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Journal of Stored Products Research 43 (2007) 323–329

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Aflatoxin contamination of pods of Indian Cassia sennaL. (Caesalpinaceae) before harvest, during drying and in storage:

Reasons and possible methods of reduction

Philip Muller, Thies Basedow�,1

Experimental Station, Institute of Phytopathology and Applied Zoology, Justus-Liebig-University, Alter Steinbacher Weg 44, D-35394 Giessen, Germany

Accepted 31 August 2006

Abstract

The presence of aflatoxins in senna plants was studied in two different areas of India using an HPLC method. Only pods of Cassia

senna angustifolia contained aflatoxins: leaves and flowers were free. Damage by insect larvae (Ephestia elutella) led to aflatoxin

formation by fungi in the pods. Fruits damaged by other factors can also contain aflatoxins. The occurrence of aflatoxins in senna pods

in South India proved to be unevenly distributed. Before harvest, 55% of samples contained less than 2 mg/kg, and 25% more than 10mg/kg, with a maximum of 255 mg/kg. Controlled sun drying of pods allowed the aflatoxin content to double, while drying in the shade was

followed by a four-fold increase. Only a very small fraction of dried senna pods carried the maximum load of aflatoxins. During storage

of Indian senna pods, the aflatoxin content usually fell.

The smallest increase of aflatoxins, but still giving rise to unacceptably high levels, was achieved using a solar dryer. The formation of

aflatoxins in Indian senna pods could also be reduced by application of NeemAzal T/Ss and even more by a fresh neem leaf water

extract, but not to below the desired level of 2 mg/kg. Northern Indian harvest methods, used in South India, resulted in a reduction of

aflatoxins, but again not sufficiently, and with a reduced economic output. Sudan senna (C. senna acutifolia), grown in South India,

showed resistance to fungal infections and the aflatoxin content was lower than in Indian senna (C. senna angustifolia), in the field and in

the laboratory, but growing Sudan senna elsewhere in India was not economical. The consequences of the findings are discussed.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Cassia senna; India; Aflatoxin; Insect damage; Drying; Harvest; Storage; Neem; Sudan senna

1. Introduction

Aflatoxins, especially aflatoxin B1, are highly toxic formammals and are carcinogenic (Eaton and Gallagher,1994; International Agency for Research on Cancer(IARC), 1993). Aflatoxins are secondary metabolitesmainly of Aspergillus flavus Link and A. parasiticus VanTieghem (Ellis et al., 1991; Davis, 1981). Due to the hightoxic action of aflatoxins, many countries have establishedmaximum residue levels, generally lying between 4 and50 mg/kg (Hansen, 1993), of these toxins in food items. The

ee front matter r 2006 Elsevier Ltd. All rights reserved.

r.2006.08.005

ing author. Tel.: +49 641 99 37580; fax: +49 641 99 37589.

ess: Thies.Basedow@agar.uni-giessen.de (T. Basedow).

t. 2006: Prof. Dr. Thies Basedow, Am Grun 15, D-24248

ies.Basedow@web.de.

EU has one of the strictest regulations with the maximumtolerated limit of aflatoxin in consumable items of 2 mg/kgfor aflatoxin B1 and 4 mg/kg for the sum of aflatoxins B1,B2, G1 and G2. Since aflatoxins are naturally occurringsubstances, it is impossible to completely eliminate themfrom products. However, the goal is to reduce them to theminimum possible level.

Cassia senna L. (Caesalpinaceae) (‘‘senna’’) is animportant medicinal plant (Harnischfeger and Stolze,1983). There are two areas of distribution. One subspecies(C. senna angustifolia) is distributed from India to Eritrea(‘‘Indian senna’’) (Hegnauer, 1996). The second subspecies(C. senna acutifolia) (‘‘Sudan senna’’) is grown in theAfrican Sahel zone (Gartner et al., 1982). Senna is one ofthe most widely used herbal laxatives (Der Marderosian,2005). The medical action of senna can be attributed

ARTICLE IN PRESSP. Muller, T. Basedow / Journal of Stored Products Research 43 (2007) 323–329324

mainly to the anthraquinone glycosides, especially senno-sides A and B (Franz, 1993). Dried leaves and pods of C.

senna contain up to 7% sennosides (Atzorn et al., 1981;Lohar et al., 1979; Lemli and Cuveele, 1978).

