mycological problems of crude herbal drugs - overview and

Indian Phytopath. 56 (1) • 1-13 (2003)

Mycological problems of crude herbal drugs -Overview and challenges*

AK. ROYUniversity Department of Botany, TM Bhaga/pur University, Bl1aga/pur 812 007

Mr. President, distinguist guests and fellowdelegates,

At the out set I would like to express gratefulthanks to the members of Executive Council ofIndian Phytopathological Society for giving me theprivilege to deliver the Jeersanidhi Award Lecturein its 55th Annual Meet. It is also a matter ofhonour that the Society has considered myresearch achievments fit for this prestigious Awardbestowed on me.

I, on this occasion would also like to rememberall the senior mycologists and plant pathologistsparticularly to my teacher late Prof. K S Bilgrami,who carved and disciplined me in the field ofresearch. He was the man who had vision inresearch and teaching deserving salutes ofappreciation not only by his students but also bythe lovers of science & scientific temperament.

I have been working on different aspects ofHerbal Drugs including Quality Control andMycotoxins Contamination for more than threedecades. During this period I got support fromvarious research sponsoring agencies like UGCand CSIR and also from my students, the sincereendeavour of whom makes me enable to deliverAward lecture before this august body of eminentscientists.

Present status of herbal drugs

Since the dawn of civilization the plants orplant parts have been in use to cure the humansufferings. Despite manifold development insynthetic medicines, the herbal drugs are stillmaintaining popularity amongst majority of

* Jeersanidhi Memorial Award Lecture

population in almost all Asian countries includingIndia. The earlier literature from Atharveda andRigveda to Materia Medica unveil the hiddenimportance of medicinal plants. As plant sciencesdeveloped, many more plants got entry in IndianPharmacopoeia. Amongst traditional therapeuticsystems i.e. Ayurveda, Unani, Siddha and Tibbi,the former grew at par with advancement of modernsystem of treatment. One of the Ancient AyurvedaTreatise 'Charak' (600 BC) has mentioned theentry of only 341 plants, fu rthermore in thebeginning of first century, Shushruta Samhita,listed 760 plants of medicinal value. Since then,bulk of literature has been published onenumeration of jneolclna: plants and theircharacterization as medicine for curing the diseasesof Homo sapians. With the advent of modernscientific techniques, many of these medicinalplants were put under chemical scrutiny leading tothe isolation and characterization of active principlesand establishment of their pharmaceutical activities(Allis, 1952; Atal & Sethi, 1962; Dhar et el., 1968;Barns et al., 1975; Aswal et ai, 1984). Thechemicals derived from plants are being used asarchitect for various synthetic products in the fieldof medicines, cosmetics and other health risks.This has also led World Health Organization (WHO)to develop an International Program to reviewavailable scientific data related to the efficacy ofmedicinal plants in the treatment of specificdiseases Despite, world wide development in plantmedicines, India which is considered earliest userof these indigenous products still occupies topmost position amongst developing countries.Recently the Govt. of India has launched severalprograms for generating awareness in mass forconservation and cultivation of indigenous medicinalplants. In addition, India is also a step ahead inearning foreign exchange by exporting crude herbal

2 Indian Phytopathology

drugs and it is estimated that the export valuewould go up more than Rs. 200 crore by 2003AD, however, we sometimes fail to achieve thetarget because of inadequate scientific &commercial infrastructures. Thus, the growinginterest in the traditional medicine in Asia,health food in Europe and North America havecreated new dimension for a number of herbalproducts.

Mycoloqical problems that include associationof mycoflora with crude herbals under storage,their role on biodeterioration and mycotoxincontamination have drawn world wide attention forquality maintenance and therapeutic potentials ofplant drugs. The threat of mycotoxin to herbaldrugs due to invasion of mycotoxin producingmoulds was first emerged in 1976 when acollaborative work was made by Udagava in JapanLater on many research stations especially intropical countries have undertaken this problem.The first report on natural occurrence of mycotoxinin crude herbal drugs was published from thislaboratory rather first from India in the year 1988.Since then an intensive work on herbalmycotoxicology has been carried out and witnessedby the publication of several research papers.WHO has also paid serious attention on mycotoxincontamination in herbal drugs considering it as aglobal problem, however, its incidence is higher intropical countries than the temperate zone; as theharvesting practices, post-harvest storage method,existing high temperature and moisture contentsespecially in monsoon season are conducive forfungal invasion, their proliferation, mycotooxinselaboration and finally in deteriorating the qualityof herbals.

Association of fungi with herbals understorage

A large number of manufacturing units somewith multi crore investments have entered in thearena of drug production of plant origin. The plantmaterials are supplied usually through varioustrade channels which some times create seriousproblems with regard to botanical identity andtherapeutic potentiality, as a result the quality ofthe plant drug in most of the cases is not fullyassured. In addition, the traditional methods ofcollection, storage and marketing coupled withhumid climatic condition make them victim to the

[Vol. 56(1) . 2003]

fungal Infestation. In continuation of earlierinvestigations reported on association of variousfungi with herbal drugs under storage (1987, 88,89, 90, 95), recently 466 samples of 39 plantshaving potential to cure liver, kidney and skindisorders have also been screened for fungalassociation. Altogether 33 fungi including mouldsof different taxonomic groups were isolated. Onthe basis of their occurrence these werecategorized to basic mycoflora- found to presentthrough out the year, periodical mycoflora -occurrence of which is varied with seasons andspecific mycoflora- fungi of selective nature. Thebasic mycoflora included Aspergillus flavus, A.niger, Aochraceus, Chaetomium globossum,Curvularia lunata, Fusarium graminearum, F.moniliforme, F. oxysporum, F .soleni, Penicilliumcitrinum, P viridicatum and Rhizopus stoloniferwhereas the periodical mycoflora were Alternariaaitternata. Aspergillus terrues, A nidulans, A ustus,Botryodiplodia theobromae, Curvularia pallescense,Chaetomium indicum, Fusarium semitectum, F.equiseti, Cladosporium herbarum, C.cladosporoides, Mucor mucedo, Microphominaphaseolina and Paecillomyces sp The highestincidence of these fungi was recorded duringmonsoon seaso' luly-Sept) when the temperatureand relative humidity (RH) ranged between 29-32°C and 79-91 % respectively (Table-1). The variationin the occurrence of fungi as basic mycoflora andperiodical mycoflora showed their appearancethrough out the year due to wide range ofendurance (Temp -5-60°C & RH-60-100%) whichhas also been reported by Armolic et al., (1956),Kennedy (1964), Lutey and Christensen (1963),Mislivec & Tuite (1970), Sinha (1979) and Dutta(1988) Casual occurrence of fungi with respect toparticular season showed their limited tolerancewith temperature and RH. Selective nature offungi has focused the specific niche of theorganism. It is actually a functional relationshipbetween organism and host linked biochemicalfactors under overall control of the environment(Bilgrami, 1987).

