Essential oils biological activity of the shrub Lippia alba (Verbenaceae)

Introduction: Lippia alba is an aromatic species belonging to the Verbenaceae family. Its essential oils have been used in different industries, because of its biological properties. Objective: Identify the perspectives of the biotechnological applications of Lippia alba essential oils. Methodology: A scoping review was conducted on the biological activity of Lippia alba essential oils registered until October, 2018 in EBSCO, Embase, Pubmed, Scopus, SciELO, and Lilacs databases. Results: Chemotypes I and III have been reported for different biological activities from the evaluations performed on microorganisms, fish, arthropods, small mammals, and cell lines; fundamentally associated with antibacterial, antifungal, cytotoxic, antioxidant, and sedative effects, among others. Records focused mainly on the health, fishing industry, and agrifood sectors. Conclusion: Studies on the effect of essential oil are promising, but do not reflect a continuity of the research toward prototypes or finished commercial products. Research groups must unify evaluation methodologies and include in all studies the relationship between phytochemical and biological activity for the meta-analyses to be possible. Likewise, they must join efforts through the National System for Agricultural Innovation (SNIA, for the term in Spanish) to generate finished products that impact upon society and facilitate progress in the country’s bio-economy.

Organic products are broadly used by the global population for essential healthcare (Plantlife International, 2004;Grand View Research, 2018). In 2017 the market of personal-care products reached $12.19-billion, and further growth is foreseen (Grand view research, 2018). Medicinal and Aromatic Plants (MAPs) are a natural source of chemical substances that provide diverse biological properties (Bouyahya, Guaouguaou, Dakka, & Bakri, 2017). That is why countries several countries (i.e. the United States, Brazil, India, Colombia, and Cuba) support scientific research that let the discussion of the biological properties of MAPs and support better uses focused on bioprospection (Tofiño-Rivera, Ortega-Cuadros, Melo- Ríos, & Mier-Giraldo, 2017).
Colombia has an outstanding floristic wealth, among which are included several promising MAPs of interest for science, such as Lippia alba (Mill.) N.E. Br, an aromatic species belonging to the Verbenaceae family, whose essential oils have been used in the cosmetic, food, and biomedical industries (Tofiño-Rivera et al., 2017;Linde, Colauto, Albertó, & Gazim, 2016) due to its biological properties. However, these properties are derived from the plant's phytochemical composition. The presence and concentration of secondary metabolites is influenced by agro-economic management applied to the production of biomass, the phenological cycle of the material harvested, edaphoclimatic conditions, extraction method of the essential oil (Olivero-Verbel, González-Cervera, Güette-Fernandez, Jaramillo-Colorado, Linde et al., 2016), and the evaluation technique employed in some cases (Ramírez & Castaño, 2009). This scoping review was conducted to identify the perspectives of the biotechnological application of essential oils (EO) of Lippia alba (Mill.) N.E. Br.
A scoping review (Armstrong, Hall, Doyle, & Waters, 2011) was carried out on the scientific literature that describes the biological activity of L. alba EO. Likewise, a search equation was elaborated by using the terms "Lippia alba and essential oil", which was used to access published articles deposited in EBSCO, Embase, Pubmed, Scopus, SciELO, and Lilacs, since the beginning of time for each database, until October 8 th , 2018. The registries recovered were filtered to eliminate duplicates using Zotero. Inclusion and exclusion criteria were applied. A select group of documents was obtained to which a detailed analysis was applied (Fig. 1). The variables of interest (year, country, author, title, institution, EO extraction technique, biological activity evaluated, evaluation method, object of study, and result obtained) were extracted and compiled in a database using Microsoft Excel 2016. IBM SPSS 23.0 was used to conduct the statistical analyses. Application of the research protocol was conducted independently by two researchers to guarantee reproducibility of the work, and the discrepancies observed were solved through a third-party concept.

Bibliometric analysis:
The search recovered 706 registries, from which 115 documents were analyzed (Fig. 1). It was identified that L. alba EO and their respective biological activity have been topics of interest in the scientific community since 1990. The publication of scientific articles has not been continuous. Countries with higher rate of published articles related with the subject are Brazil with 70 (60.5 %), Colombia with 29 (25.2 %), and India with 7 (6.2 %).The institutions that have led these investigations are Universidad Federal de Santa María with 18 (15.6 % publications), Universidad Industrial de Santander with 8 (6.9 % publications), Universidad de Cartagena with 6 (5.2 % publications), Universidade Regional do Cariri with 6 (5.2 % publications), and Universidade Federal da Bahía with 3 (2.6 % publications).