India is the major producer of this herbal medicine withan annual production of approximately 5000 t leaves and1000 t pods (statistics from Tuticorin port). More than50% of the annual production is imported by the EU.While senna leaves are almost free from aflatoxins, Indiansenna pods quite frequently contain aflatoxin concentra-tions above the acceptable limit of the EU (see below).After drying, Indian senna pods are cleaned by rotatingsieves and afterwards are normally kept in 100 kg jute bagsuntil sold. Before container shipping to internationalmarkets, senna pods are usually pressed into 180 kg bales.The storage time of dried senna pods in India lies between afew weeks and several months. The warm, humid storageconditions (3075 1C and 60 to 80% relative humidity) insouth Indian warehouses in general favour microbiologicalactivity in stored senna goods. During the rainy seasonwater damage is not unusual.

This paper describes the occurrence of aflatoxin in thepods of C. senna and evaluates the potential of aflatoxinproduction before harvest, during drying and over theperiod of storage. This is essential information forpreventing too high aflatoxin content in the pharmaceuticalcommodity. In the second part of the paper, differentexperimental approaches to reduce aflatoxins are shown,by methods of drying and harvesting, by application ofbotanicals and by use of varietal resistance.

2. Methods

Senna pods where collected from fields (a) in the maincultivation area of Tamilnadu over a period of 4 yearsduring various seasons and (b) in Gujarat in North India,where C. senna angustifolia is grown under differentclimatic and growing conditions.

Ninety-nine samples were tested for aflatoxin concentra-tion directly before and after conventional sun drying.Drying took place at Tuticorin (Tamilnadu) on a red-clay-brick floor. Low moisture contents (o10% H2O) werenormally achieved by the second or third day of drying,with regular turning of the drying pods three–four timesper day (from the second day). Whenever fresh podmaterial was analysed, a sample of the material wasweighed before and after drying at 105 1C, to enable thecomparison of aflatoxin contents of fresh and dry samples(Muller, 2005). Twenty-three samples were dried in a solar-tunnel dryer (Innotech Company, D-70599 Stuttgart-Hohenheim) for comparison.

To estimate the aflatoxin development in senna podsduring storage under south Indian conditions, 33 sampleswere tested before and after a storage period between 1 and8 months. Eight samples were pressed into 180 kg bales,while 25 samples were stored loose in jute bags.

Further experiments were conducted in the field and inthe laboratory on the effects of neem preparations,harvesting methods and varietal resistance.

2.1. Sampling

Experience showed that a representative sample had tohave a minimum size of 2 kg of the material under study,randomly taken. In fact, except when the total materialstock was less than 400 kg, more than 500 g/100 kg of podswere taken. Fresh samples were deep frozen at �18 1C andthen homogenised in a meat-mincing machine (mesh size3mm). Dried samples were ground in a lab mill to o2mm,and the fine powder was then again homogenised.

2.2. Extraction

For the extraction, the material was first homogenised inacetone for 5min in a high-speed ‘‘Ultra-Turrax’’ homo-geniser. Fifty grams of fresh minced and homogenisedmaterial were extracted in 100ml of 100% acetone, and25 g of dry powder were extracted in 125ml of 85%acetone. The suspension was then centrifuged at11,000 rpm, and 5ml of the clear extract was diluted to50ml with pH 7 buffer solutions.

2.3. Enrichment by immuno-affinity columns

Twenty millilitres of the solution were applied to theimmuno-affinity columns and filtered at a speed notexceeding 2ml/min. Afterwards, columns were washedwith 20ml distilled water. The remaining water wasremoved by pushing air through the columns with asyringe. Columns were then eluted with 1ml of freshlyprepared methanol and acetonitrile (1:1 v/v) solution. Theretention time for elution from the columns was aminimum of 30 s, with a passing rate of one drop persecond. The fractions were kept at �18 1C until HPLCanalysis.

