Substrate suitability of neem seed kernel for the growth and elaboration of aflatoxins by Aspergillus parasiticus (NRRL 2999)

Transcription

1 Indian Journal of Natural Products and Resources Vol. 3(3), September 2012, pp Substrate suitability of neem seed kernel for the growth and elaboration of aflatoxins by Aspergillus parasiticus (NRRL 2999) Kosuri Tanuja, Godugu Kavitha, Rupula Karuna and R B Sashidhar* Department of Biochemistry, University College of Science Osmania University, Hyderabad , Andhra Pradesh, India Received 18 July 2011; Accepted 4 July 2012 Neem seed kernels artificially infested with Aspergillus parasiticus (NRRL 2999) was evaluated for aflatoxin elaboration and fungal growth, and compared with groundnut, a high risk commodity for aflatoxin contamination. At optimal moisture content (10-12%) the total, individual toxins (AFB 1, AFB 2, AFG 1 and AFG 2 ) and ergosterol content increased and showed maximum levels on day 9. Crude protein and polyphenols increased while, fat and total sugar content decreased during the period of infection. The protein content correlated positively (r = 0.734) with total toxin levels, whereas fat content (r = ) and total sugars (r = ) showed negative correlation and they were all statistically significant (p<0.01). The polyphenols showed negative and non-significant correlation with total toxin levels. Azadirachtin one of the major active principles of neem seed kernel showed significant decrease on day 3 (P<0.05) and day 6 (P<0.01). Neem seed kernel has shown 54 and 74% less aflatoxin production on day 9 and 12, respectively in comparison to groundnut seeds. Ergosterol content also showed 60% decrease on day 9, conferring it a poor substrate for fungal growth and aflatoxin elaboration. Keywords: Aflatoxin, Aspergillus parasiticus, Azadirachtin, Neem seed kernel, Polyphenols, Proximate composition. IPC code; Int. cl. ( ) A61K 36/58, A61K 131/00, A61P 31/10 Introduction Aflatoxins are a group of highly toxic, mutagenic and carcinogenic polyketide compounds 1 synthesized by the fungi Aspergillus flavus, A. parasiticus and A. nominus 2. These fungi grow rapidly on a variety of natural substrates 3 and consumption of food products contaminated with toxigenic fungi causes serious health hazards to human and animals. Aflatoxins are also well known for their hepatotoxico-carcinogenic effects 4. Neem (Azadirachta indica A. Juss.) a tree belonging to Meliaceae family is familiar in India as one of the most versatile medicinal plants having a wide spectrum of biological activities. It has been extensively used in Ayurveda, Unani and Homoeopathy and the tree is still regarded as village dispensary in India. It is known for its pesticidal activity against more than 400 insect pests 5 and has many pharmacological activities, such as antiinflammatory 6, anti-malarial, anti-fertility, anti-acne 7, ascaricidal 8 and nematicidal 9. Further, several studies have shown the antimicrobial activity of neem *Correspondent author: sashi_rao@yahoo.com; Tel / FAX #: The extracts from different parts of neem tree have shown antifungal activity In a recent review by Reddy et al 3 on the control of mycotoxigenic fungi by different plant derived products, neem materials (leaves, seed /kernel and oil) were proposed to be the most economical option that showed fungal growth but inhibited mycotoxin production. Earlier studies have shown significant antifungal effects of neem leaf and seed extracts on aflatoxin-producing fungal strains Aflatoxin inhibition activity of neem leaf and seed extracts has been associated with morphological alterations in the mycelia of the aflatoxigenic fungus 22. Among different parts of the tree, neem seed kernel is the most active part, which has the maximum number of bioactive terpenoids 23 such as nimbidin and gedunin with proven antifungal activity Extracts from neem seed kernel were reported to be effective against few human pathogenic fungi 26. The moisture content required for growth and aflatoxin contamination varies with the natural substrates. A moisture content of 10-12% in groundnuts, 17-19% in wheat, corn and sorghum grains was found to be optimum 27. Variation among different sorghum genotypes stored in traditional containers in India with respect to percent mycotoxin contamination was observed by Sashidhar et al 28.

