Bioprospecting of Bixa orellana L. for the Selection of Characters with Biological Activity

1. Introduction

Bixa orellana L. (Bixaceae), known as achiote or annatto, is a native shrub from Central and South America, grown in some tropical countries of the world, such as Peru, Mexico, Brazil, Colombia, Ecuador, Indonesia, India, Kenya and Eastern Africa [1]. Both the seeds and the leaves have been used in traditional medicine to treat constipation, fever, stomach acidity, asthma, scabies, ulcers and diarrhea [2].

The seeds, as coloring, condiment and thanks to compounds like bixin and norbixin, are of interest for the food, textile, chemical, pharmaceutical and cosmetic industries [3]. In addition, tannins, flavonoids, phenolic compounds, saponins, alkaloids, terpenoids, anthraquinones and glucosides have been identified [4,5,6,7]. The content of bixin, norbixin and geranylgeraniol confers biological activity with therapeutic purposes [8,9,10].

The carotenoids, apocarotenoids, terpenes, terpenoids, sterols, and aliphatic compounds are the main compounds that are found in every part of this plant, for which a wide range of pharmacological activities have been researched [11]. Their biological activity has been demonstrated for the control of bacteria and fungi [9,12]. The antioxidant activity has been demonstrated by various studies [7,9,13], and also the anticancer activity in cell lines of medical interest [8,10,14,15]; therefore, it has been included among nutraceutical foods. Because of its broad biological activity, B. orellana L. is a source for the development of new drugs with pharmacological activity, so there is the possibility of identifying morphological and phytochemical variables with a bioprospective approach in use, under the premise that the bioprospective meta-analysis eases the identification of the genotype, its character or outstanding phytochemical variable, and the state of the character, specifying the statistical validity and reducing possible contradictions in the literature.

2. Materials and Methods

An analysis of the studies published in the Scopus, Science Direct, Scifin der, Springer and Google Scholar databases was carried out, using the search terms achiote, B. orellana L.phytochemicals, pharmacology, cancer, biological activity, antibacterial activity, anticancer, cytotoxic and antioxidant activity. From this, n=56 results were identified, and when criteria of plant organ and biological activity identified in each publication were applied, the sample was reduced to n=28. All the studies included were studies that addressed the phytochemical characterization and biological activity of extracts from B. orellana L. (Table 1).

The studies included were conducted in Africa (n=1), United States (n=1), Philippines (n=1), Ecuador (n=1), Bangladesh (n=1), South Korea (n=1), Nigeria (n=2), Yucatán Mexico (n=2), Colombia (n=5), India (n=5), and Brazil (n= 6). The last three led the phytochemical and biological activity research of B. orellana L. A database was elaborated with the information, codifying the variables and their different states (Table 2), made up by the following: organ of the plant used, extraction methods, biochemical classes, groups of compounds, phenols and phenolic acids, flavonoids, tannins, monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, alkaloids, cyanogenic glucosides, antimicrobial and anticancer activity.

Statistical Analysis

The statistical analysis was developed with two approaches. The first through a cladistics analysis that incorporates the approach of Popper’s critical rationalism through the refutation of phylogenetic hypotheses examined under a parsimonious principle [29,30]; and through non-parametric statistics using the WinClada version 1.00.08 84,85 software (free license) [31]; with the Bootstrap/Jackknife resampling methods, approaching the genotypes as populations through a random simulation until generating a parsimonious cladogram [30]. This analysis defines the stability of the clades and identifies the state of the outstanding variables. The analysis was repeated 1000 times creating the values such as support indices, consistency, and reliability in the cladograms [32]. The systematic reviews carried out in the meta-analysis were directed towards the information disseminated, to reanalyze it with approaches adapted to the present research [33]. It must be clarified that the criteria selected were those with complete, traceable data, and reproducible results to avoid biases in the study [34].

The second approach was to determine the explicative accumulated variance, the statistical weight of each variable, and its state through a descriptive multivariate analysis and multiple correspondence analysis (MCA), with the FactoMineR [35] and factoextra [36] libraries with the Rstudio statistical package [37].

3. Results and Discussion

Figure 1 shows the different organs of B. orellana L. that could have an impact on the identification of the various genotypes, while Figure 2 presents the general cladogram that indicates the distribution of the B. orellana L. genotypes analyzed in function of the characters organ of extraction, method, biochemical class, and biological activity (Table 2). In total, 12 trees were obtained to get a consensus tree. This tree showed 149 steps or changes, a consistency index of the cladogram of 50%, and a retention index that reflects the percentage of characters that retain and conserve the change of taxa of 64%.

The parsimonious distribution of the genotypes (Figure 2) is not indicative of a strict genealogical relationship, since there are no morphological and genetic characters; however, it helps to understand the adaptive specialization [38,39] of plants in face of the difference in environmental conditions different from those in their habitat. In general, reproductive isolation, selective pressure, and lack of variability create unique survival characters reflected in the content and diversity of secondary metabolites [40].