Biodeterioration of active ingredients

During storage, the fungal organisms thrive ondrug plants by utilizing various componentsincluding the active ingredients. Biodeterioration inalkaloid, phenol and protein contents of seed, fruit

[Vol. 56(1) . 2003J

Table 1. Periodical per cent incidence of fungi on herbal samples

Indian Phytopathology 3

Fungi Range of July-Sept. Oct-Dec. Jan-March April-JuneTemp/R.H. 29-32 1 79-91 18-28 1 62-70 17-27 1 54-67 29-32 1 58-82

CCI%)

2.5-2.28.4-7.3 3.3-4.76.3-7.08.8-56-- 3.8-5.6 1.5-2.2

3.4-3.87.6-8.3 5.6-5.2 3.1-1.73.6-3.8 2.5-3.54.2-3.74.4-4.6 3.7-3.22.3-2.7 2.1-1.26.3-7.8 7.2-7.0 4.2-4.12.7-2.5 2.2-2.0 1.8-1.72.3-2.0 1.7-1.53.3-3.7 2.5-2.03.4-2.5 1.7-1.5

Alternaria alternataAspergillus nidulansAterreusA ustusBotryodiplodia theobromaeChaetomium indicumCurvularia pallescensCunnighmella elataCladosporium herbarumC. cladosporoides

Fusarium semitectumF. equisetiMacrophomina phaseolinaMucor mucedoPaecillomyces sp.

and root samples of 21 plants by A. f/avus and P.citrinum (in some cases by A. niger and Fusariummoniliforme) was studied at optimum temperature(282:2 DC) and RH (902:6°/t A general trend ofdecrease (10-40%) in the concentration of all theimportant constituents (except phenols) wasrecorded under fungal infestation within 60 days ofstorage.

During storage, changes in seed content areinfluenced both by fungi as well as physical factors(Wallac et et., 1956; Mondal et el., 1981). Thedecrease in protein and phenols (at later phase ofincubation) might be due to their enzymaticdegradation into simpler components which aresubsequently utilized by fungi (Cherry et al., 1975;Mahadevan et al., 1982; Chou bey, 1984).

Nature of seeds

Table 2. Quantitative changes in alkaloid contents (I-Ig/g) of Strychnos seeds under infestation

Incubation days

Strychnine Brucine

start 30 60 90 start 30 60 90S. nux-vomica

870 60

150 135 90 21 36 207294 230 213 51 102 330420 330 84 27 177 162

S. potatorum138 465

15.75 26.25 13.0516.80

102 5.25 238 11.25

ControlInfested withA flavusA nigerP.citrinum

ControlInfested withA flavusA nigerP.citrinum


Degradation of alkaloids: Degradation of alkaloidsin Strychnos potatorum and S. nux-vomica seedswithin 90 days of infestation by A. flavus, A. nigerand P citrinum was studied. Strychnine and brucinewere identified in the seeds as the major alkaloids;their concentration was significantly reduced underinfestation (Table 2). Besides, some new alkaloidswere also detected which possibly be due to theirsynthesis by the invading fungi.

Efficacy of some antidiabetic plant samples: Itis established that fungal association with storedplant samples of medicinal value under storagedeteriorates the active ingredients (Dutta &ROY,1987; Dutta,1988) This naturally raises thequestion regarding the efficacy of samples. Weconducted experiments to determine the efficacyof antidiabetic samples. The extracts of the seedsof Strychnos and Syzygium; bark of Acacia arabica,Ficus glomerata, wood of Pterocarpus marsupiumand tuber of Cyperus rotundus were administeredto albino rats (150-200g of body wt) for glucosetolerance test. The seeds of Syzygium caused

[Vol. 56(1) • 2003]

maximum reduction (31%) in blood glucose levelafter 1 h of glucose load compared to control. Thiswas followed by P marsupium (26%,) S. potatorum(21%), F. glomerata (19%) and C. rotundus (15%).The extracts of infested samples also reduced theblood glucose level but to a limited extent (Table3). This presumably might be due to thedegradation of antidiabetic active principles by thefungi.

Mycotoxins producing fungi and theirtoxigenicity

The potential of fungal isolates to producemycotoxms also vary widely with the variation ofsubstrates as well as with the changes of strains.Initial reports from India shows that less than 10%isolates of Aspergillus f/avus are toxigenic(Shrinivasamurthy et el., 1965 and Rao et al.,1965). However, Maggon et et. (1969) havereported toxigenicity of all seven isolates of A.flavus obtained from soil of Delhi. The toxinproduction ability of fungi also varies with the

Table 3. Effect of plant extracts (healthy and infested with A. f/avus) on hyperglycaemia induced by glucose loadon albino rats

Fasting blood Post feE 'Glucose + plant Extracts)glucose blood glucose level

Herbal Extract Dose (mg/100mg) (mg/100mg)

o h % reduction 1 h % reduction2 hJeersanidhi MemorialAward Lecture % reduction

over control over control over control

H H H

Control 2.50ml/100g 84.94 120.21 85.06(Distilled water)Sttychnos 5 mg/100g 78.93 707 6.66 95.26 20.75 17.17 80AO 5A7 6.14po/atorum (seed)Sirvchnos nux- 5 mg/100g 84.72 0.25 0.36 105.54 12.20 11.06 84.96 0.11 0.23vomica (seed)Syzygium 10 mg/100g 78.06 8.09 4.72 82.79 31.12 25.85 80.31 5.58 2.37cumini (Seed)Pterocarpus 10 mg/100g 76.12 10.38 10.07 88.38 26A7 26A3 79.01 7.11 6.53marsupium (wood)Acacia arabica 10 mg/100g 82.15 3.28 3.16 104.29 13.24 13.19 84.63 0.50 0.63(Bark)Ficus glomerata 10 mg/100g 80.64 5.08 2.61 97.12 19.20 18.60 84.00 1.24 1.83(Bark)Cyperus rotendus 10 mg/100g 81.50 404 1.62 102.58 14.66 1201 83.31 1.76 1.82(Rhizome)

H - Healthy; I - Infested with A. f/avus.

