Efficacy Of Cassia Alata Leaves Powder

Published Date: 02 Nov 2017

1Department of Botany and Microbiology, King Saud University, Riyadh, Kingdom of Saudi Arabia.

2Muthaiyah Research Foundations, Thanjavur, Tamilnadu, India.

3Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK.

(Received: 03 March 2012; accepted: 15 April 2012)

Aflatoxin, a toxin produced by the fungus Aspergillus flavus Link: Fries, occurs naturally

in maize (Zea mays L.). Aflatoxin is a potent human carcinogen and is also toxic to livestock, pets,

and wildlife. When contaminated with aflatoxin, the value of maize grain is markedly reduced. In

the present study, an attempt was made to evaluate the inhibition of Aspergillus flavus growth and

aflatoxin production with using Cassia alata. The experimental design was elevated using T1

(control, Healthy maize), T2 (Healthy maize+Aspergillus flavus, T3 (Healthy maize + Aspergillus

flavus + Plant powder), T4 (Healthy maize + Plant powder). The healthy samples were inoculated

artificially with A. flavus and treated maize seeds were stored at room temperature in airtight

polyethylene bags till analysis. This stored maize was taken on 3rd, 6th, 9th and 12th (3 month

interval) month after treatment for analysis of aflatoxin production and growth of Aspergillus flavus.

Isolated colonies of A. flavus was identified based on the cultural, morphological and biochemical

characteristics. Further, 10% concentration the Cassia alata leaves powder delayed the growth of A.

flavus and complete inhibition was observed upto 120 days. The aflatoxin production was observed

in HPTLC and the highest percentage reduction inT3 compare other treatments. Hence, the present

study was concluded that the antifungal effects of the plant powder observed and has a potential for

use as a aflatoxin inhibitor.

Keywords: Aflatoxin, Aspergillus flavus,Cassia alata, Maize, HPTLC.

India is a country of vast population and

about 70% of people are engaged in agriculture.

Rapid population growth in the developing

countries leads to an ever-increasing demand for

food. Because of rising demand, production has

steadily increased over the past years. In the

production of food crops losses occur during the

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growth cycle in the field. Further losses occur

during harvest, where up to 5% of grain weight can

reduce the agricultural output

1

. Further losses occur

during storage, where found average damages of

30% in stored maize after six months storage in

Togo

2

. Storage pests are the main cause of these

losses, and under the tropical climatic conditions

the development of storage fungi, especially

species of the genera Aspergillus spp. and it

produced toxin, known as aflatoxin.

Aspergillus flavus is a soil-inhabiting,

filamentous fungus that saprophytically utilizes a

wide range of organic substrates. Though A.

flavus is considered a saprophyte, it is also an

opportunistic pathogen3 that can invade

224 ARUNACHALAM et al., Biosci., Biotech. Res. Asia, Vol. 9(1), 223-227 (2012)

agronomically important oil seed crops such as

corn, peanut and cottonseed that are under biotic or

abiotic stress. Among the toxic secondary

metabolites produced by this organism are the

aflatoxins (AFs) with aflatoxin B1 being the most

potent natural carcinogen known

4

. The foods at

highest risk of aflatoxin contamination are corn,

peanut and cotton seed

5

. Aflatoxin B1 has been

detected in 80% of maize samples obtained from

different locations in Southeast Nigeria

6

and

similarly 92 % of animal feed samples taken from

commercial sources in Thailand were contaminated

with aflatoxin B1

7

. In at least three parts of the

world, East Africa, the Philippines and Thailand,

good epidemiological evidence has been collected

showing a correlation between the incidence of

liver cancer and exposure to aflatoxins

5

.

Aflatoxins have also been identified as a

potential biological weapon for food and water

contamination

8

. Physical, chemical and biological

methods have been investigated in order to prevent

the growth of aflatoxin producing fungi and to

eliminate or reduce the levels of aflatoxins or to

degrade or detoxify aflatoxins in foods and feeds

9

.

Control by naturally produced agents is becoming

increasingly important because of consumers’

mistrust of food and feed treatments that involve

using synthetic xenobiotic substances. Natural plant

compounds have been used traditionally to preserve

foods in countries like Japan, India and Russia

10

.

Extracts and powders of various spices, herbs and

essential oils have been reported to have

antimicrobial activity against aflatoxin producing

fungi and some of them also inhibit aflatoxin

formation

11- 12

. Aim of the present study was to

evaluate the inhibition of Aspergillus flavus growth

and aflatoxin production with using Cassia alata

and has a potential for use as a aflatoxin inhibitor.

MATERIALSANDMETHODS

Sample collection

The healthy and contaminated maize (Zea

mays L.) seed samples were collected from storage

facilities at Thanjavur District, Tamilnadu, India.

Samples were placed directly in polyethylene bags

and transferred immediately to the laboratory where

they were stored in a cool place for further

investigation. The contaminated maize seed sample

was used as isolation of aflatoxin producing fungi.

Isolation and identification of aflatoxin

producing fungi

The serial dilution procedures were

followed. The potato dextrose agar was prepared

and it was poured onto the sterile Petri plates. After

solidification, the selected dilution factors 10

-2

to

10

-4

were spread on the medium respectively. Then

the plates were incubated. The isolated fungal strain

was identified based on their cultural and

morphological characteristics.

Collection of plant and preparation of powder

The plant of Cassia alata (leaves) was

collected from wild area of Thanjavur District,

Tamilnadu, India. The leaves of Cassia alata was

air dried and crushed to small piece using Mortar

and Pestle and powdered in an electric grinder.