2.4. HPLC analysis

Separation of fractions (diluted with water 1:1 v/v) tookplace by an HPLC method with a LiChrosphers RP–18column with water, methanol and acetonitrile (60:20:20; v/v/v; +119mg/lKBr and 100 ml 65% HNO3). Aflatoxin wasdetected by fluorescent measurement after derivatisation ina KOBRA Cell and quantified using calibration linesconstructed from external aflatoxins B1, B2, G1 and G2

standards. Most of the aflatoxin occurring here was B1,followed by G1 and G2. B2 was rarely found. To simplifythe data, only the sums of aflatoxins B1, G1, G2 and B2 arepresented in the results.

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Table 2

Aflatoxin content of pods of Indian senna before drying and after sun or

shade drying

Method of drying Status of

pods

Content of aflatoxins (mg/kg)

Average Range

Drying in the sun Fresh 15.9 0–255.0

(47 measurements) Dry 33.6�� 0–177.5

Drying in the shade Fresh 7.6 0–60.5

(27 measurements) Dry 28.0�� 2–80.0

��Significant increase after drying at Pp0.001 (Wilcoxon test).

Table 3

Aflatoxin contents of pods of C. senna angustifolia, and the percentage

distribution of different contents in two different areas of India, with

different climate and different harvesting methods

Region Average Distribution of aflatoxin contents (%)

(mg/kg) o4mg/kg 4–10mg/kg 410 mg/kg

Tamilnadu

(n ¼ 83)

12.17�� 23 34 43

Gujarat

(n ¼ 74)

8.15 54 20 26

��Significant difference between Tamilnadu and Gujarat at Pp0.001

(Mann–Whitney test).

P. Muller, T. Basedow / Journal of Stored Products Research 43 (2007) 323–329 325

2.5. Statistics

A sample size of 2 kg of senna pods was used as the basisfor all measurements. Due to their origin, aflatoxins are notevenly distributed within a sample (Whitaker et al., 1996),making statistical analysis difficult. Therefore in sometables the range is given in addition to the average, andsometimes the median also. Under these circumstances,non-parametric tests (Wilcoxon and Mann–Whitney;SPSS.12) were used.

3. Results

3.1. The distribution of aflatoxins in senna before the harvest

From 30 places around Tuticorin (South India), 2 kgeach of leaves, flowers, green seed pods and ripe seed podswere collected for assessment of aflatoxin content. Whileflowers and leaves were not contaminated, 23% of greenpods and 70% of ripe pods dried on the plants proved to becontaminated with aflatoxin (Muller, 2005).

3.2. The dependence of the aflatoxin content in senna pods

on insect damage

Fresh senna pods were studied with different amounts ofinsect damage (Table 1). When damaged pods wereopened, quite often mycelium was to be seen. Table 1shows that the amount of insect damage had a greatinfluence on the aflatoxin load. The insect larvae found inthe senna pods were identified as Ephestia elutella (Hubner)(Lepidoptera: Pyralidae).

3.3. The influence of drying on aflatoxin content of Indian

senna pods

The aflatoxin production in the pods of south Indian C.

senna before and after drying is shown in Table 2. Theaflatoxin concentrations in fresh samples varied betweennot detectable and 156 mg/kg aflatoxins. Most freshsamples had low aflatoxin concentrations but a very fewwere already highly contaminated before drying. Morethan 50% of fresh samples contained aflatoxin concentra-

Table 1

Aflatoxin load of pods of Cassia senna angustifolia, damaged to different

extents by insects (Ephestia elutella) in South India

Insect damage No. pods

tested

No. pods

with aflatoxin

Average

concentration

(mg/kg)

None 10 0 0

A small circular area 10 4 30

Less than half

surface

9 7 117

More than half

surface

8 8 410

tions below the acceptable limit in the EU, and more than75% of samples had less than 10 mg/kg aflatoxins.After drying, the samples had significantly higher

aflatoxin concentrations than before drying (Table 2).After conventional sun drying, less than 25% of samplesmatched EU mycotoxin standards. The average level ofaflatoxins in samples was doubled during the process ofconventional sun drying, but maximum concentrationsrose only from 156 to 177.5 mg/kg. This indicates thatsamples with low input concentrations had much higheraflatoxin increases than samples which were already highlycontaminated before drying. When pods had been dried inthe shade, the increase of aflatoxin content was even higher(four-fold, Table 2).