[Vol. 56(1) • 2003]

variation of strains. Most of the A flavus strainsare able to produce only AflB1 and B2 Similarlyproduction of Cit. by Penicillium citrinum, Ochr.Aby Aspergillus octireceus and Zear. by Fusariummonilliforme and F. graminiarum are common thanother derivatives. Reports on production ofmycotoxins by toxigenic fungi in plant drugs arequite poor. Only scanty work have been carriedout in this direction. Earlier, Roy et al. (1988), Royand Chourasia (1990) and Roy and Kumari (1992)have reported the potentials of fungi for mycotoxinproduction in plant drugs. They have isolatedseveral strains of A flavus, A .ochreceus, P.citrinum, P viridicatum, F monitiforme and F.graminearum from drug samples and werescreened for mycotoxins production potentials.Substantially a high number of isolates were foundtoxigenic. 354% isolates of Aflavus, 28.35% of Aoctireceus, 28.1 % of P. citrinum, 38% of F.graminiarum and 35.13% of F monitiforme werefound toxigenic The production level of aflatoxinB1 ranged between 0.32-3.37 ug/ml, B2--007-2.16ug/ml, G1 - 0.05-1.86 mg/ml and G2 - 0.04-0.13 mg/ml Other mycotoxins viz. ochr.A , cit. andzear were also found to synthesize by therespective fungi the range of which varied between0.09-244mg/ml, 0.05-2.31 mg/ml and 0.04-4.87mg/ml respectively (Table 4). It is well established factthat toxin producing potentials of fungi varied withstrains due to variation in their genetic make up(Bilgrami and Sinha,1992) Nishimura and Kohmoto(1983) and Lillehoj (1980) have also recordedsimilar results on the basis of which they suggestedthat aflatoxins may also act as chemical signalsbetween species in an ecological niche.

Table 4. Fungal isolates and their toxigenecity


Physical factors and mycotoxins

The encounter of deleterious moulds to plantdrugs especially during the post-harvest operationand mycotoxin contamination depends on numberof factors which may be categorized as physicalfactors - temperature, humidity, rain fall, moisturelevel of the substrate and type and duration ofstorage systems used; chemical factors- level ofCO2, oxygen, chemical nature of substrate andnutrient; biological factors- spore load, strainpotentiality and microbial interaction. All the abovestated factors are important, however, oxygenlevel, temp., moisture content of substrate andstorage periods are determining factors for themould growth as well as mycotoxin elaboration bythem. The moisture level above 13% and relativehumidity (RH) above 75% existing in storagestructures are found conducive to A Flavus growthand toxin production Our investigation also revealedthat despite physical factors, the storage periodshave also a marked impact on growth of A Flavusand aflatoxin level Of various temperature (10-40°C), RH (33-96%) and light phases (Total dark,diffuse light, electric light and sunlight) maximumlevel of afla B1 production was recorded at 25-30°C temp, 96% RH and total darkness within amonth of storage period (Roy and Kumari, 1994).Plant materials dried such as that moisture levelsremain below to the limits might salvage frommould infestation (Bewly and Blank,1985).Fluctuation in temperature also plays importantrole in this system. Plant materials being rich incarbohydrates and getting less attention for properstorage, remain in direct access of environmentalconditions leading to attack of vulnerable moulds.

Fungi Mycotoxins Range ofmycotoxins

produced (!-Ig/ml)

Aspergillus flavus

No. of Toxigenicisolates (%)

387 137 (35.4)

67 19 (28.3)167 47 (28.1)63 24 (38)148 52 (35.1)

A. ochraceusPenicillium citrinumFusarium graminearumF.moni/iforme

AfI.B1. AflB2Afl.G

1Afl.G2

Ochr.ACitrinin

ZearalenoneZearalenone

0.32-3.37, 0.07-2.160.05-1.86, 0.04-0.13

0.09-2.44

0.05-2.310.04-4.870.09-3.57


Mycotoxigenic moulds are usually found to growbetween 15-30% moisture content and between25-30DC temperatures.

Natural contamination

Natural contamination of mycotoxins instanding crops and stored food materials havebeen described from different parts of India andworld too. The list includes cereals (Hesseltine etet.. 1975; Scott, 1978; Bilgrami, 1984: Bilgrami

[Vol. 56(1) 2003J

and Sinha, 1984; Lea et a/., 1986; Wicklow et a/.,1987), pulses and oil seeds (Sinha, 1980; Mehanand Mc Donald, 1983; Sahay, 1988; Bilgrami eta/., 1989), vegetables, dry fruits and spices(Bilgrami, 1985), milk and milk-products (Yadgiriand Tulpule, 1974; Brown, 1982). However,literature related to mycotoxin contamination incrude herbals or plant drugs is still fragmentary.