Experimental Design

T1 - Control (Healthy maize)

T2 - Healthy maize + Aspergillus flavus

T3 - Healthy maize + Aspergillus flavus + Plant

powder

T4 - Healthy maize + Plant powder

The healthy samples were inoculated

artificially with A. flavus as per the method

13

. The

treated maize seeds were stored at room temperature

in airtight polyethylene bags till analysis

14

. This stored

maize was taken on 3

rd

, 6

th

, 9

th

and 12

th

(3 month

interval) month after treatment for aflatoxin analysis

and growth of Aspergillus flavus.

Fungal spore

Sample, 1 g from each flask was

serially diluted 10-fold with sterile distilled

water and 0.1 ml of each dilution was spread

uniformly on potato dextrose agar plates and

incubated in the dark for 3 days. On the 3rd day,

colonies were counted and expressed as log

colony forming units per gram (log CFU/g).

Estimation of Aflatoxins by High Performance

Thin Layer Chromatography (HPTLC)

Aflatoxin determination was based on the

Association of Official and Analytical Chemists

(AOAC) BF method (AOAC, 2001) with some

modifications. The experimental design treated maize

samples (25g) were blended with 200ml Acetone:

water (85 + 15, V/V), 2g NaCl and 5g Celite powder

and mixed. Mixture was allowed for half an hour and

then filtered. 25ml of hexane was added 2 times and

the lower layer was separated and put into a free test

tube. Lead acetate 20% in 0.3%

ARUNACHALAM et al., Biosci., Biotech. Res. Asia, Vol. 9(1), 223-227 (2012) 225

Fig 1. Comparison of spore count between T2 and T3 Treatments

acetic acid was added (Added 1 ml/10ml of extract)

and allowed for setting for 15 min and then filtered.

Filtrate was collected in a separating

funnel. 50 ml of chloroform was added and shaken

for 2 min. Lower layer was removed in anhydrous

sodium sulphate. Extract was evaporated in water

bath and the final layer of solution was transferred

to the tube containing chloroform of about 2 ml.

Samples were injected into HPTLC and florescence

intensity was calculate under long UV lamp at

365nm wavelength.

Calculation

Concentration of Aflatoxin in μg/kg =

S Y V

X W

Where,

S = μl aflatoxin standard which matches the

unknown

Y= Concentration of Aflatoxin standard μg/ml.

RESULTS AND DISCUSSION

In this present study fungal species was

isolated from the contaminated maize (Zea mays L.)

seed samples. Number of fungal colonies was isolated

from the soil samples. Among these isolated colonies

of A. flavus was identified based on the cultural,

morphological and biochemical characteristics. The

results were presented in the Fig -1. The A. flavus as a

major fungal contaminant in maize seeds and humans

are exposed to its spores

every day without any clinical outcome. But, in

some cases when persons are exposed to intense

spore dust, hypersensitivity reactions and lung

disease (primary Aspergillus pneumonia,

aspergilloma, allergic bronchopulmonary

aspergillosis, and invasive Aspergillus) may be

observed15. Many researchers have reported A.

flavus as a dominant mycoflora from different

substrates16.

The dried Cassia alata leaves powder was

used for inhibition of aflatoxin production and

growth of A. flavus. The effect of Cassia alata

leaves powder on the growth of A. ûavus and

production is presented in Table – 1. An inhibitory

effect on the A. ûavus growth production was

observed at all concentrations of the leaves powder

sample used in the study and was found to be dose

dependent. The pattern of inhibition shows that at

lower concentration of 1% inhibition of A. flavus is

relatively greater than inhibition of growth of the

fungus. 10% concentration the Cassia alata leaves

powder delayed the growth of A. ûavus and

complete inhibition was observed upto 120 days.

Efficacy of Cassia alata leaves powder to

prevent aflatoxin production in maize was

estimated to all treatments. The investigated results

were presented in Table 2. The highest percentage

reduction in aflatoxin production was observed in

T3 compare than other treatments. The percentage

reduction in aflatoxin production (%) every three

months increased in T3. Similarly T2

226 ARUNACHALAM et al., Biosci., Biotech. Res. Asia, Vol. 9(1), 223-227 (2012)

Table 1. Effect of certain Cassia alata leaves powder to prevent Aspergillus flavus growth

S. No. Compounds Concentration Spore count (log CFU/g) Inhibition (%)

1 Control - 24±2.26 -

2 C. alata 1% 16±1.85 36

2% 8±0.33 68

5% 4±1.15 84

10% 0 100

Mean ± Standard Deviation;

Table 2. Efficacy of Cassia alata leaves powder to prevent aflatoxin production in maize

S. Months Treatments Spore count Aflatoxin Percentage reduction

No (log CFU/g) (ppb) in aflatoxin production (%)

1 3 T1 ND ND -

T2 8.44±0.012 10842±118.22 -

T3 7.42±0.022 7852±122.45 27.5

T4 ND ND -

2 6 T1 ND ND -

T2 15.51±0.016 19953±124.62 -

T3 2.67±0.032 554±8.72 97.22

T4 ND ND -

3 9 T1 ND ND -

T2 21.35±0.056 23782±137.43 -

T3 0.90±0.008 160±4.66 99.32

T4 ND ND -

4 12 T1 ND ND -

T2 25.44±0.072 24658±120.62 -

T3 0.22±0.004 88±2.12 99.64

T4 ND ND -

Mean ± Standard Deviation; ND - not detected

the spore count (log CFU/g) and (ppb) Aflatoxin

were equally increased. The Fig – 1 illustrates T3

and T2 treatments were increased on opposite

direction. The fungicidal activity of some plant

extracts in controlling different plant pathogens

have been reported by several workers17-19.

Therefore, the present study is an important step

toward development of plant based pesticides,

which are eco-friendly for the management of

storage fungi, today and in near future.

ACKNOWLEDGEMENTS