3.4. Regional differences

In Gujarat, Northern India, significantly lower aflatoxincontent of senna pods were observed. Over a 4 yr period, 74samples of pods were taken in different seasons at Gujarat,and 83 at Tamilnadu, for comparison. The results areshown in Table 3. One of the reasons for different aflatoxincontent is the different method of harvest in Gujarat wherethe whole plant is cut and subsequently dried for 5 days; inTamilnadu just the pods are picked and dried. Anotherreason is the climate of Gujarat, which has low humidityand a cooler temperature in winter (Muller, 2005).

ARTICLE IN PRESSP. Muller, T. Basedow / Journal of Stored Products Research 43 (2007) 323–329326

3.5. Aflatoxin during storage

Samples stored loose in jute bags changed colour duringstorage time from green to more brownish. The numbers ofsamples with increased or decreased aflatoxin content afterstorage and the maximum concentration differences areshown in Table 4. In pressed bales, the numbers of sampleswith increasing or decreasing aflatoxin content were equal,and changes were small. In senna pods stored loosely, mostsamples showed a decrease in aflatoxin content, but therewas a large variation in results with some high increases aswell as decreases. Overall there was no significant increaseof aflatoxins during storage.

3.6. Comparing sun drying with solar-tunnel drying

The solar dryer proved more effective, pods reaching amoisture content (m.c.) of 8% within 24 h, while sun dryingtook 3 days to achieve a reduction to 10% m.c. From 23samples of each, fresh pods averaged 4.0 g aflatoxin/kg,solar-dried pods 10.5 and conventionally sun-dried pods21.8 g/kg. Hence there was a reduction of aflatoxin bysolar-tunnel drying of 50%, as compared with conven-tional sun drying. However, even with this rapid and cleandrying method, aflatoxin production took place and the

Table 4

Number of Indian senna pod samples with increased and decreased aflatoxin c

maximum differences of aflatoxin content after storage in mg/kg and %

Storage form Concentration of aflatoxins Number of sam

Pressed, n ¼ 8 Increases 4

Decreases 4

Loose in bags, n ¼ 25 Increases 6

Decreases 19

0

Afl

atox

ins

in µ

g/kg

18.000

16.000

14.000

12.000

10.000

8.000

6.000

4.000

2.000

NLWE Ne

3878

4146

1591149

Fig. 1. Box whisker plots with maxima, quartile and minima concentrations of

in Petri dishes for 4 days, after having been dipped in water (‘‘untreated’’), N

ability to keep within the EU-tolerance limit of 2 mg/kg wasdependent on the quality of the fresh raw material.

3.7. Application of botanicals

It was not possible to prevent damage by E. elutella withthe application of neem products (Azadirachta indica A.Juss) in the field, but the infestation level is mostly verylow. However, damaged pods were tested to see if neempreparations could stop or reduce the production ofaflatoxin. Two different neem preparations were used,NeemAzal T/Ss (NA) (4ml/l of water), and a neem leafwater extract (NLWE): 100 g of fresh neem leaves werechopped and added to 1 l of boiling water. Two hours later,after repeated stirring and cooling, the solution was filteredand used immediately. Three lots of 11 samples weredipped in water, in NA solution or in NLWE, respectively.These differently treated fresh pods of C. senna (12 g each)were kept for 4 days in closed Petri dishes to allow thefungi to grow, before sun drying.The results are shown in Fig. 1. The neem preparations

reduced the aflatoxin content of the Cassia pods. NLWEwas more effective in this respect than NA, but neitherpreparation lowered the aflatoxin content to the desiredlow level.

ontent after storage in different forms under south Indian conditions, and

ples Maximum differences in mg/kg Maximum differences in %

+4.2 +23

�4.4 �19

+11.4 +71

�13.6 �76

emAzal T/S water

16809

8117

2568

16492549

3777

4523

6012

aflatoxins among groups of 11 samples of C. senna angustifolia pods, kept

eemAzal T/Ss or neem leaf water extract (see text).