The biochemical and biological activity variables showed that the genotype with origin from Africa1 was located as the closest one to the basal state, hypothetically indicating due to the variables analyzed that it could have greater similarity with a genotype from the original habitat (Figure 1).

Nine clades derive from Africa1. The first formed by Nigeria2 and India3, genotypes closer to the root, which share the presence of alkaloids as plesiomorphic characters. Even when the publications do not record the time of introduction to Nigeria and India, it is presumed that they could have had some reproductive isolation, absence of variability, and agroclimatic conditions different from their geographic origin (Central and South America). Various authors mention how reproductive isolation and absence of biological variation in some organisms promotes unique characters that can be used in different sectors of society, such as the case of enzymes responsible to produce secondary metabolites with biological effect of medical, agricultural or industrial interest [41].

The second clade derives starting with India3, formed by genotypes USA1, India4, Brazil2 and Brazil3, which made up an independent evolutionary route characterized by sharing the seed as organ of extraction and which is a derived state.

The USA1 genotype shares with the rest of the genotypes from this group the presence of geranylgeraniol (apomorphic state), highlighting that it has the derived characters cis-norbixin and trans-norbixin, which are related with the biological activity against Staphylococcus aureus, also a derived state. Brazil2 and Brazil3 are sibling genotypes, and presumed to be those of greatest “evolved” specialization from this group, characterized by the presence of flavonoids that is classified as an ancestral state. When it comes to apomorphic states, the presence of terpenes, ocimene, spathulenol, isoledene, bergamotene stands out, as well as bixin and norbixin.

The genotype Brazil3 shows anticancer activity against the cell lines U251, MCF-7, NCI-H460, PC-3 and HT-29 and presents as a plesiomorphic state (ancient or primitive character). The distinction of the proliferative activity in a genotype that is in the origin center of the species proves that the specific conditions of this place favored the presence of the compounds mentioned and contributed to the anticancer activity. However, when agroclimatic conditions change outside its place of origin, it loses chemical variability.

In the case of Ecuador1, it forms an independent clade and shows an evolutionary divergence possibly due to reproductive isolation. A plesiomorphic state stands out in the group, which is the extraction method by vapor sweeping, different from the rest of the genotypes, while the presence of ocimene, pinene, germacrene, farnesol and caryophyllene, as well as the activity against Staphylococcus aureus, are new characters or derived states (apomorphic). In this group, the extraction method marked the difference in the compounds detected.

The six remaining clades derive from Ecuador1. India5, Colomba1 and Philippines1 form a group characterized by the new or derived characters represented by the farnesol compounds, saponins and carotenoids. The genotypes from Colombia and Philippines present three ancestral characters constituted by anthocyanins and polyprenol, as well as stigmaesterol and sitoesterol. Although both genotypes do not have a geographic grouping, they do present it by group of compounds, which indicates a possible displacement of the genotypes from the center of origin towards the Philippines and India, where the original compounds could be conserved, or else, there was an influence of similar agroecological conditions that impacted the production of these secondary metabolites.

Also, Ecuador1 is derived from the group constituted by India1, Brazil1, Colombia2, Brazil4 and Colombia4. It should be mentioned that the genotypes from Brazil are again those that present the highest number of plesiomorphic states (kaempherol, granatin, neostrictinin, procyanidines, ellagic acid, as well as antioxidant activity). When it comes to apomorphic characters, the presence of saponins, tannic acid, and anthraquinones stands out, which are present in the genotypes from Colombia and India. In this group, the geographic grouping is clear regarding the states that highlight the ancestral characteristics of the genotypes from Brazil, zone registered as origin center of B. orellana.

Brazil4 is attributed with antioxidant activity and can be linked to the presence of ellagic acid, situation that agrees with other [42], who determined that this compound acts as chemo-protector against different types of cancer and which shows strong anti-proliferative activity against colon, lung and prostate cancer cells.

From Colombia4, 4 subgroups are derived, an independent one formed by Nigeria3, Philippines2 and Yucatán1 which are characterized by presenting three plesiomorphic states represented by tannins, alkaloids and atropines.

From this group, the genotype located in Yucatán is the most evolved and it is proven by the derived states present, such as the presence of saponins and the hepatoprotective activity. The evolution can be due to the agroclimatic characteristics or the manipulation of the crops in the zone, since in Yucatán there are commercial crops of B. orellana L. that have been genetically improved to reach higher seed production, which can be a factor that impacts the production of secondary metabolites [43].

The other sibling arm of Colombia4 groups, on the one hand, Yucatán2, India6, India2 and Indonesia1, which, despite not having a geographic grouping was characterized by the presence of the highest number of plesiomorphic states, among which germacrene, elemene, caryophyllene and squalene stand out, as well as chemo-preventive activity.

The anticancer activity against the cell lines HeLa, A549 and MCF-7 is a simplesiomorphic state, since it also presents in Brazil3, which is a genotype close to the root.

