Procedimiento a seguir en las operaciones sujetas al Sistema

Sample mean (mg/kg) ± RSD

Imidacloprid below LOQ Chlorprofam ND

Metamitron ND Chlorothalonil ND

Thiacloprid ND Linuron ND

Thiophanate methyl

below LOQ Pendimethalin ND

Phenmedipham below LOQ Captan 2.39 ± 53.4%

Tepraloxydim below LOQ Folpet 0.25 ± 7.9%

Prochloraz below LOQ Kresoxim-

methyl ND Pyraclostrobin 0.0585 ± 80.4% Fluazifop-P- butyl ND Prothioconazole - Chloridazon ND Fluazinam ND Cycloxidum -

Using this method, one of the LC-amenable compounds (pyraclostrobin) could be detected in the bulb material. MRLs for pyraclostrobin in food range from 0.02-1 mg/kg in the EU, depending on the commodity (EU Pesticide database,

http://ec.europa.eu/sanco_pesticides). In terms of safety, the average level detected in this study is within this range. Captan and folpet have MRLs from 0.02-5 mg/kg and 0.02-10 mg/kg, respectively. For folpet a level of up to 150 mg/kg is allowed for dried hops. Even though the average levels of these two compounds may be overestimated due to matrix problems, they are still within the MRL range for food. At present there are no MRLs specified for Narcissus bulbs as raw medicinal plant material. The level of pesticide residues allowed in bulbs will depend on how they will be processed after harvest, and how the galanthamine will be extracted. This will depend on the extraction method used by the extraction or pharmaceutical company responsible.

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Overall the pesticides typically used in Narcissus cultivation do not seem to persist in or on the bulb at high concentrations. For quality control of Narcissus bulbs for

contamination above MRLs values of pesticides commonly used in their cultivation the methods described here are satisfactory. However, for more sensitive analysis of pesticide residues, more sensitive equipment is needed. The extraction method should be optimized for the volatile GC-amenable compounds. From this study can be concluded that pesticide residues are not present at levels high enough to detect using

1_{HNMR metabolomics.}

Mycotoxins in Narcissus pseudonarcissus bulbs Introduction

If a plant part is to be used as raw material for the manufacture of a pharmaceutical product, it is usually required to be free of any toxic/potentially harmful components such as pesticide residues, microbial contamination and mycotoxins. When a single chemical is to be extracted from the raw material, this may be less of a factor as when a crude preparation (e.g. powder, oil) is made out of the plant material. Nonetheless, in manufacturing pharmaceuticals, very strict quality control measures are in place and each impurity present in the final formulated product has to be analyzed, identified and known.

Mycotoxins are secondary metabolites produced by certain fungi. Their presence in food or medicinal plant material is problematic since they have been shown to be carcinogenic, teratogenic, tremorogenic, heamorrhagic and dermatitic to many organisms, and are known to cause hepatic carcinoma in humans (Aziz et al., 1998). Collectively, diseases caused by exposure to toxic fungal metabolites are collectively called mycotoxicoses (Bennett and Klich, 2003).

Mycotoxins occur in agricultural products all around the world. They can enter into the chain during the field stage, during storage or at a later stage. Mycotoxin contamination is especially problematic in conditions where shipping, handling or storage practices encourages mold growth (Bennett and Klich, 2003). For human food, three genera of fungi are mainly responsible for mycotoxin production, namely Fusarium, Aspergillis

and Penicillium (Sweeney and Dobson, 1998). Fusarium mainly affects plants while

growing in the field as a plant pathogen, and produce mycotoxins just before or after harvest, while Aspergillis and Penicillium are more common as contaminants in stages after harvest, such as during drying or storage.

All mycotoxins are low molecular weight natural products produced as secondary metabolites by filamentous fungi. Due to their large diversity of chemical structures and

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biosynthetic origins mycotoxins can be hard to define and classify. Some of the most important mycotoxins are the aflatoxins (Aspergillis spp.), citrinin (Penicillium,

Aspergillus and some other spp.), ergot alkaloids (Claviceps), fumonisins (Fusarium),

Ochratoxin (Aspergillis), Patulin (Penicillium ssp), trichothecenes (group of more than 60 sesquiterpenoids, from various fungal families including Fusarium, Myrothecium,

Phomopsis, Stachybotrys, Trichoderma, Trichothecium and others) and Zearalenone

(Fusarium spp. on grains).

Biosynthesis of mycotoxins is determined by a wide array of physical, biological and chemical factors (D’Mello et al., 1998). Primary among these are time, temperature, humidity and physical damage by other organisms such as insects. These and other factors interact in complex ways to induce mycotoxin biosynthesis. Pesticide application has also been reported to affect mycotoxin production (D’Mello et al., 1998). Production of some Fusarium toxins may be increased by sub-lethal doses of certain fungicides and herbicides (Kabak et al., 2006).

As part of the quality control of Narcissus bulbs as raw material for the extraction of pharmaceutical product, Galanthamine, it is necessary to know whether the bulbs contain mycotoxins, and if so the levels at which they are present. Narcissus has long been cultivated as an ornamental flower bulb crop. In the Netherlands, various fungal pathogens attack Narcissus plants, and these are well studied for their effect on the appearance of flowers and yield of bulbs. Since Narcissus has not traditionally been cultivated for consumption or pharmaceutical use, the presence of mycotoxins has up to now not been of much importance. Some of the fungi that infect Narcissus bulbs in the Netherlands belong to genera that are known to produce mycotoxins. For example,

Fusarium oxysporium, one of the main fungal pathogens in Narcissus, has been

reported to produce mycotoxins (D’Mello et al., 1998). Members of the genus

Penicillium also infect Narcissus and are also known to produce mycotoxins (Sweeney

and Dobson, 1998).

