MYCOBIAL CONTAMINATION AND MYCOTOXINOGENESIS OF Tinospora cordifolia : AN IMPORTANT MEDICINAL PLANT OF INDIA

An investigation of mycoflora and associated mycotoxins was carried out from dried market samples of stem portions of Tinospora cordifolia, an important medicinal plant of India. These samples were collected from various wholesale and retail shops of eight districts of Jammu and Kashmir state viz., Kathua, Jammu, Udhampur, Rajouri, Poonch, Doda, Srinagar and Leh. A total of 39 fungal species representing 18 genera were recovered by using surface washing technique. Assessment of mycobial load of T. cordifolia showed the presence of many such fungal species that are widely acknowledged as the most important mycotoxin producers. Analyses of samples for mycotoxin contamination was done by multimycotoxin detection method. The dried samples of Tinospora cordifolia were detected to be contaminated with aflatoxin B1, aflatoxin B2, ochratoxin A, patulin and citrinin. Among the various mycotoxins detected, aflatoxins were present in maximum number of samples, which is probably because Aspergillus flavus was recovered from all the investigated samples. However, fusarial species and their toxins were not detected from the investigated samples.


Introduction
Herbal medicines are used for primary health care by approximately 80 percent of the population in the developing countries (Akerele, 1993).These medicines have always remained the first choice of the people of developing countries because of low cost.Moreover, these herbal medicines have better cultural acceptability, better compatibility with human body and lesser side effects.People of the developed countries had more inclination towards chemical preparations, but during the last few years, there has been a considerable change in the medicinal system of the developed world also.Widespread toxicity and harmful after-effects associated with long-term use of synthetic drugs have forced the developed countries to think for some safe alternative healing methods (Gijtenbeek et al., 1999).They have realized that 'natural is better' and thus there is an increasing demand for 'green medicine' which is now commonly sold in health stores.
The state of Jammu and Kashmir is the sixth largest state (area wise) of India.It is situated in the north and surrounded by North-Western Himalayas and has a rich heritage of over 300 medicinal plants (Kaul, 2010).These plants are frequently harvested, dried and used either in the system of Ayurveda or in local health care traditions practiced by hakeems of Kashmir, vaidyas of Jammu and amchis of Ladakh.However, due to lack of proper knowledge and use of unscientific methods of collection, drying and storage (Kaul, 1997), these dried medicinal plants may become prone to contamination with fungal spores.Another effect is that these medicinal plants also undergo deterioration even before they are used in making drugs.Realizing the fact that mycoflora and mycotoxin contamination of dried medicinal plants has not received the attention that the magnitude of the problem warrants and since no such work has been attempted from Jammu and Kashmir State, which is a large reservoir of medicinal plants, an investigation was undertaken on one such important medicinal plant, Tinospora cordifolia Miers.
Tinospora cordifolia, which is known by the common name Guduchi, is an herbaceous vine (Fig. 1) of the family Menispermaceae (Hooker, 1897).It is used in the treatment of diabetes, high cholesterol, allergic rhinitis (hay fever), lymphoma and other cancers, hepatitis, fever, gonorrhea, syphilis, and to boost the immune system.Research has demonstrated that a combination of T. cordifolia extract and turmeric extract is effective in reducing the hepatotoxicity (Adhvaryu et al., 2008).
Alcoholic extract of the stem shows activity against Escherichia coli (Nagaprashanthi et al., 2012).A number of products of T. cordifolia are now available in the market, which are being used for curing different ailments (Table 1).
In spite of all these curative properties, it is not possible to capitalize the full medicinal wealth of T. cordifolia because of the associated fungal contamination of its market samples, which leads to altered efficacy of its drug products.Thus, an investigation was undertaken to isolate, purify and identify the mycoflora associated with the market samples of this plant and analyze the market samples for the presence of natural mycotoxin contaminants like aflatoxins (B1 and B2), ochratoxin A, patulin, citrinin, zearalenone, zearalenol and deoxynivalenol.