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Table 6

Aflatoxin content (mg/kg) of conventionally sun-dried pods of two senna

subspecies grown at three sites in South India (Tamilnadu)

Senna subspecies Black soil Sandy soil Red soil

C. senna angustifolia 9.2 5.4 15.7

C. senna acutifolia 1.6 0.7 6.9

Table 7

Contents of aflatoxins (mg/kg) of pods of two senna subspecies after 3 days

in closed Petri dishes at 30 1C

Replicate Cassia senna angustifolia Cassia senna acutifolia

1 506.2 8.7

2 789.6 35.2

3 2503.4 10.4

P. Muller, T. Basedow / Journal of Stored Products Research 43 (2007) 323–329 327

3.8. Method of harvesting

As mentioned above, in the Gujarat region, where theaflatoxin contents of senna pods are low, the whole plant iscut and dried subsequently, which takes 5 days. To testwhether this method would be of advantage in South India(Tamilnadu), six plots of 150m2 each, in a large homo-genous field of Indian senna in Tamilnadu were set aside.Three were harvested by the Gujarat method (whole plant)and three as usual for Tamilnadu (picking pods). Theresults of the aflatoxin contamination are shown in Table 5.It can be seen that the Gujarat method reduced the typicalaflatoxin content. In the second harvest, however, aflatoxinlevels increased, and although the Gujarat harvest methodgave much lower aflatoxin levels than the Tamilnadumethod (12.3–4.9 mg/kg), the EU tolerance level wasexceeded in both. Additionally, it must be mentioned thatthe yield was reduced by the Gujarat method. Only twoharvests were possible, while the Tamilnadu methodallowed three harvests of pods.

3.9. Varietal resistance

It is known in pharmaceutical companies that Sudansenna (which is not grown in fields, but collected from wildplants) is mostly free of aflatoxins. In experiments in thefield and in the laboratory, C. senna angustifolia (Indiansenna) and C. senna acutifolia (Sudan senna) werecompared in South India. In three different sites inTamilnadu (with different soils), both subspecies weregrown separately on 100m2 plots. The growth anddevelopment of Sudan senna at these sites was not asgood as that of the Indian senna. While C. s. angustifolia

gave three harvests of pods, C. s. acutifolia gave only one.The harvests occurring at the same time were compared.Table 6 shows that pods of Sudan senna at all sites had alower content of aflatoxins than Indian senna.

Fresh pods of both subspecies (grown in South India)were studied in the laboratory: 12 g (in three replicates)were placed in closed Petri dishes for 3 days at 30 1C. Afterthis period, the pods of Indian senna were covered bymycelium, while the pods of Sudan senna were still green.

Table 5

Concentrations of aflatoxins (mg/kg) in dried Indian senna pods at

Tamilnadu, after different methods of harvest

Harvest Replicate Tamilnadu harvest Gujarat harvest

First harvest I 1.4 0.8

II 0.9 0.6

III 1.1 —

Average 1.1 0.7

Second harvest I 28.2 5.4

II 5.4 3.9

III 3.4 5.4

Average 12.3 4.9

Tamilnadu harvest: picking of pods; Gujarat harvest: cutting whole plant.

The aflatoxin contents in pods of Sudan senna were verylow, while pods of Indian senna had a high aflatoxin load(Table 7).

4. Discussion

4.1. Effect of local practices

As in various other plants like peanuts, maize, pistachionuts and figs (Gourama and Bullerman, 1995; Jacobsen etal., 1993; Bhatnagar et al., 1990), the production ofaflatoxins by fungi in and around C. senna seeds can occurin the field. Observed factors leading to an increase ofaflatoxins before harvest are (a) insect damage (Borgeme-ister et al., 1994; Hell, 1997; Setamou et al., 1998) and (b)high temperatures together with high humidity, waterstress and insect damage (Diener et al., 1987). Phytopha-gous insects can damage and weaken the plants, and theycan leave faeces as substrate for the fungi (Bilgrami et al.,1992).This study has shown that in the case of C. senna, the

maximum production of aflatoxins occurred during theprocess of drying. The highly significant increase ofaflatoxin levels during the process of conventional dryingshows evidently that in Indian senna pods aflatoxin isproduced more rapidly during the process of conventionalsun drying than before harvest.It is known that A. flavus and A. parasiticus produce high

amounts of toxins especially under drought conditions(Hill et al., 1983; Stahr et al., 1990; Sanders et al., 1993).The ideal conditions are limited moisture content andtemperatures between 30 and 50 1C. Such conditionsgenerally occur during conventional sun-drying. Therefore,the first step for the reduction of aflatoxins in C. senna

should be to establish drying methods, which prevent theformation of aflatoxin. Conventional sun drying was notsufficient.

