The influence of the environment on the "active ingredients" in medicinal plants

Study focused on Tithonia diversifolia, known to have anti-inflammatory and anti-microbial properties, among others, and established a model for management of several species

By José Tadeu Arantes  |  Agência FAPESP – Tithonia diversifolia, the wild sunflower or tree marigold, is an invasive species of flowering plant in the Asteraceae family that has adapted fully to tropical and subtropical ecosystems on three continents – Asia, Africa and America.

Its therapeutic properties have long been known to practitioners of traditional medicine in many countries and are now starting to be acknowledged by researchers in rigorous scientific studies. Anti-inflammatory, analgesic, anti-microbial, anti-viral, leishmanicidal and insectidal activity have been reported in the specialist literature, among other types of action.

This wide array of therapeutic properties derives from the secondary metabolites present in the plant species. The functions of secondary metabolites in plant dynamics is not yet fully understood by science, but in general terms it can be said that while primary metabolites (protein, carbohydrate etc.) are essential to the maintenance and reproduction of life, secondary metabolites (terpenes, phenols etc.) serve as a kind of chemical interface between the plant and its environment, acting as natural anti-oxidants, fungicides and insect repellents, among other functions. Hence their usefulness to humans.

A new study entitled “Effect of the environment on the secondary metabolic profile of Tithonia diversifolia: a model for environmental metabolomics of plants” was recently published in Scientific Reports, an online journal owned by Springer Nature. Lead author Bruno Leite Sampaio has a scholarship from FAPESP to support his PhD research project “Evaluation of seasonal variation of the main secondary metabolites and anti-inflammatory activity of extracts of Tithonia diversifolia (Hemsley) A. Gray (Asteraceae)”.

“Bruno monitored the environmental factors and analyzed their correlations with the plant’s metabolic profiles. A human analogy would be monthly tracking of food intake, weather conditions, caloric expenditure etc. and correlating this kind of data with biochemical factors such as cholesterol, triglycerides, blood sugar and so on,” said Fernando Batista da Costa, a professor at the University of São Paulo’s Ribeirão Preto School of Pharmaceutical Sciences (FCFRP-USP) in Brazil, Sampaio’s PhD supervisor, and co-author of the Scientific Reports article.

“In addition to laying a sound foundation for medicinal uses of T. diversifolia, this research establishes a model that can be deployed in the management of several plant categories, from medicinal plants to food crops, poisonous plants and so on, so there are multiple possible applications,” Costa added.

To compare the effects of two different environments, Sampaio grew T. diversifolia in the School’s Medicinal Plant Garden in the city of Ribeirão Preto, 315 km from the city of São Paulo, and Fazenda Santo Antônio, a farm in the county of Pires do Rio, 145 km from Goiânia, the capital of Goiás State.

“We wanted to exclude genetic variation as a variable, so all the plants had to be identical,” Sampaio explained. “In Ribeirão Preto we produced all the specimens from a single mother plant, raising stem cuttings to seedlings and then transferring the seedlings to the Medicinal Plant Garden. We used the same procedure in Goiás, raising 48 seedlings and moving them to Fazenda Santo Antônio.”

Production of secondary metabolites in the various parts of the plants (roots, stems, leaves and flowers) was monitored month by month for 24 months. At the same time the environmental variables of interest were recorded: soil macronutrients and micronutrients, as well as other soil physico-chemical parameters; and weather data from the National Meteorology Institute, including rainfall, humidity, temperature and solar radiation.

“The study focused not on biomass variation but on production and accumulation of metabolites in different plant parts. We were especially interested in secondary metabolites, which can be understood as chemical responses to environmental variations in order to adapt to the environment. Moreover, they constitute the active ingredients that determine the plant’s pharmacological application,” Sampaio said.

“We worked with a large number of plants so as not to have to cut samples from roots, stems, leaves and flowers always from the same individuals, because that would have induced an artificial chemical response in the plants. We were interested in observing variations in normal metabolism, not false metabolic responses to mechanical injury. For this reason we collected samples by rotation, taking material from each plant only once a year.”

Samples were converted into extracts and submitted to two powerful analytical techniques: ultrahigh-performance liquid chromatography (UHPLC) coupled to diode array detection and combined with high-resolution mass spectrometry (LC-MS); and proton nuclear magnetic resonance (1H-NMR) spectroscopy. The LC-MS analysis was performed using equipment acquired with funding from FAPESP under the aegis of the Thematic Project "Morphoanatomical, metabolomic and molecular studies as contributions to the systematics of Asteraceae species and access to their pharmacological potential", led by Beatriz Appezzato da Glória, while the NMR analysis was performed at the University of Strathclyde in Glasgow, Scotland.

Put very simply, mass spectrometry measures molecular mass of specific substances, while NMR determines how hydrogen atoms participate in each substance – whether the atomic chains are open or closed, whether they have single or double bonds, whether they have aromatic rings etc. In successful cases, combining the two sets of information reveals the chemical structure of the substances concerned, and identifies them by comparison with the findings recorded in the literature.

“The major novelty of Sampaio’s research is his combination of the data obtained using these two analytical techniques in a single matrix. This experimental approach is different from the approach normally used in environmental metabolomics studies. It enabled us to reduce the number of analyses by half, made the statistical treatment much more robust, and produced results that were easier to interpret,” Costa said.

“We were able to determine how the plants’ metabolic profiles varied over a two-year period. What we found was that the different parts of the plants responded differently to environmental factors. Metabolite production in roots was influenced only by soil factors, whereas aerial parts – stems, leaves and flowers – were more sensitive to climate – rainfall, temperature, and so on.”