(i) Isolation of mycoflora and assessment of frequency (%)
Dried market samples of stem portions of Tinospora cordifolia were collected in presterilized polythene bags from various wholesale and retail shops of eight districts of Jammu and Kashmir viz., Kathua, Jammu, Udhampur, Rajouri, Poonch, Doda, Srinagar and Leh.Sample bags were brought to the laboratory and surface mycoflora associated with the market samples of dried stem portions of T. cordifolia was determined by using surface washing technique (Singh and Kainsa, 1983).For isolating maximum number of fungal propagules from the surface of each sample, three different media-Dichloran Rose Bengal Chloramphenicol agar (DRBC), Dichloran 18% Glycerol agar (DG 18) and Malt Salt agar (MSA) were used.The petriplates were incubated for 7 days at 28 ± 2 o C till the proper growth of the fungal colonies was obtained.The recovered fungal species were identified by studying their cultural and morphological characters and by using various keys and relevant literature.
Percent frequency of each fungal species was calculated by using the formula given below: Number of samples from which an organism was isolated Frequency (%) = x 100 Total number of samples tested

(ii) Extraction of mycotoxins from dried samples
Dried stem portions of T. cordifolia (Fig. 2) obtained from different markets were analysed for mycotoxin contamination by multi-mycotoxin detection method developed by Stoloff et al. (1971).In this method, 25 g of finely ground sample was taken in 250 ml Erlenmeyer flask containing 100 ml mixture of acetonitrile and 4% potassium chloride (90:10v/v).This solution was put on a mechanical shaker for 30 minutes and then filtered through Whatman no.41 filter paper.50 ml of this filtrate was taken in 250 ml separating funnel, defatted and extracted twice with 50 ml of iso-octane.The upper iso-octane layer was discarded.To the lower acetonitrile layer, 12.5 ml of water was added, shaken and extracted thrice with 20 ml of chloroform each time.The chloroform acetonitrile layer was filtered through Whatman no.41 filter paper having a bed of anhydrous sodium sulphate.The extract was collected in a beaker and then evaporated to dryness on a water bath.The residue was dissolved in 1 ml of benzene: acetonitrile (98:2 v/v) solution and stored in a clean screw cap vial for thin layer chromatography and HPLC analysis.2).Quantitative estimation of detected mycotoxins was done through high performance liquid chromatography (Table 2).The analytical equipment of HPLC (CLASS-LC10 SHIMADZU) consisted of a liquid chromatographic pump LC-10AT, an auto injection system SIL -10A with a 50 µl sample loop, a variable wavelength absorbance UV -VIS detector SPD -10 set at 365 nm.The analytical component was CLC -ODS.The mobile phase consisted of water: acetonitrile: methanol (59: 29: 12v/v).Analysis was performed at room temperature (25-30 o C) and data was recorded in HP DeskJet 670C.