Several samples of crude herbals collectedfrom different regions are found contaminated with

Table 5. Level of mycotoxin in skin curative crude herbal drugs (jJg/g)

Plants ( Parts used) A B C 0 EAfl.Bl Afl. B2 Afl. Gl Afl. G

2 Cit Ochr.A Zear.

Cassia tora 7/4 0.37-1.23 0.33-0.76 NO NO 0.47-0.51 NO 0.07-0.34(rhizome)

Cetrathesum 18/9 0167-1.47 0.88-0.31 0.08 NO 0.08-0.13 NO 0.13anthelminticum (seed)

Mesua terree 13/4 0.71-0.14 NO NO NO 0.04 NO NO(Flower)

Plumbago zeylanica 1719 0.71-1.87 0.65-1.23 0.96-0.74 0.03-0.15 0.08-0.95 0.07 0.11-0.73(stem)

Psoralia corylifolia 1718 1.77-2.14 0.34-1.04 0.14-0.39 0.05-0.24 0.08-0.17 0.07-1.1 0.43-0.56(seed)

Rauwolfia 12/7 0.42-1.23 0.08-0.37 NO NO 0.05-0.33 NO 0.38-0.77serpentina (root)

Strychnos 10/5 0.96-1.17 0.04-0.5 NO NO 0.35 NO 0.14nuxvomica (seed)

Vetivena 7/4 0.08-0.33 0.05-0.12 NO NO 0.07 0.13 0 r08-014zizanioides (root)

A - indicate No. of samples screened I No. of samples contaminated, B- level of different afls., C- level ofcitrinin, 0- level of ochr. A. E level of zear. and NO- not detected

Table 6. Level of mycotoxin in kideny curative crude herbal drugs (jJg/g)

Plants ( Parts used) A B C 0 EAfl.Gl Afl.B2 AfI.Gl Afl.G2 Cit Ochr.A Zear.

Argyreia speciosa 23/8 0.76-1.43 0.17-0.93 0.06-0.33 NO 0.46-0.84 NO 0.33-0.47(Seed)Emblica officinalis 27/11 1.02-1.87 0.32-0.91 NO NO 0.13-0.74 0.93-1.74 0.07-0.43

(Fruit)Mynstica fragrance 7/3 0.31-0.53 NO NO NO NO NO NO(fruu)

Mucuna prurienss (seed) 14/8 1.38-2.29 0.87-1.56 0.37-1.11 NO 0.43-0.78 NO 0.33-0.47

Tribulus terrestris (seed) 23/9 1.14-2.32 0.96-1.18 0.32-1.75 NO 0.78-1.13 0.14-1.3 0.93-1.37

A - indicate No. of samples screened / No. of samples contaminated, B -level of different afls., C- level of citrinin, 0 level ofochr. A E level of zear. and NO- not detected

[Vol. 56(1) • 2003] Indian Phytopathology 7

Table 7. Level of mycotoxins in liver curative crude herbal drugs (I-Ig/g)

Plants (parts used) A B C D E

Atl.B, Afl.B2 Afl.G, Cil. Ochr. A Zear

Andrographis peniculata (Leaf and Stem) 13/7 ND ND ND 0.63-0.87 0.74-1.39 0.30-0A3Asparagus racemosus (Roots) 17/10 1.57 -2A3 0.24-0.77 ND 038-104 ND 0.17-0.38

Butea monosperma (Seed) 7/5 0.67-1A7 ND ND ND ND ND

Coccinium fenestratum (Stem) 11/9 0.78-1.37 0.17-0.38 ND 0.14-0.37 ND 0.09-0.18

Ceasalpmna dyginne (Fruit) 5/3 0.16-0A3 ND ND 0.23-0A3 ND ND

Cymbopogon naradus (Fruit) 16/9 0.63-1.17 0.04-0.83 ND 0 ..07-0A4 ND O. 37

Curculigo orchioides (Stem) 8/4 078-2.14 0.08 ND 0.33 ND ND

Oiospyros embryopteris (Calyx) 6/3 ND ND ND ND 0.09-0.89 ND

Holarrhena anlidysenterica (Bark) 13/7 0.34-1A6 0.1-0.76 ND ND 1.14-2.34 ND

Mangifera mdica (Seed) 15/10- OA-0.88 0.07-0.27 ND OA2-0A8 ND 0.38

Piper longum (Fruit) 13/5 OA1-1 12 0.33-0.84 0.04-0A4 1.04-1.37 ND 1.11-1.24

Rhamnus wightii (Bark) 8/5 0.87-1.83 0.34 ND 0.52 ND ND

Tacca aspera (Rhizome) 9/6 0.37-0.76 0.05-0.12 ND OA3-0.76 0.3-0.74 0.33-0.57

Terminalia belarica (Fruit) 19/7 1.37-2.34 0.56-1A4 0.23-0A4 0.17-0.62 ND 0.31-0A2T chebule (Fruit) 20/10 0.64-1A6 0.16-1A6 ND 0.06-0.14 0.09-1.23 0.11-0.31

T .catappa (Fruit) 5/3 0.18-1.14 ND ND ND ND OA2

A - indicate No. of samples screened I No. of samples contaminated, B- level of different afls., C-Ievel of citrinin, D-level of ochr. A E-Ievel of zear. and ND- not detected.

substantial level of mycotoxin. 466 samples of 39plants having potentials to cure skin (Table 5),kidney (Table 6) and Liver (Table 7) disorder wereput under examination. Result elucidated that agood number of crude herbal drugs were foundcontaminated with mycooxins, though theconcentration varied with the samples andassociated mycotoxins producing moulds. More orless similar results regarding the incidence ofmycotoxins were also recorded in case of drugsamples used to cure kidney and skin disorders(Horry et et., 1979; Roy et a/. , 1990; Kumar andRoy, 1994). Mycotoxinspresent in crude sampleswould easily be transmitted to powdered / finishedherbal drugs. 55 samples of 11 drugs manufacturedby different registered Pharmaceutical Industrieswere found contaminated with either one mycotoxinor more than one (Table 8). The level ofcontamination was significantly higher than thehuman tolerance level. The corresponding crudeherbal samples were also found contaminated,which might be the possible reason for mycotoxinstransmission in finished products. Similar resultshave also been recorded by Horrey, 1982;Yamazaki et el., 1988; Nerrita et a/,1988;Tanaka.1988 and Roy and Chourasia,1989)

Biological assay

There are several earlier reports publishedtime to time from the different parts of the worldregarding the health risks due to consumption ofaflatoxins contaminated foods. The presence ofhigh concentration of mycotoxins both in crudesamples and finished products have drew theattention of scientists working in the field ofbiomedical research. Our investigation related toeffect of contaminated herbal medicines tolaboratory rabbits noticed remarkable changes ingeneral behaviour of animals. Drowsiness, frequenttearing of eyes, loss of hairs and reduction in bodyweight were the common symptoms appeared inthe animals exposed to afI.B,. Panda et a/. (1975)have observed the effect of afl.B, on blood featurealbino rats and guinea pigs and recorded reductionin total count of RBC, WBC and HG content of theblood. Our hematological studies also revealedthat RBC and WBC count got down considerably.HG content was decreased up to 12% in maletraits and 11.5% in female due to afl.B, exposure.

