Mycotoxin Contamination and Induced Biochemical Changes Associated with Selected Medicinal Plants

Mycotoxin contamination and induced biochemical changes in medicinal plants namely Azadirachta indica , Emblica officinalis, Plantago ovata and Vitex negundo collected from different localities of Uttarakhand (India) were investigated. Mycotoxin producing fungi like A.flavus, A.ochraceus,


INTRODUCTION
Indian forests have been a rich source of medicinal plant produce and these produce are widely used in the prevention, treatment and cure of disorders and diseases since ancient times. Literature on traditional herbal medicine preparation and survey of products of leading ayurvedic medicine manufacturer indicate use of plant produce in pharmacopoeial preparations. The concern with the quality of these natural products is due to the risk of potential fungal contamination and presence of mycotoxins. Practices used in harvesting, handling, storage, production and distribution make medicinal plants subject to contamination by various fungi, which may be responsible for spoilage and production of mycotoxins.
In this background, present study was carried out on medicinal plant produce of Azadirachta indica (Neem), Emblica officinalis (Aonla), Plantago ovata (Isabgol) and Vitex negundo (Nirgundi) to assess natural contamination of mycotoxins, incidence of mycotoxin producing fungi, and induced biochemical changes.

MATERIAL AND METHODS
Fresh samples of fruits/seeds of Azadirachta indica, Emblica officinalis, Plantago ovata, Vitex negundo (60 samples) were collected from the forests of Uttarakhand during their harvesting season whereas stored samples (166 samples) were collected from different storage centres and pharmaceutical industries.

Isolation and Identification of Mycobiota
In order to record the fungal flora associated with fresh and stored samples, blotter test as well as agar plate methods as recommended by the International Seed Testing Association [15] were followed. Isolation of mycobiota was done from the surface sterilized samples to isolate the internal fungi and unsterilized samples to isolate the surface fungi. For surface sterilization, the samples were soaked in 2% sodium hypoclorite (NaOCI) solution for 10 minutes. Subsequently the seeds, fruits were thoroughly rinsed in sterile distilled water and aseptically placed in Petri dishes containing three layered moistened blotter pads and Potato Dextrose Agar medium. The Petri dishes were then incubated for seven days at 25 ± 2°C and regularly examined under sterioscopic binocular microscope from third day and developing fungal colonies were recorded, isolated, identified and maintained on PDA. The isolated fungi were identified using research microscope with the help of standard literature and keys and matching the isolates with National Type Culture Collection at Forest Research Institute, Dehradun.

Screening of Fungal Isolates for Mycotoxin Producing Potential
The aflatoxin producing potentials of Aspergillus flavus isolates were tested in SMKY liquid medium [16]. The constituents of the medium were, Yeast extract-7g; Sucrose-200 g; Magnesium Sulphate (MgSO 4 7H 2 O)-0.5 g; Potassium Nitrate (KNO 3 -3 g; Distilled water-1lt. Methods of Schwenk et al. [17] and Davis et al. [18] were followed for testing citrinin and ochratoxin producing abilities of Penicillium citrinum and Aspergillus ochraceus isolates, respectively. The composition of liquid medium used was, Sucrose-40 g; Yeast extract-20 g; Distilled water-1lt. Zearalenone producing ability of the different isolates of Fusarium was tested on moist -rice medium as suggested by Scott et al. [19]. In all the cases mycotoxins were finally extracted with chloroform and the chloroform extract used for qualitative and quantitative detection of mycotoxins.

Natural Occurrence of Mycotoxins
The fresh and stored samples were extracted chemically for the presence of aflatoxins [20]. Few samples, in which fungi producing other mycotoxins were associated, were extracted by the method of Roberts and Patterson [21]. Samples were powdered in a grinder and 50 g flour was blended with 250 ml methanol: water (60:40,v/v) for 2 minutes at high speed. The extract was filtered through Whatman No. 1 filter paper. 125 ml of this filtrate was than extracted with 30 ml saturated sodium chloride (NaCl) solution and 50 ml n-hexane in a 250 ml separating funnel for 2 minutes. The lower methanol layer was transferred to another separating funnel. Finally, the lower methanol layer was extracted with 40 ml chloroform. The chloroform layer was drained into 125 ml flask containing 5 g cupric carbonate was allowed to settle and chloroform was decanted through Whatman No. 1 filter paper containing anhydrous sodium sulphate, extract transferred with careful washing to screw capped borosilicate vial and evaporated to dryness at 40 o C or under gentle stream of nitrogen, dissolved in 200 µl benzene -acetonitrile (98 + 2), and spotted on TLC.

Qualitative and quantitative estimation of mycotoxin
Qualitative and quantitative estimation of the mycotoxins were carried out using Thin Layer Chromatography (TLC). Silica Gel -G (with 13 % CaSo 4 as binder) was used as stationery phase for the TLC. 50 µl of chloroform extract obtained for mycotoxin screening was spotted on TLC plates. The spotted chromoplate was developed in the solvent system comprising Toluene: isoamyl alcohol: methanol (90: 32:2, v/v/v) . After developing, the plates were air dried and were observed under long (360 nm) and short (260nm) wavelengths UV-light for the detection of mycotoxins. Chemical confirmation of aflatoxin was done by Trifluoroacetic acid (TFA) as suggested by Stack and Pohland [22]. Presence of Ochratoxin on TLC plates was confirmed with ammonia fumes which changed blue green spot to a deep blue colour [18]. Confirmation of citrinin was done by spraying TLC plates with a freshly prepared mixture of 0.5 ml p-anisaldehyde in 85 ml of methanol containing 10 ml of glacial acetic acid and 5 ml of conc. H 2 So 4 and then by heating the plate at 130ºC for 10 minutes. This changed yellow streak of citrinin to yellowish green under long wave UV-light [19]. Zearalenone was also confirmed by spraying TLC plates with acidic p-anisaldehyde solution [19] by which greenish blue fluorescence turned faint brown (in visible light) and faint yellow in long wave UVlight.
Aflatoxin being most potent mycotoxin, the quantitative estimation for the same was carried out. Quantity of aflatoxin was estimated spectrophotometrically [23] with the help of UV-Spectrophotometer.