The chemical diversity of mycotoxins poses a challenge for their detection and quantitation in plant material. Many methods have been developed for different classes of mycotoxins in various substrates. Mycotoxins are usually present at levels of around µg/kg, so detection methods need to be suitably sensitive (Shephard, 2008).

Traditionally methods usually involve solvent extraction and cleanup followed by various chromatographic separation methods and detection by UV or mass spectrometry (Lin et al., 1998). Sensitive ELISA methods using antibodies have been developed for detection of certain mycotoxins in food (Trucksess and Scott, 2008). Another approach is to use PCR to detect mycotoxin-producing fungi in plant samples, targeting genes involved in mycotoxin biosynthetic pathways (Waalwijk et al., 2008).

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With the strict control measures in place in the formulation of pharmaceutical products, the presence of mycotoxins is now an issue that needs to be investigated. Due to time constraints and lack of suitably sensitive equipment, Narcissus bulbs samples heavily infected with Fusarium oxysporum were sent to an analytical laboratory that routinely screens plant samples for the presence of mycotoxins. As a control a healthy Narcissus

bulbs sample was also sent. The aim was to determine whether any mycotoxins were present in the infected tissue.

Methods

Two Narcissus pseudonarcissus cv. Carlton bulb samples (three healthy bulbs and three

bulbs heavily infected with Fusarium oxysporum) were ground in liquid nitrogen. The finely ground samples were analyzed at the RIKILT Institute of Food Safety in Wageningen according to their standard LC-MS/MS based mycotoxin screening methods (Kokkonen and Jestoi, 2009).

Results

The results of the mycotoxin screening are shown in Table 9. The plant material was tested for the presence of thirty important mycotoxins. None of the mycotoxins were detected in the healthy bulb material above the limit of detection. In the Fusarium- infected bulbs one mycotoxin, Beauvericin, was detected at 0.195 mg/kg. Beauvericin was first isolated from Baeuverina bassiana, an insect-pathogenic fungus(Hamill et al., 1969). It is a cyclic hexadepsipeptide, consisting of alternating N-methyl-phenylalanine and D-α-hydroxy-isovaleryl-(2-hydroxy-3-methylbutanoic acid) units (Figure 4).

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Beauvericin can form stable complexes with cations (e.g. Sodium, potassium, calcium etc.) as well as some neutral or charged small molecules. These complexes are

lipophilic and can be transported into a lipophilic phase (Hilgenfeld and Saenger, 1982). Beauvericin can also form dimeric complexes which can form channel pores in cell membranes through which ions can be transported (Hilgenfeld and Saenger, 1982; Kouri et al., 2003). Beauvericin was found to be toxic to brine shrimp when first isolated (Hamill et al., 1969). Antibacterial activity has been reported against various species (Castlebury et al., 1999). The compounds has been found active in various insecticidal bioassays, including mosquito larvae, blowfly and Colorado potato beetle (Grove and Pople, 1980; Gupta et al., 1991) . Toxicity to various vertebrate and invertebrate cell lines has also been reported (Çelik et al., 2010), with this bioactivity believed to be mainly due to the pore-forming properties of the beauvericin complexes (Tedjiotsop Feudjio et al., 2010). Beauvericin’s activity against human cancer cell lines has aroused some interest in investigation as its use as a therapeutic lead compound (Tedjiotsop Feudjio et al., 2010).

A simple investigation to determine whether Narcissus bulbs infected with Fusarium

oxysporum contains mycotoxins was carried out. The only mycotoxin found in this

screening was the cyclic peptide beauvericin. This compound is toxic to human cells, and therefore its presence in raw pharmaceutical material should be known. Based on these results it is also recommended that only healthy bulbs are used for the extraction of galanthamine.

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Table 9. Results of the mycotoxin screening. Compounds not detected (ND) are presented as less than the limit of detection (in mg/kg); a: <0.0005, b: <0.001, c: <0.005, d: <0.01, e: <0.02, f: <0.025, g: <0.04, h: <0.05, i: <0.1, j: <0.4.

Mycotoxin Healthy bulb (mg/kg) Infected bulb (mg/kg)

3&15-AcetylDON (sum of) NDh NDh

Aflatoxin B1 NDa NDa Aflatoxin B2 NDa NDa Aflatoxin G1 NDa NDa Aflatoxin G2 NDb NDb Agroclavine NDb NDb Alternariol NDe NDe Alternariol-methylether NDc NDc Beauvericin NDc 0.195 Citreoviridin NDe NDe Citrinin NDd NDd Deoxynivalenol NDi NDi Diacetoxyscirpenol NDd NDd Fumagillin NDg NDg Fumonisin B1 NDd NDd Fumonisin B2 NDd NDd Fumonisin B3 NDd NDd HT2 toxin NDd NDd Moniliformin NDh NDh Mycophenolic acid NDh NDh Neosolaniol NDe NDe Nitropropionic acid NDd NDd Ochratoxin A NDc NDc Penicillic acid NDh NDh Roquefortine C NDc NDc Sterigmatocystin NDb NDb T-2 Toxin NDd NDd Verruculogen NDj NDj ZON NDc NDc α-Zearalenol NDf NDf ß-Zearalenol NDh NDh

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Chapter 8. Seasonal accumulation of major alkaloids in