Isolation and identification of toxigenic and non-toxigenic fungi in samples of medicinal plants from the market

The consumption of preparations of medicinal plants has been increasing during the last decades in occidental societies. The presence of toxigenic fungi in a plant product may represent a potential risk of contamination, because of aflatoxins and ochratoxins. In this study, 12 samples of medicinal plants were analyzed in relation to the level of fungal contamination, and the presence of producers of ochratoxin A and aflatoxins was assessed by visualization of fungi using a cromatovisor in coconut milk. Most of the species found belong to the genus Cladosporium, Fusarium, Aspergillus and Penicillium. Species producing ochratoxin A were present in 2 samples (16.7%), Melissa and Hibiscus. Species producing aflatoxin were found in samples of Jacaranda decurrens (8.33%). This study suggests that herbs, if stored improperly, can provide the growth of fungi and should be examined before consumption.


INTRODUCTION
The natural products that have therapeutic activities were already being used since the dawn of civilization, have long been the primary means used for prevention, treatment and cure of human disease and animal (CHOI et al., 2002;VEIGA et al., 2005).
In Brazil, the use of medicinal plants to treat illnesses has its origin in indigenous cultures, black and immigrant Europeans, already knowing today a lot about its use by the popular wisdom.With scientific advances, this ancient practice gave way to synthetic drugs, falling into oblivion for more than 50 years (BUGNO et al., 2006), however, there has been a growing rediscovery of the value of medicinal plants not only because of some unforeseen side effects of many drugs artificial, but its high price, thus contributing to the resurgence of herbal therapy (through plants).As fungal organisms are widely distributed in the environment, may be present in soil, plants, decaying organic matter, water, air and dust, unprocessed products of animal or vegetable origin can become contaminated with a wide variety of fungal species and their toxic products (COSTA et al., 2009;PRADO et al., 2008;TASSANEEYAKUL et al., 2004).Knowing this, the consumption of natural drugs have made their use a public health problem because of the possibility of access to products without adequate conditions of use, without warranty of quality, safety and efficiency, fundamental to the recovery or preservation of health consumer.
Fungi can be dispersed by air, contamination of plants can occur both before and after harvest and during processing.Among the major genera found in Brazil include: Cladosporium, Fusarium, Aspergillus, Penicillium and Rhizopus.The presence of these fungi in herbal medicines can be harmful to human health, since these can cause mycotoxicosis, when introduced orally or other diseases when inhaled (KNEIFEL et al., 2002).The mycotoxins are considered to be among the most carcinogenic natural substances known (FREIRE; KOZAKIEWICZ, 2005).Species of Aspergillus, producers of aflatoxin, are very common in products such as medicinal plants (RIZZO et al., 2004).
According to the RDC n o 48, March 16, 2004, herbal medicines should be registered according to the norms of the National Health Surveillance Agency (ANVISA), being the search for microbiological contaminants also included in the recommendations of the World Health Organization (WHO), (ANVISA, 2004;WHO, 1992).
This study aimed to evaluate the presence of mycotoxin producing fungi in medicinal plants sold in the city of Lavras -MG.

Assessment of fungal contamination
For mycological analysis were homogenized 25 g sample of each plant with 475 ml of peptone water in stomacher equipment.After homogenization, aliquots of 1 ml were used to perform serial dilutions in three tubes containing 9 ml of peptone water.Aliquots of 0,1 ml of the dilutions were transferred to Petri dishes, in triplicate, containing the culture medium Dichloran Rose Bengal Chloramphenicol (DRBC) and incubated at 25 °C for 7 days.After incubation, fungal colonies were counted, recorded and the number of colony forming units (CFU) per gram were calculated as Samson et al.(2004).
For species identification we used the manual identification described by Klich (2002) and Samson et al. (2004).The culture media used were Czapek Yeast Agar (CYA), Malt Extract Agar (MEA), Yeast Extract Sucrose (YES), at the temperatures recommended by the manual of identification.The toxigenic potential of isolates was evaluated in coconut-agar medium, second Lin and Dianese (1976), and incubated at 25 ± 1 ºC for 5 days.
The frequency of occurrence of fungi found in the microbiota of the samples was calculated as in equation 1, second Mandeel (2005).