Results and Discussion
During the period of investigation, 22 market samples of Tinospora cordifolia were collected in pre-sterilized polythene bags.These samples were screened for the mycobial load by using surface washing technique and three media (Dichloran Rose Bengal Chloramphenicol agar, Dichloran 18% Glycerol agar and Malt Salt agar) of different chemical composition.While recovering surface mycoflora, Dichloran Rose Bengal Chloramphenicol agar medium could trap maximum number of fungal species, Dichloran 18% Glycerol agar medium helped to recover a wide range of non-fastidious xerophilic fungi including most of the Penicillium and Aspergillus species, whereas, malt salt agar was more useful in recovering the members of Aspergillus glaucus group.This indicates that nutritional requirements of various fungi differ and there is no single medium, which can help in the recovery of all the fungi.During the present investigation, 39 fungal species representing 18 genera were recovered from the dried stem portions of T. cordifolia (Table 3).Assessment of surface mycoflora associated with T. cordifolia showed the presence of many such fungal species that are widely acknowledged as the most important mycotoxin producers.In view of this, an investigation was undertaken to verify contamination of 8 major mycotoxins from the market samples.These included aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), ochratoxin A (OTA), patulin (PAT), citrinin (CIT), zearalenone (ZEN), zearalenol (ZOL) and deoxynivalenol (DON).
Other mycotoxins viz., ochratoxins A and citrinin were detected form samples of only 2 districts, whereas patulin was detected from samples of only 1 district of Jammu and Kashmir.Presence of aflatoxins in maximum number of samples is probably because Aspergillus flavus was recovered from all these samples.In addition, no contamination of Fusarium species and their toxins viz., zearalenol, zearalenone and deoxynivalenol was detected from the investigated samples.This study clearly indicates that xerophilic aspergilli and penicillia are commonly associated with the dried stem of Tinospora cordifolia.
Among the various Aspergillus species, Aspergillus flavus was detected from all the samples.In addition, some other xerophilic species belonging to Eurotium, Phyllosticta, Geotrichum, Emericella, Scopulariopsis, Trichoderma, Paecilomyces and Sepedonium were also recovered from the investigated stem of the medicinal plant.Realizing the importance of the quality of dehydrated medicinal plants, a large number of workers have recently engaged themselves in the study of surface mycoflora of various herbal drug plants during storage and marketing (Toma and Abdullah, 2013;Stevic et al., 2012).These workers also reported diverse range of fungal species belonging mainly to Aspergillus, Penicillium, Rhizopus, Chaetomium, Fusarium, Eurotium and Cladosporium.The presence of mycoflora, which are known to produce toxic metabolites (mycotoxins) were detected from the dried samples of T. cordifolia and many of them have hazardous effects (IARC, 1993).A survey of literature indicated occurrence of mycotoxin contaminants from dried medicinal plants of various Indian states viz., Bihar (Roy and Chourasia, 2001), Uttaranchal (Singh, 2003), Jammu and Kashmir (Sharma et al., 2013).
In the present investigation, dried stem samples of T. cordifolia were detected to be contaminated with very high concentration of aflatoxins B1 and B2 in comparison to the permissible tolerance limits.However, the magnitude of aflatoxin contamination varies with the type of dehydrated medicinal plant, storage practices, geographical factors, seasonal changes and varying aflatoxigenic potential of associated A. flavus strains (Bilgrami, 1984) Few other Indian researchers have also reported aflatoxin contamination in amounts exceeding permissible limits from varied types of dehydrated medicinal plants and their formulations (Roy and Chourasia, 2001;Singh, 2003).Similarly, there are reports of dried medicinal plants with aflatoxin contamination from other countries also (Elshafie et al., 2003;Wongwiwat et al., 2004).
Very few samples of dried stem portions of T. cordifolia were detected to be OTA contaminated (0.48 to 0.54 mgkg -1 ) and they belonged to Rajouri and Doda districts.Reports of OTA contamination from other medicinal plants and their crude products have been provided by other ethanomycologists also (Aziz et al., 1998;Efuntoye, 1999;Santos et al., 2013;Singh, 2003).IARC (1993) has classified OTA in group 2B because of its toxicity on humans.Committee on Toxicity of Chemicals in Food, Consumer Products and Environment (COT) considered OTA as a genotoxic carcinogen and proposed that its level in food be reduced to the lowest (COT, 1997).The joint expert committee of Food Additives of the WHO and FAO set a provisional maximum intake of 100 µgkg -1 body weight.However, few countries have legislative limits ranging from 5 to 50 µgkg -1 (Webley et al., 1997).
Another mycotoxin detected from the dried sample of T. cordifolia consisted of patulin.There is no earlier report of patulin contamination from dried medicinal plants.However, very high patulin contamination has been reported from rotted rosaceous fruits and their products (Beretta et al., 2000).
In the present investigation, detection of aflatoxins B1 and B2, ochratoxin, patulin and citrinin from market samples of dried stem of T. cordifolia clearly indicates that its powdered formulations are not completely safe for direct human consumption.In view of the detected mycotoxin contamination, an urgent need for proper drying and storage of crude herbals is urgently required.

Fig. 1
Fig. 1 Habit of Tinospora cordifolia Fig. 2 Dried stem portions of Tinospora cordifolia For qualitative estimation, known amount of sample extracts and standards of investigated Int.J. Agril.Res.Innov.& Tech. 3 (2): 16-21, December, 2013 mycotoxins obtained from Sigma Aldrich Co. were spotted on activated TLC (Thin Layer Chromatographic) plates and developed with different solvent systems (Table

Table 2 .
Table1.Various valuable economic products of Tinospora cordifolia Qualitative and quantitative estimation of mycotoxins

Table 3 .
Percentage frequency of mycoflora recovered from dried market samples of Tinospora cordifolia stem portions collected from various districts of Jammu and Kashmir state

Table 4 .
Analysis of mycotoxin contamination in market samples of dried stem portions of Tinospora cordifolia -, Not detected