Xanthotoxin reverses Parkinson's disease-like symptoms in zebrafish larvae and mice models: a comparative study.

BACKGROUND: The aim of this study is to preliminary evaluate the antiparkinsonian activity of furanocoumarin-xanthotoxin, in two behavioral animal models, zebrafish larvae treated with 6-hydroxydopamine and mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in order to compare both models. METHODS: Xanthotoxin was isolated from Pastinaca sativa L. (Apiaceae) fruits. Then, the compound was administered by immersion to zebrafish 5 days after fertilization (dpf) larvae or intraperitoneally to male Swiss mice, as a potential therapeutic agent against locomotor impairments. RESULTS: Acute xanthotoxin administration at the concentration of 7.5 µM reversed locomotor activity impairments in 5-dpf zebrafish larvae. In mice model, acute xanthotoxin administration alleviated movement impairments at the concentration of 25 mg/kg. CONCLUSIONS: The similar activity of the same substance in two different animal models indicates their compatibility and proves the potential of in vivo bioassays based on zebrafish models. Results of our study indicate that xanthotoxin may be considered as a potential lead compound in the discovery of antiparkinsonian drugs.

Imperatorin as a Promising Chemotherapeutic Agent Against Human Larynx Cancer and Rhabdomyosarcoma Cells.

Naturally occurring coumarins are bioactive compounds widely used in Asian traditional medicine. They have been shown to inhibit proliferation, induce apoptosis, and/or enhance the cytotoxicity of currently used drugs against a variety of cancer cell types. The aim of our study was to examine the antiproliferative activity of different linear furanocoumarins on human rhabdomyosarcoma, lung, and larynx cancer cell lines, and dissolve their cellular mechanism of action. The coumarins were isolated from fruits of Angelica archangelica L. or Pastinaca sativa L., and separated using high-performance counter-current chromatography (HPCCC). The identity and purity of isolated compounds were confirmed by HPLC-DAD and NMR analyses. Cell viability and toxicity assessments were performed by means of methylthiazolyldiphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, respectively. Induction of apoptosis and cell cycle progression were measured using flow cytometry analysis. qPCR method was applied to detect changes in gene expression. Linear furanocoumarins in a dose-dependent manner inhibited proliferation of cancer cells with diverse activity regarding compounds and cancer cell type specificity. Imperatorin (IMP) exhibited the most potent growth inhibitory effects against human rhabdomyosarcoma and larynx cancer cell lines owing to inhibition of the cell cycle progression connected with specific changes in gene expression, including CDKN1A. As there are no specific chemotherapy treatments dedicated to laryngeal squamous cell carcinoma and rhabdomyosarcoma, and IMP seems to be non-toxic for normal cells, our results could open a new direction in the search for effective anti-cancer agents.

Antioxidant Activity of Pastinaca sativa L. ssp. sylvestris [Mill.] Rouy and Camus Essential Oil.

In the last decade, there has been growing interest in the food industry in replacing synthetic chemicals with natural products with bioactive properties. This study's aims were to determine the chemical composition and the antioxidant properties of the essential oil of Pastianica sylvestris. The essential oil was isolated with a yield of 0.41% (w/v) by steam distillation from the dried seeds and subsequently analysed by GC-MS. Octyl acetate (78.49%) and octyl hexanoate (6.68%) were the main components. The essential oil exhibited an excellent activity for the inhibition of primary and secondary oxidation products for cold-pressed sunflower oil comparable with butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), which were evaluated using peroxide and thiobarbituric acid values. The antioxidant activity of the essential oil was additionally validated using DPPH radical scavenging (0.0016 ± 0.0885 mg/mL), and ß-carotene-linoleic acid bleaching assays. Also, the amounts of total phenol components (0.0053 ± 0.0023 mg GAE/g) were determined.

Substrate-specificity of cytochrome P450-mediated detoxification as an evolutionary strategy for specialization on furanocoumarin-containing hostplants: CYP6AE89 in parsnip webworms.

