevalonate utilization leads to steroidal alkaloids, iridoids, alkaloids, sesquiterpene lactones, etc. In comparison with the polyketides and terpenoids of less advanced plant groups mentioned above, these compounds all show a high state of oxidation." [40].
Regarding the distribution of the major secondary metabolites in Rubiaceae, indole alkaloids are indicated as the main chemical markers of this family [42][43][44][45][46]. Iridoids, anthraquinones, triterpene glycosides, flavonoids, lignoids, terpenes and phenols derivatives, were also reported [47]. Indole alkaloids occur just in families belonging to the Gentianales order (Loganiaceae, Rubiaceae, Apocynaceae and Naucleaceae), where one observes monoterpene indole alkaloids mainly [48]. The occurrence of indole alkaloids out of Gentianales order is quite rare and when found they are usually simple indole alkaloids.
A good correlation between the biosynthetic pathways and morphological aspects of the Ixoroideae, Cinchonoideae and Rubioideae subfamilies is obtained by evaluating chemical data, combined with the parameters cited by Robbrecht [8]. Each one of these subfamilies presents a different and typical profile of indole alkaloids, iridoids and anthraquinones which are considered as Rubiaceae chemotaxonomic markers [49]. Other studies based on chemotaxonomic data obtained by gas chromatography coupled to mass spectrometry show that the iridoid glycosides are present in several different species belonging to the Rubiaceae subfamilies [50][51][52]. Monoterpene indole alkaloids, especially which are derivatives of tryptamine and monoterpene (iridoid) secologanin are another predominant class in Rubiaceae. Quinoline alkaloids, which are products from the monoterpene indole and isoquinoline alkaloids rearrangement, yielding emetine-type alkaloids, are also characteristic of Rubiaceae, however, strychnine class alkaloids are not present in this family. Other alkaloid types are quite heterogeneous leading to a hard chemotaxonomic correlation [53].
Several studies have reported the use of chemical data to assist plant taxonomy [53]. Interest in this area increased due to the appearance of fast and accurate analytical techniques. However, there are still limitations on the application of chemical data in systematics. Even with a growing number of phytochemical studies, there are still many plants that remain without any chemical study.

Data Obtained Through the Bibliographic Survey
The present study sought to survey phytochemical studies of all species of Rubiaceae published in ScienceDirect and CAS SciFinder websites between 1990 and 2014. The data compiled in this review show the distribution of the studied species classified by their respective tribes and subfamilies as well as the isolated compounds and their chemical classes (Table 2).
Based on the obtained data, the main occurrence of iridoids, anthraquinones, triterpenes, indole alkaloids and alkaloids belonging to different chemical subclasses, was observed. The chemical profile, as expressed by the occurrence of major categories of secondary metabolites (alkaloids, anthraquinones and iridoids) showed to be quite different for each subfamily. Furthermore, the study of specific classes may contribute to chemotaxonomic correlations, since there are compounds with restricted distribution [54]. These same classes of substances served as a distribution pattern to create and modify plant classification systems as proposed by Dahlgren [54].
In Ixoroideae subfamily, the iridoids are found as chemotaxonomic markers, in Cinchonoideae the indole alkaloids predominate over other substances and in Rubioideae the anthraquinones are the major class of secondary metabolites ( Figure 3). These global findings corroborate those found in the Brazilian Rubiaceae chemotaxonomic study by Bolzani [15].
Other studies also describe indole alkaloids as the class of substances of major occurrence in Cinchonoideae, especially in Guettardeae tribe [50,55]. Studies by Wijinsma and Verpoorte [56] and Bolzani et al. [15] describe the occurrence of standardized chemical markers: iridoids in Ixoroideae; indole alkaloids in Cinchonoideae and anthraquinones in Rubioideae. These data corroborate the one presented in this review.
Therefore, it was observed triterpenes widely distributed in all subfamilies, therefore a chemotaxonomic correlation cannot be established. The occurrence of a common pattern in secondary metabolism may suggest, strongly, taxons having a common ancestor. Thus, if there are morphological similarities, they can either be due to a common ancestry or convergent evolution [54]. Furthermore, the seco-iridoids are iridoids precursors and also participate in the biosynthesis of monoterpene indole alkaloids, so they may be involved in two distinct chemotaxonomic subdivisions [57,58]. Thus, different species may exhibit different chemical substance classes, but having the same precursor, which may indicates a phylogenetic relationship [59][60][61][62][63][64].
However, studies establishing a chemotaxonomic classification of plants are quite complex, since there are different types of secondary metabolites that can be distinct in correlated species. These differences in the production of secondary metabolites can be attributed to a number of factors such as genetic mutation, blocking of a biosynthetic pathway and changes in the metabolism due to infection. Soil and climatic variations such as altitude, soil type, macronutrients, micronutrients and water availability, plant age, ultraviolet radiation, rainfall, seasonality and circadian rhythm, also have great influence on the production of metabolites. Besides the fact that the chemical composition can be variable in accordance with the plant organ, it is necessary to study the plant as a whole, to be able to infer a degree of similarity [59][60][61][62][63][64].
Considering the chemical profile of the Rubiaceae family and the metabolic pathways used to produce it, Rubioideae is the most ancient subfamily from an evolutive point of view [16], then it was subdivided into Ixoroideae and finally into Cinchonoideae. The chemical biosynthetic pathway now supports this botanical conclusion. In Rubioideae, anthraquinones are the main metabolites and the pathways are not so specific, being iridoids and indole alkaloids produced also in a large amount.
In Ixoroideae, the most active biosysthetic pathway is the one that produces iridoids; while in Cinchonoideae, it is the one that produces indole alkaloids together with other alkaloids.

Conclusions
This review has encompassed phytochemical studies of Rubiaceae species for the past 24 years. These substances have been isolated mainly from Uncaria, Psychotria, Hedyotis, Ophiorrhiza and Morinda genera. From the Rubioideae subfamily, 139 species were studied; 80 from the Ixoroideae, and 74 from the Cinchonoideae. Some correlations between iridoids, triterpenes, alkaloids and anthraquinones occurrence and distribution between tribes and subfamilies could be observed, providing chemotaxonomic clues. From an evolutionary point of view, the Rubioideae is the most ancient subfamily [16], then it was subdivided into the Ixoroideae and finally into the Cinchonoideae.