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The arnica extract

A study proves the analgesic and anti-inflammatory properties of the Brazilian species and indicates how to develop safe phytotherapies

NORBERTO PEPORINE LOPES ArchiveIt is common to use the Brazilian arnica (Lychnophora ericoides) in the form of teas and infusions against itching, mosquito bites, cuts, pains and inflammations – and it works. Researchers at the University of São Paulo (USP) in Ribeirão Preto decided to try to find out why – and they did find. They found three substances, two anti-inflammatory and one analgesic, with pharmacological active proven on laboratory animals or in vitro, directly on proteins. They also discovered what each part of the plant produces. Both the root and more intensely the leaf produces anti-inflammatory substances, while the analgesic is only in the root. The stalk, from what has been observed, does not produce substances of any pharmacological interest.

The team of professor Norberto Peporine Lopes, who coordinated this work, didn’t want only to isolate the main active ingredients for new medicines. Their objective was wider: to find the best conditions for the cultivation of the plant and the production of quality phytotherapies and at low cost, with the least quantity possible of constituents that might have collateral effects. It is a challenge that included a revision of the indications in the use of arnica, which must be used only externally. “We don’t recommend ingestion under any circumstances” , alerts Lopes. “Some substances could be toxic to the liver”.

According to him, nobody knows for sure what they are and what they could do, these substances in the commercial preparations made from arnica and sold as panaceas against mosquito bites, bruises or sprains. Among the more than 50 substances already found in different extracts, two, one an anti-inflammatory derived from quinic acid, from the internal tissue of the leaves, and the other the lignan named “lignana cubebina”, with a potent active analgesic from the roots, displayed satisfactory results in tests done with Swiss mice. The second was described in an article of the November/December edition of the magazine Phytochemistry. Other anti-inflammatory substances called goyazensolide and centraterin have arrived at a more advance stage.

They were tested directly on the proteins associated with the inflammatory process, also with good results. Professor Lopes considers the goyazensolide and centraterin, stored principally in the structure of the leaf called glandular trichome, a species of modified skin, the most potent furanoheliangolides (the class to which these substances belong ) inhibitors of the so-called NF-kB factor, the cellular messenger responsible for the start of the inflammation. To impede that this factor links to the DNA (deoxyribonucleic acid), the NF-kB avoids the formation of the proteins that start the inflammation.

However, goyazensolide and centraterin can cause allergic reactions on the skin, an undesired consequence as well of European arnica (Arnica montana). For this reason they are looking to perfect the extraction and purification methods. “We already know how to separate these substances, or at least, how to reduce the concentration of these lactones”, says professor Lopes. “Afterwards, we want to pass on these processes to small factories that, this way, will know how to produce with quality and know what they are selling.”

The researchers began their work in 1998. In rocky country such as the plateaus of Parecis (MT), of the Veadeiros (GO) and Diamantina (BA) and on the hills of Cipó and of Canastra (MG), they took samples and spoke with the herbalists, root extractors, and home-made medicine dealers. Their objective was to get to know the plant and the medicine preparations.

In the field, they verified that the herbalists sold both the upper parts, leaves, flowers and stalks, as well as the roots, all indicated as anti-inflammatory and analgesic, while the commercial preparations made use of only the leaves. Asking themselves why, the USP team decided to study the therapeutic activities of each part of the plant and found in the leaves just substances with anti-inflammatory activity and in the roots mainly the analgesic activity, although the roots also have secondary anti-inflammatory properties.

Italian immigrants
Professor Lopes states that there are commercial products from Brazilian arnica made by backyard companies, unregistered with the Ministry of Health and not even with proven chemical contents, although a series of reports confirm their therapeutic effects. On the other hand, homeopathic medicines are made from the European arnica, a well-studied species with proven effects.The Brazilian arnica, found onlly in Brazil and also called arnica of the sierra, false-arnica or candeia, began to be used in the 18th century by Italian immigrants in substitution of the European variety, here non-existent. Due to the similarity of the smell and oil of essence, they tested the species Lychnophora ericoides, of the same family, the Asteraceae, and the anti-inflammatory effect was the same.

Consequently, there was an increase in the use of this bush of up to two meters tall. It is found in regions of vegetation on rocks, or high plateaus with rocky and not very humid soils. The indiscriminate extraction and the destruction of its natural habitat put the arnica into the condition of a threatened plant, according to the Brazilian Botanical Society. One more reason why Lopes decided to study it.