RESULTS AND DISCUSSION
The risk from the presence of microorganisms in herbal drugs or other product intended for human consumption is due to the potential damage it might cause to consumers.In order to guarantee the quality and safety of these products in Brazil, the RDC Resolution n o 48, March 16, 2004(BRASIL, 2004), states that the search for microbiological contaminants in herbal medicines should comply with pharmacopoeial specifications.Both the Brazilian Pharmacopoeia (2010) and U.S. Pharmacopeia (2005) set a maximum contamination by fungi of 2 x 10 2 CFU/g per product, to products for oral use.
Table 1 lists the fungi isolated from each sample with its respective level of contamination and toxigenic potential.
The frequency ranged from 3,7% to Boldo do Chile, Guaco and Quebra-Pedra to 14,8% for Carobinha that presented contamination by 4 fungal species found.Mandeel (2005) evaluated the frequency of fungal contamination in pepper with the results ranging from 0,9% to 23,75%.
Through visualization of fungi in cromatovisor amid coconut milk, A. ochraceus (one per sample) producer of ochratoxin A was isolated from Melissa and Hibisco, and isolates from Carobinha, A. flavus (two in the sample), producer of aflatoxin.
Publications relating to the identification and quantification of fungi in herbal medicines in Brazil are scarce.Bugno et al. (2002), found contamination by Aspergillus and Penicillium in tea infusion, powder (guarana), fluid extract, tablets and capsules.However, show profiles similar to those of other countries.In Portugal, Martins et al. (2001) found the presence of filamentous fungi such as Aspergillus, Penicillium, Absidia, Mucor, Cladosporium and Paecilomyces in 62 samples of 7 different herbal medicines.Similar results were seen by Abou-Arab et al. (1999), Egypt andEfuntoye (1999), in Nigeria.
Food borne fungi are responsible for a high global incidence of mycotoxins, which are secondary metabolites produced by fillamentous fungi and involved in a toxic response called mycotoxicosis in human and higher animals when contaminated (FREIRE; KOZAKIEWICZ, 2005).Contamination of medicinal plants by toxigenic fungi and their mycotoxins pose a special hazard to health and can cause acute poisoning or chronic case of ingestion  et al., 1999;IARC 1993).
In Brazilian legislation, mycotoxins have not yet maximum permissible levels (LMT) to its presence in medicinal plants according to RDC n o 7, February 18, 2011, which establishes maximum permissible levels (LMT) for mycotoxins in food.
Currently, it is known that aflatoxin can cause, among other problems, cirrhosis, acute liver necrosis, hemorrhage, kidney, hepatitis B and serious injury to the skin.In addition, aflatoxin is considered a teratogenic agent, therefore, in pregnant women can cause irreversible harm to the fetus, and mutagenic, because the products of their metabolism in the body react with DNA at the cellular level by interfering with the immune system of individual infected, reducing thereby its resistance to diseases.
The herbal medicine in general is susceptible to fungal contamination during the process of planting and harvesting.In addition, improper storage and handling of these products can be a source of secondary contamination.Some cares in the steps of the processing of medicinal plants are needed to reduce the microbial load, proper hand hygiene of handlers of medicinal plants, the material collected should be placed on a clean surface, the collection container should be cleaned, should to seek to remove impurities that may accompany the body or newly collected plant, drying the plant should be completed as quickly as possible, making it difficult to microbiological contamination, since the raw material is manipulated after harvest again.The site should be cleaned dry, well ventilated, protected from attack by insects and other animals and also without light.After, the material of packaging must be properly cleaned, stored in a dry, ventilated and protected from sunlight and the incidence of the entry of rodents and insects.There should be no direct contact of the pack with the floor, and this should be placed on a pallet to prevent moisture transfer and possible contact with animals (REIS; MARIOT, 2001).The training of farmers by trained professionals is necessary.Facts observed in practice, as the presence of fragments of insects, earth, wood, and microbiological contaminants, indicate that should take urgent education measures in the interests of efficiency and therapeutic safety.

CONCLUSION
T h e s a m p l e s w i t h h i g h e r f u n g a l contamination were the Sete Sangria, Quebra-pedra and Guaco, and 41,66% in this study of medicinal plants did not meet the maximum acceptable by law.Among the fungi, the presence of Aspergillus was predominant (83,3%).In the samples of Melissa and Hibisco detected the presence of Aspergillus ochraceus producing ochratoxin A and the sample Carobinha was detected the species Aspergillus flavus producing aflatoxin.Although the presence of toxigenic fungi in a product does not imply the detection of mycotoxins, their presence represents a potential risk of contamination.Knowing the increase in consumption of herbal products as alternative medicines and the risk of acquiring and using natural products contaminated with fungi and mycotoxins, it is necessary to establish appropriate standards for toxigenic fungi and mycotoxins in medicinal plants in order to reduce the risks to consumer health.
of fungi (%) were estimated to determine the abundance of isolates within a given sample of plant in relation to all species isolated from all samples.

TABLE 1 .
Distribution of fungal contaminants, the level of contamination in the samples and toxigenic potential of medicinal plants.
FIGURE 1. Occurrence frequency of fungal microbiota of all medicinal plants.