Chemotype I EO can be used to enhance aquaculture techniques because they maintain the freshness of fish stored in ice (Veeck et al., 2018), do not modify the organoleptic characteristics (Cunha et al., 2010;Toni et al., 2014;Hohlenwerger et al., 2017), or affect fish quality (Sena et al., 2016). However, further studies are needed, given that Veeck et al. (2018) reported that concentrations from 30-40 µl/L do not represent antimicrobial activity against mesophiles and psychrophiles, and Veit  response to stress (Toni et al., 2015;Cárdenas et al., 2016;Batista et al., 2018); 2) prior sedation is not recommended with 200 µL/L during three minutes with L. alba E Oto reduce initial agitation, nor is treatment recommended with 30-40 µL/L EO for transport because it does not avoid oxidative stress in liver (Salbego et al., 2014); for Argyrosomus regius, 15 μL/L is not recommended to transport live fish, given that it does not inhibit stress (Cárdenas et al., 2016); 3) concentrations of 1 600 and 3 200 μL/L of chemotype III oils are effective against Anacanthorus spathulatus, Notozothecium janauachensis, and Mymarothecium boegeri, natural parasites of Colossoma macropomum; but these dosages are toxic to fish gills, therefore, there is a need to propose strategies to take advantage of the antihelmintic effect of these products without affecting the host organism, using cutting-edge biotechnological techniques, like nanotechnology (Soares et al., 2016). Chemotypes I and III seem to be the most common. Chemotype I has more reports on the biological effect; however, it must be considered that the cell and molecular characteristics of each microorganism or organism also interfere with the EO spectrum, given that on Tribolium castaneum and Sitophilus zeamais parasites, chemotype III was more effective than chemotype I (Peixoton et al., 2015a), but on Candida krusei and Aspergillus fumigatus, chemotype I was more effective (Mesa- Arango, et al., 2009). Additionally, chemotype I had anesthetic effect on Hypsiboas geographicus (Salbego et al., 2017a), but not on Neohelice granulata (Souza et al., 2018), and chemotype III was anti-inflammatory in the RAW 264.7 murine cell line (Sepúlveda-Arias et al., 2013), but this effect was not registered on Oreochromis niloticus (Rodrigues- Soares et al., 2018).
One of the critical variables associated with energizing an industry through L. alba EO corresponds to the phytochemical characterization of the plant material from which it is extracted, given that the availability of raw material with standardized conditions constitutes a critical point for the bio-industry (BIOinTropic, Universidad EAFIT, & Silo, 2018). We can highlight the lack of a comprehensive productive model for the agroindustrial use of L. alba because only specific studies are identified on a single management practice and for a specific area of the country (Zambrano, Buitrago, Durán, Sánchez, & Bonilla, 2013).
Regarding this work, it was found that 18.3 % of the articles did not relate the biological activity of the EO with its composition, given that no indication is provided of the chemotype or majority components. In this sense, under a general vision of the registries recovered, exploratory or preliminary works were observed without the continuity of the evaluation, according to the Colombian technical standards and norms by the WHO to advance in the formulation of bio-products (Decree 677, 1995;Decree 2510Decree , 2003WorWHO, 2004;ICONTEC, 2011), which is reflected in the lack of patents registered or pending, although research groups exist in Colombia that include L. alba as study model (Tofiño-Rivera et al., 2017). A vector and confluent effort as a country is needed here through strategies defined within the National Agricultural Innovation System (SNIA, for the term in Spanish) to encourage development based on bio-economy and, thus, take advantage of the diversity of L. alba chemotypes registered throughout the country -citral in Bolívar and Cesar, limonene in Arauca, and carvone in Cundinamarca, Tolima, Boyacá, Valle del Cauca, Santander, Antioquia, Quindío, and Cesar (Durán et al., 2007;Mesa-Arango et al., 2009;Olivero-Verbel, Guerrero-Castilla et al., 2010;Tofiño-Rivera et al., 2016)-, considering the specific uses referred by the scientific literature and which have been systematized in this document.