The parsnip webworm, Depressaria pastinacella, is restricted to two hostplant genera containing six structurally diverse furanocoumarins. Of these, imperatorin is detoxified by a specialized cytochrome P450, CYP6AB3. A previous whole-larva transcriptome analysis confirmed the presence of nine transcripts that belong to the CYP6AE subfamily. Here, by examining midgut-specific gene expression patterns we determined that CYP6AE89 transcripts were highly expressed and furanocoumarin-inducible. Computer docking and energy-minimization of a CYP6AE89 model with all six furanocoumarins showed that 5-methoxylated bergapten and 8-methoxylated xanthotoxin had the smallest distances from the heme to the proton-donor residue in the catalytic I-helix, and that the 5,8-dimethoxylated isopimpinellin and bergapten had the smallest energy-minimized distance from the heme oxygen to the furan ring double bond. To evaluate this prediction, we expressed the CYP6AE89 protein in an Escherichia coli system, and used it to detect high catalytic activity against the two mono-methoxylated linear furanocoumarins - bergapten and xanthotoxin - and weak activity against isopimpinellin. Thus, CYP6AE89, like CYP6AB3, is probably specialized for detoxifying only a subset of hostplant furanocoumarins. A maximum-likelihood tree built with six representative lepidopterans with manually annotated cytochrome P450s shows that CYP6AE89 may have evolved much faster than the other CYP6AE proteins, possibly indicative of host selection pressure.

Evaluation of furanocoumarins from seeds of the wild parsnip (Pastinaca sativa L. s.l.).

Although the wild parsnip (Pastinaca sativa L. s.l.) fruits are known to contain linear and angular furanocoumarins, the individual components of the seeds have not been fully identified and quantitated, and, in the case of immature seeds, reported. In view of this, the main furanocoumarin compounds were extracted using pyridine, and were isolated using semi-preparative high-performance liquid chromatography. The structural elucidation of isolated compounds was done based on detailed spectral analysis conducted by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI/MS), 1H and 13C NMR and, where possible, by gas chromatography-mass spectrometry (GC-MS). The quantitative analysis of furanocoumarin compounds in the wild parsnip was conducted by analytical ultra-performance liquid chromatography (UPLC-DAD), calculated against the standard curves of isolated compounds. The total yields of furanocoumarin compounds from the seeds after extraction with pyridine were 107.2-222.8 mg g-1 (fresh weight) and 50.2-66.4 mg g-1 (soluble dry matter). Thirteen furanocoumarins were identified. The main compounds (percentage in FW) in immature seeds were bergapten (40.8), pimpinellin (10.5), methoxsalen (5.7), isopimpinellin (4.3), imperatorin (3.2), and phellopterin (7.2). Seven constituents previously not described in P. sativa seeds and its products were identified, namely, byakangelicol (14.4), heraclenin (8.5), isobergapten (2.5), byakangelicin (1.3), heraclenol (0.5), psoralen (0.3), and isobyakangelicin (0.8). The latter is a new compound of the Apiaceae family. Extraction of immature seeds using pyridine gave a much higher yield and a greater variety of furanocoumarins. This indicates that the wild parsnip, along with other Apiaceae family plants, may be an important source of bioactive compounds.

Endemic Balkan parsnip Pastinaca hirsuta: the chemical profile of essential oils, headspace volatiles and extracts.

The present study for the first time reports the chemical composition of the endemic Balkan parsnip Pastinaca hirsuta Pancic essential oil and headspace (HS) volatiles, obtained from fresh roots, stems, flowers and fruits, as well as fresh fruits n-hexane and diethyl ether extracts. According to GC-MS and GC-FID analyses, ß-Pinene was one of the major components of the root and stem HS volatiles (50.6-24.1%). (E)-ß-Ocimene was found in a significant percentage in the stem and flowers HS volatiles (31.6-57.3%). The most abundant constituent of the fruit HS, flower and fruit essential oils and both extracts was hexyl butanoate (70.5%, 31.1%, 80.4%, 47.4% and 52.7%, respectively). Apiole, accompanied by myristicin and (Z)-falcarinol, make up over 70% of the root essential oils. γ-Palmitolactone was the major component of the stem essential oils (51.9% at the flowering stage and 45.7% at the fruiting stage). Beside esters as dominant compounds, furanocoumarins were also identified in extracts. (Dis)similarity relations of examined plant samples were also investigated by the agglomerative hierarchical cluster analysis. The obtained results show there is difference in the composition of volatile components from different plant organs, while the stage of growth mainly affects the quantitative volatiles composition.

A characterization of content, composition and scavenging capacity of phenolic compounds in parsnip roots of various weight.