Aflatoxins exposure also induced a variety ofchanges in the tissues profiles of liver, kidney,skin, intestine and genital organelles. Presence of


Table 8. Mycotoxin incidence in pharmaceutical products

[Vol. 56(1) • 2003]

Medicines

Zear.Mycotoxin (J.Jg/g) mean Conc.Cit. Ochr.A

Talisadi churnaAvipattikar churnaAshwagandhadi ChurnaAjmodadi churnaSitopaladi ChurnaAbhayasanLivotritChausasthi pippalShivksarEladi churnaPippalyadi churna

0.080.070.0250.400.45

0.06NONO0.070.07NO

NONONONONONO0.07NONONONO

0.028NONONONO0.230.47

NO0.76

NONO

NONONONONONONONONONO0.87

NO -not detected

papilloma, crystallization of skin cells, developmentof necrosis and non- differentiated hyperchromaticnuclei were noticed in the tissues of skin, liver andkidney in animals treated with afls. contaminatedmedicines. Besides, these impaired function ofglomerules, crystallization in Bowman's capsuleand necrotic patches in intestine mucosa wereseen. Ovary and testis exhibited loss of germepithelial layer as well as degeneration of folliclesand spermatogonial cells. Presence of fibrosis inseminiferous tubules and the bile duct proliferationwere noticed in aflB1 fed rabbits. Earlier Allcrotand Lewis (1963) have also recorded similarchanges in the histology of liver of cattle due toaflatoxicoses. Bullaer et al. (1969), Newborne andButler (1969) and Newborne and Rogers (1973)have noticed abnormal activities of cells of thevital organs in laboratory animals due to ingestionof mycotoxin contaminated food. Bilgrami andSinha (1988) have observed chromosomalabnormalities like clumping fragmentation, meioticpolyploidy and euploidy in mice fed with afls.contaminated food, which may also be linked withthe genetic disorder in reproductive units.

Prevention and detoxification

Segregation and Decontamination: Medicinalplant samples may contain mycotoxins beyond thetolerance level (30ppb) fixed by WHO, but in a lot,not all are contaminated and in some cases toxicityis in a very small quantity. Sorting of crude samples

by visual examination is not very effective becausehealthy appearing samples may have concealeddamage i.e. propagules inside substrate. The mosteffective way to remove off colour suspect samplesparticularly seeds by means of electronic coloursorting. Blanching followed by means of electroniccolour sorting and hand-picking are found effectivein detecting aflatoxin contaminated samples(Tiemestra,1977).

Chemical segregation: Where removal andsegregation of toxic samples are not effective orpartially successful, there still remains the possibilityof destroying or inactivating the aflatoxins in crudesample (seeds) by chemical treatment. It istechnically referred to as detoxification. It is alsoeconomically viable and meet the criteria listed bythe FADIWHO/UNEP Conference on Mycotoxinsheld in Nairobi, Kenya (1977).

The polar solvents such as methanol or ethanolused in the extraction methods for processingmedicinal samples remove most aflatoxins fromthe extracted finished product. However, onenegative feature in these procedure is the removalof desired portions of the samples which in turnlowered net value of the final drugs (Detroy et aI.,1971). Rayner et al. (1977) reported that 80%aqueous isopropanol completely removed aflatoxinin cotton seed and pea nut meal. Cotton seedextracted with aqueous acetone (25-30%) hasbeen reported to remove most aflatoxins with onlynegligible removal of natural oils and proteins(Goldblatt, 1971).

[Vol. 56(1) : 2003]

Various chemicals viz. Methoxymethane(Aibara and Yano, 1977), formaldehyde (Codiferet al.,1976), calcium hydroxide (Giddey et aI.,1977), ethylene oxide (Deqesch, 1978), ~odiumhydroxide (Mann et.a/., 1969), sodium hypochlorite(Natrajan et aI.,1975), hydrogen peroxide (SrinivasaMurthy et aI., 1967) and ammonia (Coker, 1989)have been tested for destruction of aflatoxin incontaminated samples.

Physical detoxification: Aflatoxins are stable upto their melting point of around 250°C (Fenell,1966). A number of heat treatment have beentested in aflatoxin detoxification studies (Rehanaet al.,1979; Seenapa and Nyaquahunqu, 1982).Steaming under pressure and other cookingtechnique often reduce aflatoxin levels but do noteliminate the toxin completely (Schreder et aI.,1985) Under appropriate conditions aflatoxin isdegraded by light, particularly ultraviolet radiation.Sunlight is utilized to reduce aflatoxin levels incontaminated peanut cake and vegetable oils(Samarajeeva et et., 1977; Shantha and SreenivasaMurthy, 1981). Ultraviolet Irradiation of oil seedmeals to destroy aflatoxin has been patented(Arthur and Robertson, 1970). Aflatoxin is alsodegraded by gamma radiation (Van Dyck et et.,1982).

Thus, several methods of detoxification in theliterature, however, none of these is commerciallyviable and economically acceptable, particularly incase of herbal drugs. An attempt has been madein this laboratory earlier to find out a simplemethod to detoxify the aflatoxin contaminated crudeherbals (Table 9). The % detoxification varied


from 57-78%, 21-40 and 81-90 % when thecontaminated samples were exposed under directsunlight 8 hrs, oven heat (60°C), UV light(8hrs.daily) and UV light (12hrs.daily) respectivelywithin 30 days of incubation period. Detoxificationof aflatoxin by radiation has also been reported byBilgrami et et. (1984).