Estimation of Total Alkaloid
Total alkaloid was estimated following the methodology of Waldi et al. [24] gm of powdered sample was soaked with 28% ammonium hydroxide solution and little dried up. Subsequently, the sample was soxhleted with a mixture of chloroform: ethanol (3:1, v/v) for 8 hrs.The extract was acidified with N/2 H 2 SO 4 . The acid extract was collected. The process was repeated thrice for complete extraction of alkaloids. The combined acid extract was made alkaline with dilute NH 4 OH. The alkaloids were extracted from alkaline extract with 20 and 15 ml of chloroform. Chloroform was distilled on water bath, solvent was completely dried up and the residue was weighed on monopan balance to calculate the crude alkaloids. The crude alkaloids of each sample were dissolved in 0.5 ml of methanol. 50 µl of the solution was spotted on a TLC plate with the help of a micropipette. A mixture of cyclohexane, chloroform and diethyl amine (5:4:1, v/v/v) were used as solvent system. The air dried chromatoplates were then sprayed with Dragendroff reagent.
Statistical analysis of the results was carried out using SPSS package. Results of control and contaminated samples were tested for their differences by paired t-test using SPSS package.

Mycotoxin Contamination of Medicinal Plant Produce
Natural occurrence of mycotoxin contamination in fresh and stored samples was analyzed by following the standard procedures. Mycotoxin contamination in stored samples of E. officinalis and V. negundo showed aflatoxin B 1 as natural contaminant. Twenty eight percent (28%) samples of E. officinalis exhibited higher concentration of aflatoxins, up to 0.98 µg/g whereas in case of V. negundo 6% samples were found naturally contaminated with aflatoxin B 1 and mycotoxins were not detected in case of A. indica and P. ovata (Table 3).

Changes in the Alkaloid Content of Medicinal Plant Produce
Changes in the level of alkaloid content in medicinal seeds/ fruits due to infestation of toxigenic strain of A. flavus have been carried out. A. flavus caused considerable changes total alkaloid content of the medicinal seeds/ fruits under study during their infestation (Table 4).
There was an indication of inhibition in the total alkaloid content due to infestation with toxigenic strain of A. flavus. The percent amount of alkaloid in healthy medicinal seeds/fruits was 0.065%, 0.020%, 0.004%, 0.042% in, A. indica, P. ovata, E. officinalis, and V. negundo respectively. On the other hand, in infested fruits/seeds of A. indica, P. ovata, E. officinalis, and V.negundo, the percent (g/100 g) amount of total alkaloid recorded was 0.062%, 0.020%, 0.003%, and 0.039% respectively. Statistical   analysis of the results showed that a decline in the level of total alkaloid content due to fungal infestation in the substrates is significant at 5% level of significance.

DISCUSSION
The results of the present investigation indicate that a large number of fungi were associated with the medicinal plant produce of Azadirachta indica, Emblica officinalis, Plantago ovata and Vitex negundo, Association of fungi as well as their incidence are governed by the nature of the substrates, methods of storage and prevailing environmental conditions. Earlier reports also indicate varied pattern of fungal incidence with E. officinalis [25,13] and Azadirachta indica [26].
Four mycotoxigenic fungi viz. Aspergillus flavus, A. ochraceus, Fusarium verticillioides and Penicillium citrinum were commonly associated with the stored samples, however their incidence in fresh samples was comparatively low. The range of toxin production by these fungi in liquid medium varied with the type of the substrate. It was also noted that aflatoxin B 1 was produced by majority of the toxigenic isolates of A. flavus, however, the frequency of aflatoxins other than B 1 was comparatively low.  [27,28].
The variation in natural occurrence of mycotoxins may be due to differences in their moisture contents and constitutional make up. Earlier reports also indicate association of large number of mycobiota and mycotoxins in edible and medicinal fruits/seeds of forest origin [3][4][5][6][7][8]13]. There is an indication of decline in the level of total alkaloid in infested substrates. Inhibition in the level of total alkaloid content might be due to their utilization or degradation into simpler forms. Dutta and Roy [29] worked on deterioration in total alkaloid content of Strychnos nux-vomica by some fungi and reported that A. flavus and P. citrinum significantly inhibited the level of alkaloid content. Bilgrami et al. [30] also reported significant biochemical changes in dry fruits during aflatoxin elaboration by A. flavus.

CONCLUSION
Presence of mycotoxin producing fungi and high concentration of aflatoxins in the medicinal plant samples is a matter of great concern as these raw materials are commonly used for the preparation of herbal drugs. Due to fungal infection, quality of the product is deteriorated and use of aflatoxin contaminated herbal drugs may cause severe health hazard. Therefore, it is necessary for the Indian herbal industries to set up appropriate standards for screening the crude herbal drugs and medicinal plant produce to be used as raw material in the pharmacopoeial industry.