The contents, composition and radical scavenging capacity of phenolic compounds from parsnips of various root weights grown in Serbia were examined. The content of phenolic compounds depended on root size, and the highest content was in the group where the samples with the lowest root size were grouped. The content of phenolic compounds varied from 109.7 to 125.3 micromol of chlorogenic acid per g dry extract, i.e.5470.8 to 6245.1 mg per kg fresh weight. Among the identified compounds were quercetin, kaempferol, apigenin and genkwanin glucosides and hydroxycinnamic and caffeoylshikimic acids. The highest antioxidant capacity was found for the group with the lowest root weight; the IC50 values ranged from 1.59 to 2.49 mg mL(-1). There was positive correlation between the total phenolic content and scavenging capacity, and the kaempferol glucosides content could be an indicator of DPPH scavenging capacity of parsnip roots.

Absolute configuration of falcarinol (9Z-heptadeca-1,9-diene-4,6-diyn-3-ol) from Pastinaca sativa.

Falcarinol (9Z-heptadeca-1,9-diene-4,6-diyn-3-ol; (1) is a polyacetylene commonly found in several plant families. The absolute configuration of naturally occurring 1 is not clear and contradictory results have been reported in the literature. Determination of the absolute configuration of 1 from Pastinaca sativa L. was carried out. Isolation of 95% pure 1 was performed via successive fractionation and preparative-HPLC. A racemic mixture comprised of 3R-1 and 3S-1 was synthesized in order to confirm the absolute configuration of the isolated natural product using chiral HPLC. Based on a combination of chiral HPLC and specific rotation, 1 present in P. saliva was found to have a 3R absolute configuration (i.e. (3R, 9Z)-heptadeca-1,9-diene-4,6-diyn-3-ol).

Development of selective comprehensive two-dimensional liquid chromatography with parallel first-dimension sampling and second-dimension separation--application to the quantitative analysis of furanocoumarins in apiaceous vegetables.

Various implementations of two-dimensional high-performance liquid chromatography are increasingly being developed and applied to the analysis of complex materials, including those encountered in the analysis of foods, beverages, and nutraceuticals. Previously, we introduced the concept of selective comprehensive two-dimensional liquid chromatography (sLC × LC) as a hybrid between the more conventional, but extreme opposite sampling modes of heartcutting (LC-LC) and fully comprehensive (LC × LC) 2D separation. The sLC × LC approach breaks the link between first dimension ((1)D) sampling time and second dimension ((2)D) analysis time that is faced in LC × LC and allows very rapid (as low as 1 s) sampling of highly efficient (1)D separations, while at the same time allowing efficient (2)D separations on the timescale of tens of seconds. In this paper, we improve upon our previous sLC × LC work by demonstrating the ability to perform the processes of (1)D sampling and (2)D separation in parallel. This significantly improves the flexibility of the technique and allows targeted analysis of analytes that elute close together in time in the (1)D separation. To demonstrate the value of this added capability, we have developed a sLC × LC method using multi-wavelength ultraviolet absorbance detection for the quantitative analysis of six target furanocoumarin compounds in extracts of celery, parsley, and parsnips. We show that (2)D separations of (1)D effluent containing the target compounds of interest reveal the presence of unanticipated interferent peaks that would otherwise compromise the quantitative accuracy of the method. We also demonstrate the application of the chemometric method iterative key set factor analysis with alternating least-squares to sLC × LC to mathematically resolve target compounds that are only slightly separated chromatographically but not sufficiently resolved for accurate quantitation.

Screening of the in vitro amoebicidal activities of Pastinaca armenea (Fisch. & C.A.Mey.) and Inula oculus-christi (L.) on Acanthamoeba castellanii cysts and trophozoites.

The aim of this study is to evaluate the in vitro amoebicidal activity of the aqueous extracts of Pastinaca armenea and Inula oculus-christi from Turkey. In the presence of aqueous extracts (ranging from 1.0 to 32.0 mg/ml), numbers of the viable Acanthamoeba castellanii trophozoites and cysts were decreased during the experimental process. Both extracts showed a time- and dose-dependent amoebicidal action on the trophozoites and cysts. Among the extracts tested, I. oculus showed the strongest amoebicidal effect on the trophozoites and cysts. In the presence of 32.00 mg/ml extract solution in the media, no viable trophozoites were determined from the time of 24 h. Moreover, in the presence of 16.00 mg/ml I. oculus-christi extract, no viable trophozoites were detected in 72 h of the experiment. Effectiveness of I. oculus-christi extract was found moderate against the cysts. In the presence of 32.00 mg/ml extract, only 25.3% of the total cysts were killed effectively. In the case of P. armenea, 40.3% and 23.0% of the trophozoites and cysts were killed in the presence of 32.00 mg/ml extract at the end of the experimental process (72 h), respectively.