Tissue culture
Conscious that arnica awakens commercial interest that could accelerate its extraction and increase the risk of its extinction, the USP team began to test the tissue culture of the plant with micro propagation in vitro and techniques of seed germination. The most important result, obtained in collaboration with Suzelei França and Ana Maria Soares Pereira, of the University of Ribeirão Preto (Unaerp), was obtaining of callus tissue, in a disorganized cell culture, which produces goyazensolide. In this way they could obtain at least one of the substances via biotechnology processes, without plant extraction.

The germination study of arnica indicated something more determinant than the soil. The plant only grows in symbiotic interaction with microorganisms of the soil, the so called arbuscular mycorrhizal fungi, as demonstrated by the agronomy engineer Dr. Marcos Eduardo Paron during his doctorate.

Dr. Paron, now a professor at the Federal University of São Carlos (UFSCar) in Araras, isolated 21 species (of the genres Glomus, Scutellospora, Entrophospora, Gigaspora and Acaulospora) which favored the growth of the arnica. “The commercial growing of arnica could depend on strategies such as inoculation with these fungi”, he says. It seems to be relatively simple to make the plant grow in places which are not uplands. It is enough to cultivate the fungi on trapping-plants (in this case the sorghum) in vases with sterile sand and then to mix in the soil used for the seedlings.

The conclusions have practical implications. “We concluded that we can produce the arnica in any place, not only on the hills as it was thought, as long as we have in the soil the type of fungus population that the shrub needs to carry out the symbiosis”, concludes Lopes. It only remained to know the period in which the plant contains more of the substances with analgesic and anti-inflammatory activity. The herbalists said that the arnica is only good a little before and a little after flowering, when the leaf gives out a sticky oil. Could it be true? For a year, Professor Lopes studied an adult plant on the Serra da Canastra. In the end, all said and done, the production of the anti-inflammatory goyazensolide is in fact greater during the time mentioned by the herbalists.

Lopes was still a boy when he began to enjoy field studies with plants. At 12 years of age, he used to follow his father, José Norberto Callegari Lopes, who was a professor of organic chemistry at USP in Ribeirão Preto. After graduating from the School of Pharmaceutical Sciences, he did his masters at USP in São Paulo under the orientation of Dr. Massayoshi Yoshida of the Otto Gottlieb group, one of the leaders in this area. A Czech naturalized Brazilian, Dr. Gottlieb developed here the research into lignoids, a group of chemical compounds which belong to the analgesic substances studied in Ribeirão Preto. Since March, Lopes has been doing his post doctorate specialization at Cambridge University in England. He is working with liquid chromatography coupled to a mass spectrometer, a technique which, on his return early next year, he intends to apply in the study of biosynthesis of anti-inflammatory and analgesics substances. The plan goes beyond the arnica. In his view, this work could result into techniques in quality and production of phytotherapeutic medicines in a manner, not only of the arnica shrub.

While this is going on, a pharmaceutical-technical study is in progress, in collaboration with Newton Lindolfo Pereira and Osvaldo de Freitas, also of USP of Ribeirão Preto, which will permit the production of the phytotherapeutic in the case that the toxicology tests don’t reveal any undesirable side-effects. Also a strategic question is being taken care of, the patenting of the results of the research, vital for surviving in a growing world market, in the United States the phytotherapeutics gross close to U$ 4 billion per year. Attentive to reality, Lopes, together with Yoshida, Massuo Kato and Sérgio Albuquerque, has already patented the production process of two neolignans, extracts of the virola (Virola surinnamesis), which are 50 times more active than the violeta de genciana, a commercial product used in prophylaxis of Chagas’s disease.

Lopes places great importance to the patent, remembering that certain Brazilian native plants, such as the espinheira-santa (Maytenus ilicifolia), indicated in the treatment of ulcers, were transformed into patented medicines by foreigners, stimulated even by work published by Brazilians. “If we don’t regulate our conditions of research, we will have to pay royalties to other countries in order to sell material which is ours in origin.” He considers the patenting of the native phytoterapeutic also to be fundamental as it is an effective and cheap medicine, which would benefit mainly people who normally don’t buy the allopathic medicines due to their high price.

The Project
Monitoring of the Biosynthesis of Lactonas Sesquiterpenicas in the Upper Parts in Cell Cultures of Lychnophora ericoides and Evaluation of the Anti-Inflammatory and Analgesic Activities (nº 98/06157-7); Modality Regular line of research assistance; Coordinator Professor Norberto Peporine Lopes – PharmaceuticalSciences Faculty of USP in Ribeirão Preto; Investment R$ 50,000.00