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chemistry

Samples of health

Study on propolis results in patenting a method for analyzing this resin and in an oral antiseptic

MIGUEL BOYAYANFlasks with propolis solution: diversity in each region of the countryMIGUEL BOYAYAN

The antiseptic, healing and anti-inflammatory properties of propolis, a resin produced by bees to seal and sterilize the hives – hence the Greek origin, the word made up of pro (defense) and polis (city) – have been the subject of studies for over two decades. Even so, when researcher Maria Cristina Marcucci Ribeiro went to Spain, in 1995, and took with her samples of Brazilian propolis to draw up comparisons with European propolis, she discovered important differences among the main chemical components.

From then until now, Maria Cristina, currently a professor at São Paulo Bandeirante University (Uniban), has carried out several research studies in which the main subject was propolis. As a consequence, the works resulted in two patents registered with the National Institute for Industrial Property (INPI) and financed, in 2002, by FAPESP’s Nucleus for the Patenting and Licensing of Technology (Nuplitec).

The first, called by the researcher a “mother-patent”, chemically classifies Brazilian propolis from the traditional producing regions, such as the states of Minas Gerais, Rio de Janeiro, São Paulo, Paraná, Santa Catarina and Rio Grande do Sul. The patent deals with the process carried out by the researcher to analyze and to determine the type of propolis according to chemical markers. “This survey will make it possible to set standards for the propolis obtained from each region, facilitating the production of medicines”, Maria Cristina explains.

The second patent deals with formulation based on this plant resin for use in dentistry, without alcohol, which makes it completely different from similar products available on the market. It was in the course of the development of the oral antiseptic that Maria Cristina realized the need for companies to have standardized products, capable of offering the same chemical components, even though they are obtained from different suppliers. The work of typifying propolis has clocked up more than 2,000 samples analyzed. To arrive at the oral antiseptic dissolved in water, which has a patent requested in Brazil, tests were carried out on cariogenic bacteria (that cause cries), with the participation of Professor Walter Bretz, from the University of Pittsburgh, in the United States.

“Formulation with propolis inhibited all the cariogenic bacteria”, reports the researcher. The tests consisted of collecting saliva with a high concentration of bacteria, which was incubated with the mouthwash. It was left to Bretz to study the application of propolis in inflamed tissues of the gums and in recovering teeth, “with excellent results”. Tests with animals showed that the product, which has a slightly yellow color, does not show any toxicity. The study of its stability is being carried out by the University of the South of Santa Catarina (Unisul). “One of the last stages of the process, this test is fundamental for assessing for how long the product remains stable, without creating any fungus”, Maria Cristina explains.

The importance of the study becomes obvious when one learns that Japanese researchers have isolated and requested a patent for two chemical components (acids), which are active against tumors, found only in the product that originates from Brazil. Maria Cristina recalls a congress in Belo Horizonte in 2001, at which Japanese researchers were present. One of them, physician Katsumichi Matsushige, from Toyama University Hospital, told her that, in Japan, they have a sector intended for alternative therapies in the treatment of cancer. And, inside this sector, there is one section just for treatment with Brazilian propolis. Massive doses of the product, intended to strengthen the immune system, are given to patients during the process of traditional treatment for cancer: surgery, radiotherapy and chemotherapy. According to the Japanese doctor, the results have shown that in many cases there is no recurrence of the disease. For the researcher, this report is one more proof that she is on the right track.

Maria Cristina’s career until arriving at Uniban to work on the teaching staff and to take part in the recently formed research nucleus in 1998, started at the Chemistry Institute of the State University of Campinas (Unicamp), where she did her undergraduate, master’s and doctoral studies, with a spell of 13 months in Spain. In the last few years, she has been funded by FASPESP, under the Young Researcher Support Program.

She took over one hundred samples to the Soil Science and Applied Biology Center of Segura, located in Murcia, Spain, for the chemical composition to be studied. “But, after four or five months, I had not managed to identify hardly anything, just one or two compounds”, Cristina says, recalling her initial frustration for not having compound standards isolated with which she could compare our propolis. She explains that European propolis has, on average, 20% to 25% in flavonoids, chemical compounds that occur in the form of plant pigments, responsible, for example, for the color of orange rind and strawberries. It was also believed that Brazilian propolis had a similar composition. “But ours only has 2% to 4% in flavonoids”, she says.