Influence of Sous Vide and water immersion processing on polyacetylene content and instrumental color of parsnip (Pastinaca sativa) disks.

The effect of blanching (95 +/- 3 degrees C) followed by sous vide (SV) processing (90 degrees C for 10 min) on levels of two polyacetylenes in parsnip disks immediately after processing and during chill storage was studied and compared with the effect of water immersion (WI) processing (70 degrees C for 2 min.). Blanching had the greatest influence on the retention of polyacetylenes in sous vide processed parsnip disks resulting in significant decreases of 24.5 and 24% of falcarinol (1) and falcarindiol (2) respectively (p < 0.05). Subsequent SV processing did not result in additional significant losses in polyacetylenes compared to blanched samples. Subsequent anaerobic storage of SV processed samples resulted in a significant decrease in 1 levels (p < 0.05) although no change in 2 levels was observed (p > 0.05). 1 levels in WI processed samples were significantly higher than in SV samples (p

Metabolism of myristicin by Depressaria pastinacella CYP6AB3v2 and inhibition by its metabolite.

Although methylenedioxyphenyl (MDP) compounds, such as myristicin, are useful in the management of insecticide-resistant insects, the molecular mechanisms for their action in mammals and insects have not been elucidated. In this study, GC-MS analyses of methanol extracts of foliage of wild parsnip (Pastinaca sativa) have identified myristicin as a substrate for CYP6AB3v2, an imperatorin-metabolizing cytochrome P450 monooxygenase from Depressaria pastinacella (parsnip webworm). In contrast with its strong inhibitory effects on many mammalian P450s, myristicin is effectively metabolized by CYP6AB3v2 (V(max) and K(m) of 97.9 pmol/min/pmol P450 and 17.9 microM, respectively) at a rate exceeding that recorded previously for imperatorin, the only other known substrate for this highly specialized enzyme. The myristicin metabolite of CYP6AB3v2 is 1-(3',4'-methylenedioxy-5'-methoxyphenyl)-2,3-epoxypropane. Molecular dockings have indicated that, unlike other epoxide metabolites of furanocoumarins, this epoxide metabolite is likely to remain in the CYP6AB3v2 catalytic site due to its low binding energy (-31.0 kcal/mol). Inhibition assays indicate that myristicin acts as a mixed inhibitor of this insect P450 and suggest that the epoxide metabolite may be an intermediate involved in the formation of P450-methylenedioxyphenyl complexes.

Allelic variation in the Depressaria pastinacella CYP6AB3 protein enhances metabolism of plant allelochemicals by altering a proximal surface residue and potential interactions with cytochrome P450 reductase.

CYP6AB3v1, a cytochrome P450 monooxygenase in Depressaria pastinacella (parsnip webworm), is highly specialized for metabolizing imperatorin, a toxic furanocoumarin in the apiaceous host plants of this insect. Cloning and heterologous expression of CYP6AB3v2, an allelic variant identified in D. pastinacella, reveals that it metabolizes imperatorin at a rate (V(max) of 10.02 pmol/min/pmol of cytochrome P450 monooxygenase (P450)) significantly higher than CYP6AB3v1 (V(max) of 2.41 pmol/min/pmol) when supplemented with even low levels of cytochrome P450 reductase. Comparisons of the NADPH consumption rates for these variants indicate that CYP6AB3v2 utilizes this electron source at a faster rate than does CYP6AB3v1. Molecular modeling of the five amino acid differences between these variants and their potential interactions with P450 reductase suggests that replacement of Val(92) on the proximal face of CYP6AB3v1 with Ala(92) in CYP6AB3v2 affects interactions with P450 reductase so as to enhance its catalytic activity. Allelic variation at this locus potentially allows D. pastinacella to adapt to both intraspecific and interspecific variation in imperatorin concentrations in its host plants.

