19

EUGENOL, AN ANTIFUNGAL COMPONENT IN CLOVE THAT CHECKS THE CONTAMINATION OF ASPERGILLI IN RICE

Reddy CS, Reddy KRN, Mangala UN, Muralidharan K

Fungal deterioration of stored seeds and grains is a chronic problem in the Indian storage system because of the tropical hot and humid climate. Harvested grains are colonized by various species of Aspergillus under such conditions leading to deterioration and mycotoxin production. Infection of Aspergillus spp. was found on most paddy samples collected from different rice growing areas in India, either at or immediately after harvest or during storage. An investigation was carried out to isolate and characterize the antifungal component in clove, the extracts of which were found effective against Aspergilli in our earlier studies. Eugenol, a major component in clove was identified on TLC plate as dark colored spot with Rf 0.5 along with standard. This compound was purified by column chromatography. HPLC of this sample showed the eugenol peak at RT 5.03 with 79.3 percent purity. Based on the HPLC results, 1 g of powdered cloves contained 40 mg of eugenol. The proton and 13C NMR confirmed the identity of eugenol. IR spectrum of this sample showed OH functional group at 3462 cm-1 and the molecular weight was estimated at M/z 165 (m+1 peak) by mass spectrometry, which agreed with the eugenol composition, C10H12O2. For bioautography test, TLC plates were spray inoculated with Aspergillus flavus, A. parasiticus, A. niger and A. ochraceus. Eugenol on TLC inhibited mycelial growth of all four species of Aspergillus. In agar diffusion tests, eugenol produced inhibition zones ranging from 30 to 55 mm at 4.8 mg/disc depending on the Aspergillus spp. tested. On rice grains treated with eugenol at 2.4 mg/g aflatoxin B1 biosynthesis was completely inhibited. This is the first report on the inhibition of Aspergillus flavus, A. parasiticus, A. niger and A. ochraceus by eugenol obtained from clove. Clove is expensive, but eugenol can be commercially extracted from other sources such as Ocimum gratissimum, O. tenuiflorum, O.sanctum, Cassia fistula, Zieria smitii, Clarkia breweri, Ageratum conyzoides and whole tobacco, which are also known to be useful sources for eugenol.

showposter

EUGENOL, AN ANTIFUNGAL COMPONENT IN CLOVE THAT CHECKS THE CONTAMINATION OF ASPERGILLI IN RICE

C. S. Reddy*, K. R. N. Reddy, U. N. Mangala and K. MuralidharanDirectorate of Rice Research, Rajendra Nagar, Hyderabad – 500 030, India.

INTRODUCTION

Fungal deterioration of stored seeds and grains is a chronic problem in the Indian storage system because of the tropical hot and humid climate. Harvested grains are colonized by various species of Aspergillus under such conditions, leading to deterioration and mycotoxin production. Infection of Aspergillus spp. was found on most paddy samples collected from different rice growing areas in India, either at or immediately after harvest or during storage. An investigation was carried out to isolate and characterize the antifungal component in clove, the extracts of which were found effective against Aspergilli in our earlier studies.

MATERIALS AND METHODS

Characterization of Eugenol: The crude extract of cloves was loaded on to column of silica gel and eluted with a gradient of hexane-dichloromethane. The fractions were spotted on TLC strip and developed to detect fluorescence under UV. Fractions (8-21) that showed fluorescence were combined, concentrated and the purity of eugenol was analysed by HPLC. The purified eugenol was re-suspended in methanol and analyzed by NMR, FTIR and EIMS to identify the chemical structure.

Bioautographic assay of Eugenol: For bioautographic assay of eugenol, the TLC plates were sprayed with the spore suspension of Aspergillus flavus (DRAf009), A. parasiticus (DRAp032), A. niger(DRAn014) and A. ochraceus (DRAo013) and incubated in a moist and dark chamber at 250 C. The inhibitory zones were observed on the TLC plate and visualized under UV light at 254 nm. To detect antifungal activity, eugenol was applied to sterile filter paper discs at 4.8, 3.2, 1.6, 0.6, 0.54, 0.48, 0.32 and 0.16 mg eugenol / disc. The discs were placed on the surface at the two sectors of PDA plates that were already inoculated with a fresh inoculum of spores. The plates were then incubated and the inhibition of fungal growth was measured as zone diameter (mm). The effect of eugenol on aflatoxinB1 biosynthesis was evaluated by treating the A. flavus inoculated rice grains with eugenol at 0.8, 1.6 and 2.4 mg / g of rice and estimating the aflatoxin (µg / kg of paddy or milled rice) by indirect ELISA.

.

RESULTS AND DISCUSSION

Identification and purification of eugenol: The eugenol was tentatively identified based on the presence of a dark colored spot on TLC plates under UV at Rf 0.5. HPLC of the elute from this dark spot that was further purified on column chromatography showed eugenol peak at RT 5.03 with 79.3 per cent purity (Fig. 1). Based on the HPLC results, clove was estimated to contain 40 mg of eugenol/ g dry powder.

SUMMARY AND CONCLUSIONS

Rice grains are colonized by Aspergilli at or immediately after harvest. The antifungal component of cloves was isolated, characterized and tested for efficacy against Aspergilli. The major component, eugenol was identified on TLC plate as dark colored spot with Rf 0.5 along with standard. This compound was purified by column chromatography. HPLC of this sample showed the eugenol peak at RT 5.03 with 79.3 % purity. Based on the HPLC results, 1 g of powdered cloves contained 40 mg of eugenol. The proton and 13C nmr confirmed the identity of eugenol. IR spectrum of this sample showed OH functional group at 3462 cm-1 and the molecular weight was estimated at M/z 165 (m+1 peak) by mass spectrometry, which agreed with the eugenolcomposition C10H12O2.