ARTICLE IN PRESSP. Muller, T. Basedow / Journal of Stored Products Research 43 (2007) 323–329328

Also, lowered plant health and stress factors can increasethe formation of aflatoxins (Jones and Duncan, 1981;Sander et al., 1985; Payne et al., 1989). The increase ofaflatoxins in pods in the end of the season, when plantshave been stressed by harvesting (Tamilnadu method) andpossibly by drought stress, is in line with these observa-tions.

For many goods with aflatoxin problems, storageconditions and time are important factors controlling theincrease of aflatoxin concentrations (Fonseca et al., 1995;Hell, 1997). In contrast to other crops, the aflatoxincontent in pressed senna pods was quite stable duringstorage in pressed bales. When senna pods were stored inloose bags, decreases of aflatoxin concentration duringstorage were detected more frequently than increases. Ingeneral, the results here showed that the nearly air tightpressing of senna pods into bales helped to preserve thequality of pods during long storage periods in India, whilekeeping pods loose in jute bags reduced quality aspectssuch as colour and smell but did not increase aflatoxinlevels. Dried senna pods may contain anti-fungal sub-stances (El-Ballal et al., 1989).

The high humidity of about 60–80% in the Tamilnaduarea, combined with a temperature of 30 1C, allows furthermicrobial activity in dried goods (Diamante, 1995;Weidenborner, 1998). It is known that aflatoxins can bedegraded by Aspergillus spp. (Doyle and Marth, 1978;Tsubouchi et al., 1983) and by other microbes (Bol andSmith, 1989; Faraj et al., 1993). So, it is interesting, here,that for pods stored loosely in bags an overall decrease inaflatoxins was observed. On the other hand, the loosestorage method cannot be recommended, because it leadsto a decrease of quality.

4.2. Effects of other measures

It has been seen that it is possible to reduce the contentof aflatoxins in senna pods considerably by four methods(solar drying, addition of neem preparations, differentharvesting methods and varietal resistance). These meth-ods, though, did not reduce the aflatoxin load sufficientlyto meet the EU tolerance level. Additionally, the methodshad economic drawbacks.

According to Chang and Markakis (1981) and Cueroet al. (1987), the production of aflatoxins by A. flavus

increases in situations of strong competition for water withother microorganisms, in order to prevent the growth ofcompetitors. So the aim must be to dry senna even fasterthan by solar drying.

It is known that neem leaf extracts can reduce aflatoxinproduction by A. flavus and A. parasiticus (Bhatnagar andMcCormick, 1988; Bhatnagar et al., 1990; Zeringue andBhatnagar, 1990), an effect attributed to C3- to C9-alkenals (Zeringue and Bhatnagar, 1994). The timing isimportant, since the start of secondary metabolism in thefungi has to be prevented (Bhatnagar et al., 1993). Thisobservation should be followed up in future. In the current

tests the neem leaf extract did not suppress aflatoxinproduction completely, but it was interesting to note thatNeemAzal T/S (based on neem seed) had some effect.Thus, neem seed also contains some effective ingredients.Resistance of subspecies/varieties seems to be very

promising. Cassia senna acutifolia (Sudan senna) is knownto contain anti-fungal substances (Ismail and Babikir,1986). But there is still a lot of work to be done to fullyunderstand the anti-fungal mechanism of C. senna acuti-

folia and to transfer it to the closely related C. senna

angustifolia.For further progress, possibly the introduction of non-

toxigenic strains of A. flavus as competitors (Brown et al.,1991; Cotty and Bhatnagar, 1994; Cotty, 1994) could helpto prevent pre- and post-harvest aflatoxin formation in C.

senna. Also, since aflatoxins are very unequally distributedamong pods, a simple selection method, which eliminatesonly the most highly contaminated pods, could be aneconomic solution.For the time being, it is good that producers of senna

medicines can avoid problems by switching over to the useof senna leaves which are free of aflatoxins (Muller, 2005),but do contain the pharmacologically effective sennosids(Lemli and Cuveele, 1978; Atzorn et al., 1981).

Acknowledgements

We are grateful to roha arzneimittel GmbH (Bremen,Germany), which financed this study.

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