Pigments from fluorescent flowers can have clinical applications

Researchers at the University of São Paulo's Chemistry Institute prepare compounds that can be used as tools to diagnose and treat diseases.

By Karina Toledo

Agência FAPESP – The petals of fluorescent flowers are pigmented with betalains, a class of colored natural product that is also present in the common beet (Beta vulgaris) and in bougainvillea plants.

Researchers at the University of São Paulo’s Chemistry Institute (IQ-USP) in Brazil are studying naturally occurring betalains to develop methods for the preparation of betalain derivatives that can be used in the diagnosis and treatment of diseases, such as malaria and cancer.

The research project “Floral betalamic pigments: fluorescence and antiradical capacity”, which is supported by FAPESP, was coordinated by Erick Leite Bastos, a professor at IQ-USP. Bastos is also leading the project “Intermolecular interactions involving betalains”, which is scheduled for completion in late 2016.

“The amount of betalain in the fluorescent flowers of plants like moss rose (Portulaca grandiflora) is too small for a viable research project, so we extracted betalain from red beetroot, which has plenty of the substance but isn’t fluorescent, and converted it into the floral pigment. This process is known as semi-synthesis or partial synthesis,” Bastos said.

The next step was the investigation of how this betalain would interact with living animal cells. “We wanted to know whether floral betalain would also accumulate in animal cells, given that it’s found in plant cells. However, when the substance was incubated with human erythrocytes, i.e., red blood corpuscles, which are very simple cells, no staining was observed,” he said.

Because the properties of floral betalain did not favor their accumulation in the model cell, the group developed an artificial betalain, which they called betacoumarin-120 (BtC-120). This has the same core material as the natural substance but accumulates in some types of cell.

In assays described in papers published by the journal PLoS One, the group applied BtC-120 to cultured erythrocytes infected with Plasmodium falciparum, one of the protozoans that cause malaria. The synthetic (and non-toxic) betalain crossed several membranes and accumulated inside the live parasite, which became fluorescent. Now, their aim is to evaluate whether BtC-120 can distinguish the parasite from other cells. “The previous assay was done with erythrocytes, an excellent model system. Our challenge is to modify the compound to obtain a specific marker for the parasite amid other cell types,” Bastos said.

Several anti-malaria drugs bind easily with betalains, enabling these pigments to be used as tools for monitored drug delivery, he explained.

Cancer targeted

Ongoing studies with BtC -120 have also shown that it is possible to use artificial betalains to stain only tumor cells.

“We’re exploring the differences between tumor cells and healthy cells to create fluorescent compounds that accumulate only in tumors to facilitate their effective surgical removal,” Bastos said.

In partnership with Renata Tonelli, a professor at the Diadema campus of the Federal University of São Paulo (UNIFESP), the IQ-USP research group are studying the relationship between the structure of betalain and its interaction with cultured cells.

“Our work includes seeking to understand the transport of betalains into cells and the sub-cellular compartment in which they accumulate,” Bastos said.

For example, betalain’s high antioxidant activity may also influence oxidative processes that are harmful to cells. “Right now we don’t know how internalization of antioxidant betalain affects cells, but it may influence the pathways of cell death,” Bastos said.

The mechanism by which betalains act as antioxidants is being investigated by Karina Nakashima, who has a master’s scholarship from FAPESP.

Antiterror tool

In addition to interacting with cells, betalains also have high affinities to metal cations. The IQ-USP research group began by preparing chemical compounds of rare-earth cations and betalains with the aim of creating luminescent substances for applications in electronics.

“We were surprised to find that the complexes formed between betalain and rare earths, especially lanthanides, aren’t luminescent. But the orange complex formed between the pigment from red beetroot and the europium (III) cation was used to create a quick method to detect spores of Bacillus anthracis, the bacterium that causes anthrax and is used in terrorist attacks,” Bastos said.

The study was performed by Letícia Gonçalves, a student who worked as an intern at the US National Institute of Standards and Technology (NIST) while preparing her PhD with a scholarship from FAPESP. The results were published in PLoS One.

In 2001, several letters containing large amounts of B. anthracis spores were mailed in the US, resulting in five deaths and 17 other cases of infection.

“In the presence of pure calcium dipicolinate or when B. anthracis endospore germination is chemically induced, the orange complex of the compound turns red. This change could be a red alert not to open the letter,” Bastos said.

Although the method can be used to count the number of endospores based on red shift, false positives may occur when a highly complex matrix, such as soil, is analyzed.

“Despite this limitation, the method offers a rapid, low-cost way of monitoring the effect of nutrients on the speed with which endospores germinate in vitro,” Bastos said.

Leaving aside the various possible applications, Bastos stressed the basic science focus of the research. “We set out to understand how changes in the structure of betalains affect their properties and how they interact with other chemical species, including biomolecules,” he said.