The other chemical compounds in the resin produced in Brazil are prenylated acids (on average, from 8% to 10%), aromatic acids and compounds that are now the object of several studies. “Like the flavonoids, the prenylated acids have an antiviral, antibacterial, antioxidant and antitumor action”, Maria Cristina explains. The chemical composition of propolis is very complex. In some kinds, over 200 substances have already been described, with functions still unknown in the human organism.

Her discouragement on finding that the immense European literature on propolis would not be of much use for the research was transformed when she began to isolate each substance. “To start the work, I chose two different Brazilian propolises and isolated 13 compounds”, she says. “Then I realized that there were not a hundred different propolis samples from Brazil, but that were grouped by region”. The main composition varies according to the flora available in the area. But, generally speaking, the bees collect the resin in one species that is very abundant in Brazil, the lantana (Baccharis dracunculifolia).

Typically Brazilian, this species of Baccharis is invasive and develops rapidly, but it does not easily adapt itself to other regions, as the Japanese have found out, having taken cuttings from here. This interest is justified by the fact that at the moment they and other purchasers from Asia take almost all the crude propolis produced in Brazil.

Chemical marker
The map of Brazilian propolis drawn up by Maria Cristina, made following the groups of chemical markers, lists two large groups and one subgroup. From the north of Minas to the south of São Paulo, the BRP group is found (the initials stand for Brazil and prenylates). In the southern states (Paraná, Santa Catarina and Rio Grande do Sul), BRG appears (the G refers to conifer aldehyde, on account of species of conifers, like the araucaria, existing in the region). The BRP group is also found in the state of Paraná, with a submarker.

One other, called BRPG (with markers from BRP and BRG), has a presence in Paraná and in Santa Catarina, while one type of propolis with a large quantity of flavonoids, something rare in Brazilian territory, is found in Rio Grande do Sul, in the regions of the frontier with Argentina and Uruguay. All the groups are mapped and accompanied by figures, which act as indicators of the chemical concentration. The oral antiseptic, for example, was developed with propolis from the BRP1 group. The same study that confirms these markers, but done with other techniques, is being carried out by the group led by Professor Marcos Eberlin, from the Chemistry Institute at Unicamp, in which Maria Cristina also takes part.

The analysis of the propolis samples is done using a technique for fractionation called High Performance Liquid Chromatography (HPLC), a tool used to separate, isolate and identify chemical compounds. This data is then put into a data management program called Tip, for typification, developed to order. Tip has a database will all the patterns and the corresponding concentration. “You only have to indicate on the spreadsheet the peak of each compound, and it does the quantification. This software for classifying and quantifying propolis can be applied to any natural product, provided that it is done before analyzing the chemical components and the areas of the corresponding patterns are entered onto the spreadsheet.

One of the propolises typified, was tested on Staphylococcus aureus bacteria resistant to the oxacillin antibiotic, isolated at the São Paulo Hospital of the Federal University of São Paulo (Unifesp), by a student from Uniban, Patricia Laguna Miorin, in her dissertation for a master’s degree, to be defended at the Biomedical Sciences Institute (ICB), of the University of São Paulo (USP). These bacteria cause hospital infection, with a high mortality rate. The results achieved showed that this antibiotic, without any more activity in combating these bacteria, went back to being effective, when associated with propolis. With this novelty, Maria Cristina and Patricia were encouraged to ask Nuplitec to register the patent for the synergetic effect of this kind of propolis with oxacillin, in partnership with Uniban and ICB-USP.

At the moment, Maria Cristina is working on the relationship between the chemical composition of the propolis and the respective biological activity. “Once the chemical types are established, we will be putting them into experimental biological models, to see what type serves for a given disease”, she says. This is the way that the propolis with the best results for Helicobacter pylori , a bacterium that cause gastric ulcers and tumors in the digestive tract, for example, may be transformed into a medicine by the pharmaceutical industry. “And, instead of running after the discoveries made abroad with Brazilian raw material, we will be able to get a head start and patent their active principles”. To do so, she invites other researchers in the area to set up a database on Brazilian propolis.

The projects
1. Study of the Antimicrobial Activity of Samples of Brazilian Propolises on Cariogenic Bacteria Present in Saliva (nº 95/09306-5); Modality Young Researcher Support Program; Coordinator Maria Cristina Marcucci Ribeiro – Uniban; Investment R$ 32,722.25 and US$ 74,615. 12
2. Process for Identifying Types of Brazilian Propolises and Propolis Based
Formulations for Odontological Use (nº 00/10031-0); Modality Intellectual Property Support Program (PAPI); Coordinator Maria Cristina Marcucci Ribeiro – Uniban; Investment R$ 9,530.00 and R$ 6,000.00

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