Can dietary furanocoumarin ingestion enhance the erythemal response during high-dose UVA1 therapy?

As phototoxic skin reactions caused by psoralen are induced by wavelengths within the UVA1 spectrum, we assessed the potential of the small amount of psoralen in a normal diet to provoke phototoxicity in volunteers with skin types I and II. Threshold erythema was unaffected by ingestion of a 200-g portion of parsnip.

Parsnip webworms and host plants at home and abroad: trophic complexity in a geographic mosaic.

Due to differences in the structure of communities in which interactions are embedded, the intensity of interactions between species may vary with location; thus, what results from differences in outcomes and in degree of specialization is a geographic mosaic, which provides the raw material for divergent coevolutionary trajectories. Where selection intensity is great, reciprocal responses are likely in so-called "hotspots"; in contrast, where selection pressures are relaxed, reciprocal responses in "coldspots" are far less likely to occur. There are few if any studies examining how a gradient of increasing trophic complexity might influence the probability of phenotype matching and, correspondingly, the "temperature" of the coevolutionary interaction. This study was conducted to compare outcomes of the interaction between wild parsnip (Pastinaca sativa) and parsnip webworm (Depressaria pastinacella) in its indigenous area, Europe, to its area of introduction, the midwestern United States. Specifically, we tested the hypothesis that increasing trophic complexity, represented by alternate host plants or the presence of natural enemies, reduces the selective impact of parsnip webworms and hence diminishes linkage between host plant chemistry and webworms that would be expected in coevolutionary hotspots. This comparison of a two-species interaction in its area of introduction and its area of indigeneity revealed common patterns that are more reflective of interaction temperature than of continental origin. Where webworms are rare, parsnips produce lower levels of xanthotoxin and bergapten in both the midwestern United States and Netherlands populations. However, the most striking result from this intercontinental comparison is that what is a ubiquitous two-species interaction in North America is in fact exceptional in Europe; webworms could more reliably be found infesting H. sphondylium even where P. sativa was available as well. This preference for H. sphondylium exists despite the comparatively high probability of parasitism associated with this host plant and may reflect the overall lower furanocoumarin content of H. sphondylium. The interaction of parsnip webworms and wild parsnips at home and away demonstrates clearly the potential for rapid contemporary evolution of chemical traits upon re-association with a coevolved enemy, a potential evolutionary outcome that should be considered in the design and implementation of future weed biological control programs.

Polyacetylenes from the Apiaceae vegetables carrot, celery, fennel, parsley, and parsnip and their cytotoxic activities.

A dichloromethane extract of root celery yielded falcarinol, falcarindiol, panaxydiol, and the new polyacetylene 8-O-methylfalcarindiol. The structure of the new compound was established by one- and two-dimensional (1D and 2D) NMR, mass spectrometry, and optical rotation data. Nonpolar extracts of roots and bulbs of carrots, celery, fennel, parsley, and parsnip were investigated for their content of polyacetylenes by high-performance liquid chromatography with diode array detection (HPLC-DAD). All five species contained polyacetylenes, although carrots and fennel only in minor amounts. Additionally, the cytotoxicity of the four polyacetylenes against five different cell lines was evaluated by the annexin V-PI assay. Falcarinol proved to be the most active compound with a pronounced toxicity against acute lymphoblastic leukemia cell line CEM-C7H2, with an IC(50) of 3.5 micromol/L. The possible chemopreventive impact of the presented findings is discussed briefly.

Genetic variation in primary metabolites of Pastinaca sativa; can herbivores act as selective agents?

Although insect herbivory has been shown to act as a selective agent on plant secondary metabolism, whether primary metabolites contribute to resistance and can respond to selection by herbivores remains untested. In the wild parsnip (Pastinaca sativa), its principal herbivore, Depressaria pastinacella, acts as a selective agent on furanocoumarin resistance factors. In this study, we determined whether webworms can, by causing differential reductions in fitness, act as selective agents on parsnip primary metabolites. Estimates of narrow-sense heritabilities were significantly different from zero for C18 fatty acids in buds and developing fruits, fructose and sorbitol in buds, fructose, myo-inositol, bergapten, and psoralen in fruits. Wild parsnips protected from webworms by insecticide produced 2.5 times as much seed biomass as unsprayed plants; that webworms accounted for this difference in plant fitness was indicated by a significant negative relationship between reproductive effort and an index of webworm damage. Only a handful of metabolites influenced resistance to webworms; these included osthol, sorbitol, and protein in developing fruits as well as previously documented furanocoumarins. Osthol, a coumarinic compound, enhanced resistance, as did protein content, while sorbitol lowered resistance. Other primary metabolites may affect resistance to webworms, but their effect was context-dependent, that is, their effect depended on concentrations of other metabolites (epistasis). Susceptible plant phenotypes were found to have average chemical compositions. Although there was genetic variation in some of the primary metabolites in parsnips, the epistatic nature of their involvement in resistance and the lack of genetic variation in some suggest that selection on them from webworms will be either inconsistent or ineffective.