The derived character has to do with the presence of geranylgeraniol, carotenoids, bixin and norbixin, in addition to the activity against P. aeruginosa, E. coli and S. aureus. The biological and anticancer activity is determined by the variety of phytochemical compounds present in B. orellana L. and by the capacity of geranylgeraniol to induce apoptosis in A549 cells [26,44,45]. In this group, it can be inferred that there was a flow of plants from Yucatán towards India and Indonesia and the antiproliferative activity was conserved.

The last two groups derive from the branch coming from Colombia4. The node formed by Nigeria1 and Bangladesh share the apomorphic states represented by saponins and the biological activity against E. coli. Only an ancestral state is present (tomentosin).

The genotypes SouthKorea1, Colombia3 and Colombia5 are the last group and share four apomorphic states integrated by carotenoids, cis-norbixin, trans-norbixin and bixin. In addition, they present butein, catechins and chlorogenic acid, as well as cytotoxic activity as ancestral characters. The genotypes located in Colombia were considered the most evolved, compared to Africa1, Nigeria2 and India3, whose evolution can be due to pressure processes, such as the manipulation, edaphoclimatic conditions, or the genetic flow between genotypes. It should be highlighted that the activity found in the genotypes present in this bioprospective study is consistent with that found authors [1,46,47], who determine that the tannins, quinones and terpenoids have biological activity; in addition, lipophylic flavonoids can be disruptive for the cell membrane [48].

Table 3 shows the apomorphic characters present in the genotypes studied, observing sinapomorphic characters (shared characters) among the genotypes USA1, India2 and India4, such as geranylgeraniol, while India4 and India5 share farnesol; Brazil2 and India5, steroids; Colombia2 and Colombia4, tannic acid; Colombia4, India6 and Bangladesh1, saponins; India5 and Indonesia1, carotenoids; Brazil2 and Indonesia1, bixin and norbixin; Brazil3 and Ecuador1, ocimene; USA1 and Ecuador1, activity against S. aureus; and India6 and Colombia3 activity against P. aeruginosa. On the other hand, cis-norbixin, trans-norbixin, spathulenol, isoledene, bergamotene, germacrene, farnesol and caryophyllene are autopomorphic states (unique characters) because they are present in a single taxon or genotype.

Table 4 also shows that apomorphic characters (new or derived) are related with the antimicrobial activity against P. aeruginosa, E. coli, S. aureus and biochemical class, primarily the carotenoids bixin, norbixin, 9’-cis-norbixin, transnorbixin, saponins and monoterpenes. The plesiomorphic characters are related more with the hepatoprotective, chemo preventive and anticancer activity against the cell lines MCF-7, NCI-H460, PC-3, HT-29, HeLa and A549; as well as the presence of flavonoids (naringenin, kaempherol, anthocyanins, procyanidines, ellagic acid), triterpenes (stigamesterol and sitoesterol), tannins (granatin, neostrictinin), sesquiterpenes (elemene, caryophyllene) and coumarins. It stands out that some compounds from B. orellana in this bioprospective analysis act on microorganisms that can cause public health problems, highlighting characters for a possible program for genetic improvement.

It is important to highlight that the non-detection of a compound does not mean it is absent, since it could have been undetected because of the plant organ used, extraction method, or seasonal time of sample harvesting. For future studies, the proposal is to elucidate the “absent” compounds to secure the grouping; something to remember is that since it is a meta-analysis, comparative biases between taxa can be originated due to the various methods of sampling, extraction and analysis. Over-studied and under-studied taxa can bring biases to the analysis.

Multivariate Analysis

The multivariate analysis allowed identifying the variables that explain the highest explicative variance, in addition to exploring the correlations and reducing the dimension of the analysis with new indices (Córdoba et al.2012). It was determined that in four principal components (PC), the accumulated value is 86.31% (Table 5).

Table 6 indicates the variance by dimension that suggests that the antiproliferative activity is statistically significant for the cell lines U251, MCF-7, HeLa, PC-3, NCI-H460 and HT-29 in CP1 and CP4, where the presence of atropine, bixin, norbixin, naringenine, anthraquinones, alkaloids, coumarins, phenylpropanoids, tannins and some triterpenes is also significant, which suggests a relationship between the presence of some of these compounds or their synergy for B. orellana to present biological activity against the cell lines mentioned and some bacteria. In the rest of the dimensions, no statistical significance is present for the anticancer activity.

4. Conclusions

There is scientific evidence for the use of B. orellana L. as agent with anticancer activity, primarily against the cell lines U251, MCF-7, HeLa, NCI-H460, PC-3, A549 and HT-29, as well as biological activity against S. aureus, E. coli and P. aeruginosa. The antimicrobial and anticancer activity is related primarily with biochemical compounds such as geranylgeraniol, ellagic acid, carotenoids (bixin and norbixin), naringenin, and alkaloids. The conditions of reproductive isolation of the genotypes from Brazil, Yucatán, India and Indonesia provided the ideal agroclimatic conditions to produce compounds with biological activity because they produce those metabolites. This analysis can be used as reference for additional studies, genetic improvement programs, and revaluation of the species.