For bioautography test, TLC plates were spray inoculated with A. flavus, A. parasiticus, A. niger and A. ochraceus. Eugenol on TLC inhibited mycelial growth of all four species of Aspergillus. In agar diffusion tests, eugenol produced inhibition zones ranging from 30 to 55 mm at 4.8 mg / disc depending on the Aspergillus spp. tested. On rice grains treated with eugenol at 2.4 mg / g, aflatoxin B1 biosynthesis was completely inhibited. This is the first report on the inhibition of A. flavus, A. parasiticus, A. niger and A. ochraceus by eugenol obtained from clove.-----------------------------------------------------------------------------------------------------------------------------------------------------

Presented at:*E-mail: drcsreddy@scientist.com (or) drcsreddy@gmail.com

Fig. 1. HPLC spectrum of purified Eugenol from clove

Characterization of eugenol. The chemical characterization of eugenol was made by following the method of Saito et al (2004). The proton nmr spectrum of eugenol showed aromatic proton peaks at δ 6.6 and δ 6.75; aromatic alcoholic proton peak at δ 5.0; allyl protons between δ 4.9 to δ 6.3; methoxy protons at δ 3.8; and benzylic protons at δ 3.2 (Fig. 2). The C13 nmr spectrum showed aliphatic carbon peaks at δ 48.4 and δ 56.3, and aromatic carbons between δ 113.9 to δ 149 (Fig. 3). The FTIR spectrum of eugenol showed the presence of alcohol group at 3462 cm-1 and methoxygroup at 1149 cm-1 (Fig. 4). Saito et al (2004) had also reported similar results of all such spectral features in eugenol samples. The molecular wt. (Fig. 5) of eugenol isolated from clove was estimated at M/z 165 (m+1 peak), and the molecular formula is derived as C10H12O2. Faria et al (2006) had reported that the molecular wt. of eugenol purified from O. gratissimum was 164, very similar to M/z 165 (m+1 peak) estimated for eugenol from clove in our study.

Fig. 4. IR spectrum of purified Eugenol from clove

Fig. 5. Mass spectrum of purified Eugenol extracted from clove

Bioautography: The elute in methanol from the dark spot at Rf 0.5 on TLC plate completely inhibited mycelial growth in all the four species of Aspergillus (Fig. 7). This is the first report of bioautography of Aspegillus spp. on TLC plates.

Bioassay: The purified eugenol at 4.8 mg / disc, produced inhibition zones varying from of 30 to 55 mm depending on the Aspergillus spp. tested (Fig. 6, Table 1). On rice treated at 2.4 mg eugenol / g of grains, the inoculum of A. flavus failed to grow and thus aflatoxin B1 biosynthesis on rice was prevented (Fig. 4, Table. 2).

Eugenol peak at RT 5.03 with 79.3 % purity.

Based on HPLC results, clove was estimated to contain 40 mg of Eugenol / g dry powder.

FTIR (KBr) spectrum showed presence of Alcohol group at 3462 cm-1 and Methoxy group at 1149 cm-1.

1149 cm-1

3462 cm-1

Molecular weight was estimated at M/z 165 (m+1 peak) by Mass spectroscopy, which agreed with composition C10H12O2 for Eugenol.

M/z 165 (m+1 peak)

Fig. 2. 1H NMR spectrum of purified Eugenol from clove

1H NMR (200 MHZ) spectrum of Eugenol showed Aromatic proton peaks at δ 6.6 & δ 6.75; Aromatic alcoholic proton peak at δ 5.0; Allyl protonsbetween δ 4.9 to δ 6.3; Methoxy proton at δ 3.8; and Benzylic proton at δ 3.2.

δ 6.75 δ 5.0δ 6.3

δ 3.8δ 4.9

δ 6.6

δ 3.2

δ 113

δ 149

δ 48.4

δ 56.3

13C NMR spectrum showed Aliphatic Carbon peaks at δ 48.4 & δ 56.3, and Aromatic Carbons between δ 113 to δ 149.

Fig. 3. 13C NMR spectrum of purified Eugenol from clove

Fig. 6: Zone of Inhibition produced by purified Eugenol Fig. 7: Effect of Eugenol on the growth of A. flavusEugenol EugenolControl Control

Eugenol

Control

A. flavus A. parasiticus A. niger A. ochraceus

Inhibition zone (mm)Eugenol(mg / disc) Af Ap An Ao Control0.16 0 0 0 0 00.32 0 0 0 0 00.48 0 0 0 0 00.54 0 0 0 0 00.60 0 0 0 0 01.6 0 0 0 0 03.2 0 0 0 0 04.8 44 55 38 30 0

Rf 0.5 on TLC

Inhibition

A. flavus growth

Table 1: Antifungal activity of Eugenol by Agar diffusion

2.421

1.335

0

2.88

00.5

11.5

22.5

3

AFB1

( m

g / K

g S

eed)

0.8 1.6 2.4

Untreated

Eugenol (@ g / Kg Seed)

Af = A.flavus; Ap = A.parasiticus; An = A.niger; Ao = A.ochraceus Fig. 8: Effect of purified Eugenol on AfB1 biosynthesis