A Gautam, S Sharma, R Bhadauria
fungal contamination, mycotoxins, powdered herbal drugs, storage
A Gautam, S Sharma, R Bhadauria. Detection of toxigenic fungi and mycotoxins in medicinally important powdered herbal drugs. The Internet Journal of Microbiology. 2008 Volume 7 Number 2.
This investigation was designed to through light on the microbial status of some powdered herbal materials used in Triphala preparation. A total of 68 powdered samples
The use of Ayurveda is one of the oldest, richest and most diverse traditions, associated with the use of medicinal plants in India (Tandon
As the case with other herbal drugs, raw material and powdered ingredients of trifla i.e.
Moreover, the occurrence of mycotoxin contamination in trifla churn seems to be very inevitable. The climatic conditions of Gwalior, where the average temperature and relative humidity are relatively high beside poor conditions and prolong duration of herbal drug storage, could also promote fungi i.e
Materials and methods
Source of samples
A total of 68 powdered samples of
The standards of Alfatoxins B1, B2, G1 & G2 (Sigma, Chemical, St. Louis, USA) were used.
Measurement of moisture content
Moisture content was measured prior to rinsing of raw materials in distilled water. For moisture content, weighed amount of individual samples were dried at 100 O C for 24 h and the difference in weight was calculated according to Essono
MC = [(Wi – Wf)/Wi] × 100
Where MC = Moisture content; Wi = Initial weight and Wf = Final weight
One gram of each powdered sample was mixed aseptically in 9 ml of sterile distilled water and shaken vigorously. Appropriate serial dilutions were made and 0.1 ml of the dilution was transferred aseptically to sterilized petri plates containing growth media. For mycobiota analysis, freshly prepared potato dextrose agar (PDA) and czapek dox agar (CZA) medium were used (Gautam and Bhadauria, 2008). Triplicate of each sample were incubated at 25±2oC for 7 days and examined daily. The mean number of fungal colony-forming units (cfus) was recorded. After incubation, the plates were examined visually and under a microscope. Identification of fungal species was done by culture and morphological characteristics (Gilman, 1975).
Incidence of fungal species
The incidence of different fungal spp. was assessed calculating the percentage relative frequency relative density and incidence. The relative frequency is defined here as the percentage of samples within which a given species was found at least once. The relative density (%) is related to the number of isolates of genus or species was observed to occur in the samples analyzed. Their values were obtained according to Giridher and Ready (1997); Marasas
Mycotoxin standard preparation
The standard solutions were prepared by dissolving the pure aflatoxins (AfB1, AfB2, AfG1 and AfG2) in acetonitrile: water (1:1, v/v) to give concentrations of 1 mg/ml each for AfB1, AfB2, AfG1 and AfG2. The solutions were stored at 40C. Aflatoxin standards were obtained from the Sigma Chemical Co. (St Louis, MO, USA).
Detection of mycotoxins
Natural occurrence mycotoxins in collected herbal drug samples were examined by thin-layer chromatography (TLC) (Singh, 1988). TLC technique was employed in 20x20 cm glass plates with silica gel (0.25mm thickness). The plates were dried in oven at 1100C for about one hour and stored in dust and moisture proof containers. For mycotoxins analysis, fifty grams of each powdered samples were extracted with chloroform. About 50 µl of extracts were applied in silica gel plates with the help of micropipette, together with specific standards, developed with a mobile phase containing benzene: methanol: acetic acid (24:2:1), were observed under long wavelength UV-light at 365 nm. For mycotoxins identification, the fluoresce and Rf value of the samples spot on TLC plates were matched with the fluorescent intensity and Rf value of standards.
The analysis of data was performed with Microsoft Excel 2007 (Window XP) for Mean and standard deviation. The statistical analysis was performed using student’s t-test. The p-value <0.05 was considered significant.
Fungal contamination and water content of herbal powdered samples
All samples were found to have a water content ranging from 5.84±2.68 to 7.14±0.38. However, significant differences could be noted between the mean water content of the different kinds of samples. Highest mean water content was found in Amla powder samples followed by Haritiki and Baheda powder samples. The mycological examination of powdered samples revealed that 100% fruit samples of
A total of 771 isolates were identified and recorded during the present study among all samples.
Fungal load of samples, determined by fungal colony count on agar plates revealed strong differences between samples. Table 2 is showing mean fungal load of the samples.
Occurrence and incidence of fungi in powdered herbal drugs
Relative density value (%) was estimated to determine the abundance of fungal isolates of given species among all samples (Table 2). The highest percentage relative density was shown by
Like in case of relative density, the highest relative frequency occurrence was recovered in
Based on incidences of occurrence,
Mycotoxin contamination of samples
It is known that, under favourable conditions, some fungi can synthesize orally toxic metabolites – mycotoxins. Hence in our present study, we paid our attention to herbal powdered samples contamination with mycotoxins also along with mould contamination. A total of ninety six powdered samples were analysed for mycotoxin detection through thin layer chromatography (TLC). No significant mycotoxin contamination was recorded in the analysed powdered samples. Only 8 (14.03%) out of 96 samples analyzed were found positive for mycotoxins.
Total six mycotoxins namely aflatoxin B1 & B2, aflatoxin G1 & G2, citrinin and sterigmatocystin were detected during mycotoxins analysis of powdered samples. Table 4 showing the number of samples contaminated and percentage frequency of mycotoxins occurrence in different powdered samples. Among the six mycotoxins detected, more frequently mycotoxins were aflatoxins G1, G2 & Citrinin (21.42% each) followed by AfB1 & B2 (14.28 each) and then sterigmatocystin with 7.14% frequency of occurrence. Maximum numbers of contaminated samples (7/25) as well as mycotoxins (4/6) were found in
The presence of higher fungal counts and high moisture contents in powder
Most of the identified fungal species like
In the present investigation, although a considerable mould contamination was detected but mycotoxins contamination was detected only in 14.03% powdered samples suggesting good storage conditions of herbal powder samples. This is because the mould presence itself does not imply that the stored material is also contaminated with mycotoxins. Certain mycotoxin-producing fungi can be present without mycotoxins being present itself (Brodnik
We wish to thank the Head, School of Studies in Botany, Jiwaji University Gwalior, Madhya Pradesh, India, for providing necessary laboratory facilities to carry out the present investigation successfully. This work was financed by Madhya Pradesh council of Science and Technology (MPCST), Bhopal, India.