Microwave-facilitated extraction of furanocoumarins onto paper substrates: an imaging technique to analyse spatial distribution and abundance in leaves.

An impediment to studying the implications of fine-scale variation in the distribution of secondary compounds within plants has been an inability to map their distribution. Conventional approaches require fine-scale dissections, followed by extraction and analysis, a strategy which is often limited by the large number of extractions required and by the difficulty in quantifying compounds in very small samples. A method has been developed which allows the microwave-facilitated transfer of furanocoumarins from fresh leaves to two-dimensional substrates (phase separation paper). The fluorescence of these compounds under UV light is enhanced by treatment with base, which probably opens the lactone ring of the compounds. The developed prints are then photographed under UV light and the images analysed with commonly available software programs. Images so obtained for furanocoumarins in wild parsnip foliage revealed that the total fluorescence from extracted furanocoumarins is correlated with the amount of furanocoumarins remaining in the leaf. Thus, the images provide information not only on spatial distribution but also quantitative data.

Influence of the extraction mode on the yield of some furanocoumarins from Pastinaca sativa fruits.

Analysis of plant material is an important task in chemotaxonomical investigations, in search of plants with pharmacological activity or in standardisation of plant drugs. The choice of optimal conditions for the analysis of plant material and effect of extraction method on the yield of furanocoumarins from Pastinaca sativa fruits were examined. The following extraction methods were used in experiments: exhaustive extraction in Soxhlet apparatus, ultrasonification (USAE) at 25 and 60 degrees C, microwave-assisted solvent extraction in open and closed system (MASE) and accelerated solvent extraction (ASE). In most cases, the yield of furanocoumarins was highest by use of ASE method as well as by ultrasonification at 60 degrees C.

Effects of storage conditions on furocoumarin levels in intact, chopped, or homogenized parsnips.

Furocoumarins represent a family of natural food constituents with phototoxic and photomutagenic properties. They are found mainly in plants belonging to the Rutaceae and Umbilliferae such as celery, carrots, and parsnips. Parsnips (Pastinaca sativa L.) have become more and more popular as a vegetable, e.g., as a constituent of or ingredient in baby food. Previous work has shown that microbial infection of parsnip roots can result in a dramatic increase in furocoumarin levels. In this study, freshly harvested parsnips were stored as whole roots, pieces (cubes), or homogenate at +4 degrees C or -18 degrees C over various time periods under standard conditions. It was found that furocoumarin concentrations (sum of five furocoumarins: angelicin, isopimpinellin, 5-methoxypsoralen, 8-methoxypsoralen, and psoralen) in freshly harvested parsnips, analyzed by HPLC after extraction with diethyl ether and sequential solid phase (reversed-phase and silica) extraction, was generally lower than 2.5 mg/kg, and storage of parsnips in any form investigated at -18 degrees C over up to 50 days did not lead to a marked increase in furocoumarin levels. In contrast, storage of whole parsnips, but not of cubes or homogenate, at +4 degrees C resulted in a marked biphasic increase of furocoumarin concentrations after 7 and 38 days of storage up to levels of about 40 mg/kg. A dramatic increase in furocoumarin concentrations up to 566 mg/kg was observed when whole parsnips obtained from the market were kept at room temperature over 53 days, resulting in a visible microbial (mold) infection. Baby food products from the German market containing parsnips as an ingredient or constituent showed furocoumarin levels < or =0.41 mg/kg, suggesting that properly stored roots/preparations have been used. It is recommended that, after harvesting, parsnips be kept at -18 degrees C or under other conditions that prevent microbial infections.