Microbial detoxification: Many microorganismsincluding fungi, actinomycetes, bacteria and algae,have been tested for their ability to destroy ortransform aflatoxin (Ciegler et el., 1966; Roy andChaurasia, 1990; Chourasia and Roy, 1993;Chou rasia, 1995).During the course of severalinvestigations it was noticed that A. niger is astrong antagonist of A. tlevus (Table 10) and couldbe expected to restrict the synthesis of aflatoxins.Earlier Wicklow et al. (1979) have also recordedthe similar results while working with interferencecompetition and aflatoxins level in corn. They havealso suggested that biosynthesis of kojic acid byA. flavus under competitive environment may notnecessarily be favouring afls. production. Abacterium Flavobacterium aurantiacum has beenfound to remove aflatoxin from solutions andpreparations to pea nut milk (Ciegler et al.,1966)

Control in storageltransit

Storage of crude herbal seed samples underhygienic conditions with low moisture content (about8%) and low temperature protect them from mouldInsect infestation should avoid moulding of thesesamples and consequently reduce the risk ofaflatoxin contamination (Roy and Kumari, 1994).

Table 9. Percent detoxification of afl B, in some medicinal seeds

Treatments Incubation % of detoxificationperiods(days) A. speciosa E. ribes N. nucifera

Sunlight 15 30.26 30.33 26.28(8 h daily) 30 70.77 62.54 57.00

Oven heat 15 17.17 14.46 11.59(at 60°C, 8 h daily) 30 39.90 28.88 21.55

UV - light 15 39.09 36.59 30.00(8 h daily) 30 62.11 56.00 49.88

UV - light 15 60.55 53.06 49.99(12 h daily) 30 90.00 86.07 80.84

10 Indian Phytopathology [Vol 56(1) 2003]

Table 10. Evaluation of interaction between Aspergillus flavus and other fungal colonies associated with crudesamples (piper fruits) for antagonism and aflatoxin production

AntagonistsType of

interactionAflatoxin production"

(mg/ml)Percent inhibition of

growth of A. flavus topotential antagonist

B, G,

Aspergillus niger van Tiegh.A. nidulens (Eidam) WinterA. ochraceus WilhelmA. sydowii (Fres.) Thom & ChurchAlternaria alternata (Fr.) KeisslerChaetomium globosum Kunzeex Fr.Curvularia lunata (Wakker ) BoedijinFusarium oxysporum Schlecht.Penicillium citrinum ThomTrichoderma viride Pers. ex Fr.Control (Aspergillus flavus (Link.) Fr.)

oBABACAABE

97.5045.2050.0035.2045.2540.2550.6553.1547.1060AO

0.09OA70.50OA50.600.590.650.15OA40.391.00

0.100.520.500.270.20OA70.300.220.120.150.75

"Value represent the mean aflatoxin levels of three replicates. (Roy and Chourasia,1990)A- Mutual intermingling of two organisms; B- Mutual inhibition on contact; C- Mutual Inhibition on distance;0- Inhibition on contact the antagonists continues too grow unchanged and reduced rate; E- Inhibition at adistance, the antagonists continues to grow resulting clear zone at unchanged or reduced rate (Johnson &Curl, 1972)

Various ways In which stored crude samples absorbmoisture have been highlighted (Chourasia, 1995).These include direct wetting by rainfall and byleakage through covers and through seepage ofground water and by direct absorption of moisturefrom humid atmosphere. Moisture may also begenerated in stored samples / seeds fromrespiration of insects and rodents. Modification ofstorage environment at high scientific level andtransportation under safe condition will be effectivein reducing risk of mycotoxin contamination incrude herbal seeds.

Epilogue

It is documented elsewhere that 80% of worldpopulation have faith on traditional medicines,particularly in plant drugs for their primary healthcare. Therefore, popularity of herbal drugs isgradually gaining impetus in both developing anddeveloped countries due to their availability atlower price and having least side effects. However,the most derogatory part of such medicines is thelack of proper attention on their quality control.There is unhindered possibilities of biodeterioration

and mycotoxin contamination during collection,storage and processing. It is well established thatmycotoxin contaminated herbal drugs may causehealth complications instead of curing diseases.Thus, it is worthwhile to undertake an intensivesurveillance upon this problem. The naturalvegetation of our country is considered as richdepository of a large number of medicinal plantsand therefore, future of herbal drugs holds greatpromise in India. Moreover, sincere efforts are tobe required for their conservation, cultivation,propagation and quality control of finished products.It is expected in the coming years that variousmethodologies would be adopted to bringrevolutionary progress, which include geneticengineering, better field and storage managementand processing technologies, so that India couldcompete in the International market in a big wayThe credibility of this traditional system which istime tasted would certainly go up and enhancetheir efficacy, reputation as well as acceptability ifquality control parameters are developed andfollowed strictly in comparison to the syntheticmedicines.

[Vol. 56(1) : 2003]

REFERENCESAlles, G.A. (1952). A Comparative study of the

Pharmacology of certain Crytopine alkalines. J.Pharmacology 104: 1253-1263.

Ahmad, S.K. and Roy, A.K. (1995) Microbial interactionand mycotoxin production. Recent Advances inPhytopathological Researches. pp.171-177. M.D.Publ. New Delhi.

Armolick, N., Dickson, J.G. and Dickson, A.D. (1956).Minimum humidity requirement for germination ofconidia of fungi associated with the storage ofgrains. Phytopath. 46: 451-461.

Aswal, B.S., Bhakuni, D.S., Goel, A.K., Mehrotra,B.N. and Mukherjee, K.C. (1984). Screening ofIndian plants for biological activities. Indian J. Exp.BioI. 22: 312-332.

Atal C.K. and Sethi, P.O. (1962). Proteolytic activity ofsome Indian plant II. Isolation, properties and kineticstudies of Calotropain. Planta Medica 10: 77-90.

Barnes C.S., Price, J.C. and Huges, R.L. (1975). Anexamination of some reputed antifertility plantsLyoydia 38: 135-140

Bewly, J.D. and Black, M. (1985) Seed: Physiology ofDevelopment and Germination. Plenum Press, NewYork and London, pp.349.

Bilgrami, K.S. (1984). Mycotoxin in food J. Indian Bot.Soc. 63: 109-120.