Unlike synthetic substances (Célis, 2018) and other natural standardized substances in which the concentration used over the same or different organisms will permit a meta-analysis for a finer verification of the biological effect of the substance in context (Valero et al., 2011), this possibility would not be rigorous in the case of the set of documents recovered in this research on the biological activity of L. alba EO because although many of the investigations refer to the chemotype or the majority component, the same concentration of EO, or the same chemotype, may have large variations in the concentrations of its majority or minority components. This variation is dependent on the equation that explains the phenotypic expression of an attribute F = G + A + GXA: chemotype -genetics G-, bioclimatic offer of the plantation -A-, and the genotype x environment interaction -GXA-, which are conditioners of the plant's physiological response over the secondary metabolism, upon the bioclimatic offer, agro-economic management, and harvest time (Palacio-López & Rodríguez-López, 2008). In addition to this heterogeneity exhibited by the raw material, the extraction method also affects the final composition of the EO, which hinders the possibility for a meta-analysis for the bioactivity of L. alba from a set of studies with high variability in the concentrations of the bioactive components of the EO (Durán et al., 2007;García-Perdomo, 2015;Barrientos, Reina, & Chacón, 2012;Delgado, Sánchez, & Bonilla, 2016). It would, then, be important to have a set of studies with detailed phyto-chemistry of the EO and which use the individual standardized bioactive components as controls (Ortega-Cuadros, Tofiño-Rivera, Merini, & Martinez-Pabón, 2018). Other exploratory revision works have registered the variable biological activity of the L. alba EO, specifically in the results of microbial control, dependent on the extraction technique used and the quality of biomass harvested. This work also highlighted high heterogeneity in assessment techniques of the biological activity of the L. alba EO used by the scientific community (Ortega-Cuadros & Tofiño-Rivera, 2019).
It is concluded that L. alba EO is a substance of broad spectrum of use, which is why industrial development is feasible based on this raw material. Nevertheless, it is necessary to establish within the SNIA research programs that prioritize the most consistent effects registered in this review, in accordance with national and global demands. Besides, progress is necessary on preliminary tests at pilot scale established by the Colombian norms to advance in the generation of commercial formulations. One of the first steps in this respect corresponds to the methodological harmonization, given that in some studies there was no appropriate control of the variables interfering on the biological quality of the L. alba EO, no detailed phyto-chemical analyses were presented, nor were adequate controls involved to facilitate a subsequent meta-analysis that guides the prioritizing of investment resources in the most consistent application lines. Another significant element is the generation of strategic alliances to elaborate protocols to standardize agro-economic management conditions of the plants in production zones, as well as articulation with the industry for scaling of the protocols formulated.
Ethical statement: authors declare that they all agree with this publication and made significant contributions; that there is no conflict of interest of any kind; and that we followed all pertinent ethical and legal procedures and requirements. All financial sources are fully and clearly stated in the acknowledgements section. A signed document has been filed in the journal archives.

ACKNOWLEDGMENTS
The authors thank the Library at Universidad de Antioquia, the Biblioteca Agropecuaria (BAC) and AGROSAVIA for funding the research "Development of production systems based on aromatic and medicinal species in agro-ecological associations with improved vegetable varieties (hot pepper, bean, and eggplant) for degraded soils of the Caribbean", "Development and validation of sustainable control strategies for parasitic diseases with the greatest productive impact on livestock systems in Colombia". Also, as the project: "Restoration of soils degraded by mining by using rhizo-remediation strategies based on the use of native aromatic species that promote the development of regional micro-economies", of the international mobility call -Argentina chapter, 2014 call by COLCIENCIAS. Finally, thanks also go to FONTAGRO, for its project, "Development of regional micro-economies in the production of essential oils harvested in mining soils", from the 2016 call. RESUMEN Actividad biológica de los aceites esenciales del arbusto Lippia alba (Verbenaceae). Introducción: Lippia alba es una especie aromática perteneciente a la familia Verbenaceae, cuyos aceites esenciales han sido empleados en diferentes industrias dada sus propiedades biológicas. Objetivo: identificar las perspectivas de aplicación biotecnológica de los aceites esenciales de Lippia alba. Métodos: se realizó una revisión sistemática exploratoria de la literatura sobre la actividad biológica de aceites esenciales de Lippia alba registrada hasta octubre 2018 en las bases de datos EBSCO, Embase, Pubmed, Scopus, SciELO, y Lilacs. Resultados: los quimiotipos I y III han sido reportados para diferentes actividades biológicas a partir de evaluaciones realizadas en microorganismos, peces, artrópodos, pequeños mamíferos, y líneas celulares; fundamentalmente asociadas con efectos antibacterial, antifúngico, citotóxico, antioxidante y sedante, entre otros. Los registros se enfocaron principalmente a los sectores salud, industria pesquera y agroalimentaria. Conclusión: los estudios sobre el efecto del aceite esencial son promisorios, pero no reflejan una continuidad de la investigación hacia prototipos o productos comerciales acabados. Los grupos de investigación deben unificar metodologías de evaluación e incluir en todos los estudios la relación entre fitoquímica-actividad biológica, para que los metaanálisis sean posibles. De igual manera, deben aunar esfuerzos por medio del Sistema Nacional de Innovación Agropecuaria (SNIA), para generar productos acabados que impacten en la sociedad y faciliten el avance de la bioeconomía del país.