Bilgrami K.S. and Sinha, K.K. (1984). Mycotoxincontamination in food and its control. Indian Rev.Life Sci. 4: 19-36.

Bilgrami K.S. (1985). Mycotoxin in dry fruits and spicesFTR, UGC. Project Bhagalpur Univ. Bhagalpur.

Bilgrami, K.S. and Sinha, S.P. (1988). Mutageniceffects of mycotoxins. FTR, UGC. Project BhagalpurUniv. Bhagalpur.

Bilgrami, K.S. and Sinha, K.K. (1992). Incidence ofmycotoxin in maize and its impact on physiologicalprocesses of seed. In Mycotoxin incidence andeffects. (Eds. K.S. Bilgrami and K.K. Sinha). CBSPub. New Delhi, pp.73-82.

Brown C.A. (1982). Aflatoxin M in Milk. Food Technol.Australia, 34:228-231.

Chourasia H.K. (1995) Mycobiota and mycotoxins inherbal drugs of Indian Pharmaceautical Industries.Mycol. Res. 99: 697-703.


Chourasia H.K. and Roy, A.K. (1993) Growth andaflatoxin production by Aspergilljus parasiticus withco-occurring fungi and bacteria J. Ind. Bot. Soc.72: 131-134.

Ciegler, A.R., Peterson, E., Laqoda, A.A. and H.H.Hall (1966) Aflatoxin production and degradationby Aspergillus flavus in 20 litre fermentors. ApplMicrobiol. 14: 826-833.

Ciegler, A., Liliehoj, E.B., Peterson, R.E. and Hall,H.H. (1966). Microbial detoxification of aflatoxinproduction of fungi. J. Sci. Food Agric. 27: 324-230.

Codifer L.P., Mann, G.E. and Doliear, F.G. (1976)Aflatoxin Inactivation: Treatment of Peanut mealwith formaldehyde and calcium hydroxide. J. Assoc.of Anal. Chem. 53: 204-206.

Coker R.D., Jones, B.D. and Nagler, M.J. (1989).Mycotoxin Training Manual, Tropical Developmentand Research Institute, London.

Dhar M.L., Dhar, M.M., Bhawan, B.N., Malhotra, B.N.and Ray, C. (1968). Screening of Indian plants forbiological activity. Part I. Indian J. Exp. BioI. 6:232.

Dutta G.R. (1988). Pathological and Pharmacologicalstudies of some plants having hypoglycemicproperties. Ph.D. Thesis. Bhagalpur Univ.Bhagalpur.

Dutta, G.R. and Roy, A.K. (1987). Mycoflora associatedwith Strychnos seeds and deterioration of theiractive principles under storage. Indian Phytopath.40: 520-524.

Fenell, A.J. (1966). Aflatoxin in ground nuts IX Problemof detoxification. Tropical Science 8: 61-70.

Giddey C., Brandt, J. and Bunter, G. (1977) Thedetoxification of oil seed cakes polluted byaflatoxins: Research and development of anindustrial process. Agricultural Technology Age27: 331-338.

Goldblatt, L.A. (1971). Control and removal of aflatoxin.J American Oil Chem Soc 48: 605-610.

Heselltine, C.W., Bothast, R.J. and Shotwell, D.L.(1975). Aflatoxin occurrence in some white cornunder loan, 1971: IV Mold Flora. Mycologia 67:392-408.

Horie, Y., Yamazaki, M., Itokawa, H. and Kinoshita,H. (1979). On the toxigenic fungi contaminatingherbal drugs as raw materials in pharmaceauticalindustries. Trans. Mycol. Soc. Japan, 23: 435-447.


Kennedy, B.W. (1964). Moisture content, mould invasionand seed viability of stored soybeans. Phytopath.54: 771-774.

Kumar, S. and Roy, A.K. (1994) Mycotoxincontamination of some kidney curative herbalmedicines. J. Indian. Bot. Soc. 73: 341-342.

Kumar S. and Roy, AK. (1995) Incidence of mycotoxinsin traditional herbal medicines from India. Proc.Nat. Acad. Sci., India 65(B)1: 67-71.

Lea, U.S., Jang, H.S. Tanaka, T., Toyasaki, N.,Sugiura, N.J. Oh, Choand, C.M. and Ueno, Y.(1986). Mycological Survey of Korean cereals andproduction of mycotoxins by Fusarium isolates.Appl. Environ. Microbiol. 52: 1258-1260.

Liliehoj, E.B. (1980). Proceedings, Sixth Inter Fed,Symp., London, Advances in Biotechnology (Des.Muo-young, M.C. Vezina and K. Singh). PergamonPrers, Elmsford, N.Y. p. 397.

Lutey R.W. and Christensen, C.M. (1963). Influenceof moisture contents , temperature and length ofstorage upon survival of fungi in barley kernels.Phytopath. 53: 713-717.

Maggon K.K., Vishwanathan, L.,Venkitasubramanian, T.A and Mukherji, K.G.(1969). Aflatoxin production by some Indian strainsos Aspergillus flavus. J. Gen. Microbial. 59: 119-125.

Mislivec, P.O. and Tuite, J. (1970). Mycologia 42: 75-88.

Mondal G.C., Nandi, D. and Nandi, B. (1981). Studieson deterioration of some oil seeds in storage I:variation in seed moisture infection andgerminability. Mycologia 83: 157-166.

Narita, N., Suzuki, M., Udagawa, S., Sekita, S.,harada, M., Aoki, N., Tanaka, T., Hasegawa, A,Yamamoto, S., Toyazaki, N. and Matsuda, Y.(1980). Af!atoxin potential of Aspergillus flavusisolates from Indonesian herbal drugs. Proc. Jpn.Assoc. Mycotoxin Col. 27: 21-26.

Natarajan K.R., Rhee, K.C., Cater, C.M. and Mattil,K.F. (1975). J. American Oil Chem. Soc. 52: 160-163.

Newborne, P.M. and Butler, W.H. (1969). CancerRes. 29: 236.

Newborne, D.M. and Rogers, A.E. (1973). J. Nat!.Cancer Inst. 50: 439.

Nishimura, S. and Kohmoto, K. (1983). Toxins andPlant pathogenesis (Eds., Daly, J. M. and Deverall,B.J.), Academic Press, N.Y.

[Vol. 56(1) : 2003]

Panda, P.C., Murthy, AS. Sreenivasa Murthy, V. andMulti, I.A.S. (1975). Indian J. Exp. Bioi. 13: 569.

Rao, K.S., Madhawan, T.V. and Tulpule, P.G. (1965).Incidence of toxigenic strains of Aspergillus flavusaffecting ground nut crop in certain coastal districtsof India. Indian J. Med. Res. 53: 1169-1202.

Roy, AK. and Chaurasia, H.K. (1989). Aflatoxinproblem in some medicinal plants under storage.Int. Jour. Crude Drug Res. 27: 156-160.

Roy, AK. and Chaurasia, H.K. (1990). Inhibition ofaflatoxin production by microbial interaction. J.Gen. Appl. Microbiol. 36: 59-62.

Roy, AK. and Chaurasia, H.K. (1990). Effect of relativehumidity on fungal assiciation and aflatoxinproduction in Piper longum fruits. Ind. Bot. Reptr.8: 138-140.

Roy, AK. and Chaurasia, H.K. (1990). Mycotoxinincidence in root drugs. Int. J. Crude Drug. Res.28: 157-160.

Roy, AK. and Chaurasia, H.K. (1990). Aflatoxinproduction on Piper longum fruits under differenttemperatures. Int. J. Crude Drug. Res. 28: 233-235.

Roy, AK. and Vineeta Kumari (1992). Effect of lighton fungal population and aflatoxin B, contaminationEmblica ribes seed. J. Ind. Bot. Soc. 72: 243-245.

Roy, AK. and Vineeta Kumari (1994). Aflatoxin problemin medicinal seeds under storage, In Vistas inSeed Biology, (Eds. T. Singh and P.C. Trivedi),PP.381-393, Printwell, Jaipur, India.

Roy, AK. and S. Kumar (1995). Traditional herbalmedicines, moulds and mycotoxins- A Review.Recent Advances in Phytopathological Researches.pp.25-37. M.D. Pubs., New Delhi.)

Roy, AK. and Kumar, S. (1997). Microbes for theprevention of aflatoxin contamination in herbalmedicines. Proc. Nat. Symp. on Plant Diversification& Human Welfare, pp. M.D. Pub. New Delhi.

Seenapa, M. and Nyaqahanqu, I.K. (1982). Retentionof aflatoxin in uqali and bread made fromcontaminated flour. J. Fd. Sci. Technol. 19: 64-65.

Sreenivasa Murthy, Parpia, V., Srikantia S.H.AB.and Shankar Murthy, A (1967). Detoxification ofaflatoxin in peanut meal by Hydrogen peroxide. J.Assoc. Off. Anal. Chem. 50: 350-354.

[Vol. 56(1) : 2003]

Shantha, T. and Sreenivasa Murthy, V. (1981). Use ofsun-light to partially detoxify groundnut (peanut)cake flour and casein contaminated with aflatoxinB1. J. Assoc. Off. Anal. Chem. 64: 291-293

Tiemestra, P.J. (1977). In. Mycotoxin In Human AndAnimal Healths (Eds Rodricks J.Y., C. W. Heseltineand MA Mehlman) Pathotok Park Forest SouthIllinois USA pp 121-137.

Rayner, E.T., Koltun, S.P. and Dollear, F.G. (1977).Solvent extraction of aflatoxins from contaminatedagricultural products. J. American Oil Chem. Soc.52: 160-163.

Rehana, F.S., Basappa, C. and Murthy, V.S. (1979).Destruction of aflatoxin in rice by different cookingmethods. J. Fd. Sci. Technol 16: 1196-1202.

Roy, A.K., Prasad, M.M., Kumari, N. and Chaurasia,H.K. (1988). Studies on association of mycoflorawith some drug plants and aflatoxin producingpotentiality of A. flavus Ind. Phytopath. 41: 261-262.

Wallac H.A.H., Sinha, R.N. and Mills J.J. (1976).Fungi associated with small wheat bulks duringprolonged storage in Manitoba, Can.J. Bot. 54:1332-1343.

Sahay, S.S. (1988). Occurrence of Aflatoxin in mustardseeds, cakes and oils. J. Indian Bot. Soc. 67: 188-190.


Sinha, M.K. (1979). Pathological and Physiologicalstudies of seed borne fungi of some pulses ofBihar. Ph.D. Thesis, Bhagalpur Univ., Bhagalpur.

Sinha, K.K. (1980). Survey and study of aflatoxinproducing isolates of A. flavus associated withmaize grains In storage and standing Crops. Ph.D. Thesis, Bhagalpur Univ. Bhagapur.

Tanaka, T., Hsegawa, A., Yamamoto,S., Aoki, N.,Toyazaki, N., Matsuda, Y., Udagawa, 5., Sekita,5., Narita, N., Suzuki, M. and Harada, M. (1988).Natural occurrence of aflatoxins in traditional herbaldrugs from Indonesia. Proc. Jpn. Assoc.Mycotoxicol. 28: 33-35.

Van-Dyck P.J., Tobback, P., Feys, M. and Van deVoorde, H. (1982) Sensitivity of aflatoxin B1 toionizing radiation. Appl. Environ Microbiol. 43: 1317-1319.

Wicklow, D.T., Horn, B.W. and Shotwell, O.L. (1987).Aflatoxin formation in pre harvest maize earscoinoculated with Aspegillus flavus and A. niger.Mycologia 79: 679-682.

Yadqiri B. and Tulpule, P.G. (1974). Aflatoxin inBuffaow milk. Indian J. Dairy Sci. 27: 293-297.

Yamazaki, M., Horie, Y. and Itokawa, H. (1980). Onthe toxigenic fungi contaminating crude drugs. I.Mycotoxin of commercially available crude drugsand productivity of mycotoxins in some species.Yakugaku Zasshi 100: 61-68.

mycological problems of crude herbal drugs - overview and

Documents