2 396 INDIAN J NAT PROD RESOUR, SEPTEMBER 2012 Earlier, influence on biochemical composition of sorghum genotypes during Aspergillus parasiticus infestation was studied by Ratnavati and Sashidhar 29. This study indicated decrease in starch and fat contents and an increase in protein and polyphenol contents during the course of infection. An increase in phenolic compounds in some groundnut cultivars as a resistive mechanism against fungal infection was shown 30. However, this aspect has not been elucidated in the neem seed kernels. Among the various active principles of the neem, azadirachtin from the neem seed kernel is important due to its well established insecticidal 31-33, antibacterial and antifungal properties 34. Azadirachtin has the advantage of being a natural insect control agent 35 and it is the major active metabolite from the neem seed kernels 36. Recently, Akhisa et al 6 reported the anti-melanogenesis effect of Azadirachtin B in mouse skin in vivo. In order to assess the presence of antifungal compounds in neem seed kernels, it is important to ascertain as to whether the neem seed kernel is a suitable substrate for toxigenic Aspergillus species infestation and aflatoxin elaboration. Several studies showed the antimicrobial activities of certain isolated active principles from neem seed kernels However, not many studies have focused on neem seed kernel as a substrate for Aspergillus parasiticus growth and associated biochemical changes. In the present experimental investigation, the optimal moisture content of the neem seed kernel that supports maximum aflatoxin elaboration by Aspergillus parasiticus NRRL 2999 was envisaged. Ergosterol, a predominant cell membrane sterol of fungal origin 37, was monitored during various time points of fungal infection, as an index of fungal growth. The alterations in the proximate biochemical composition (crude protein, fat and total sugar content) and protein precipitable/bioactive polyphenols at optimal moisture level at various time points of fungal infection are reported. Keeping in view of its importance, the azadirachtin content of the neem seeds at different time points of fungal infestation was studied, as it is a major bioactive component of the neem seed kernels. Groundnut was used as a positive control for comparative study throughout the experimental investigation, not only because it is a high risk agricultural commodity for aflatoxin elaboration, but also due to its relative similarity in proximate composition with neem seed kernel Materials and Methods Chemicals Neem seed kernels were sourced from the local market vending Ayurvedic and Herbal materials (Hyderabad, Andhra Pradesh, India) and groundnut kernels (DRG 12) were procured from Acharya N. G. Ranga Agricultural University (Rajendranagar, Hyderabad, Andhra Pradesh, India). Aflatoxin reference standards (AFB 1, AFB 2, AFG 1 and AFG 2 ), Ergosterol standard and polyester silica gel-g TLC plates (thickness 250 µm; particle size 2-25 µm) were sourced from Sigma Chemicals Co., St. Louis, USA. Azadirachtin reference standard was obtained from Chem Services, Pennsylvania, USA. All other chemicals and reagents used were of analytical grade. Strain A highly toxigenic fungus strain, Aspergillus parasiticus (NRRL 2999) was obtained from United State Department of Agriculture (USDA), Peoria, Illinois, USA. Cultures were maintained on potato dextrose agar (PDA) slants for eight days at 28.0 ± 1 C in a BOD incubator (Kalorstat, Dwaraka Equipment (P) Ltd., Mumbai, India). Estimation of moisture content The moisture content was estimated as per the procedure of AOAC 43, in semi-dried and dried neem seed kernels and groundnut kernels after procurement. These seeds were subsequently dried to a constant weight in an oven at 70 C. Inoculation of neem seed and groundnut kernels with A. parasiticus In order to establish an optimum moisture content of the neem seed kernels for aflatoxin elaboration in comparison with groundnut kernels, the neem seed and groundnut kernels (50 g) dried to a constant weight earlier were washed 2-3 times with sterilized glass distilled water and surface sterilized with sodium hypochlorite solution (1% v/v) and washed thoroughly with distilled water 2-3 times to remove any residual sodium hypochlorite solution. The neem seed and groundnut kernels were separately presoaked in sterilized glass distilled water in a clean Erlenmeyer flask (250 ml) to retain a moisture content of 8-10, and 12-14% in neem seed kernels and 10-12% in groundnut kernels. The seeds (10 g) were transferred into a clean Erlenmeyer flask (250 ml) pre-sterilized in a laboratory autoclave at 1.05 kg/cm 2 (103 kpa) pressure at 121 C for 15 minutes.

3 TANUJA et al: SUITABILITY OF NEEM SEED KERNEL FOR THE GROWTH OF AFLATOXINS 397 Fungal spore inoculum s in a volume of 500 µl containing spores prepared in 0.01% Tween- 20 was added to 10.0 g of kernel samples, in the laminar flow hood under aseptic conditions. These samples were incubated at 28 ± 1 C and the fungus was allowed to grow for a period of 12 days. At 5 different time points of fungal infection i.e., 0, 3, 6, 9 and 12 days, samples were drawn and processed for further analysis. The samples were maintained in duplicate flasks and two sub-samples were drawn from each of the duplicate flasks for analysis. The samples were dried in vacuum oven at 20 kg/cm 2 and at C for 48 hours. After drying, samples were powdered in a mechanical grinder to a particle size of 0.4 mm. The seed samples were analyzed for the presence of aflatoxins and ergosterol before inoculation of fungal spores, in order to avoid any back ground interference. Extraction and estimation of aflatoxins The infected seeds were ground in a high-speed mechanical blender (Sumeet, Mumbai, India) to a fine powder. The powdered neem seed and groundnut kernel samples were defatted using a Soxhlet apparatus (Borosil, Mumbai, India) using n-hexane as a solvent. The defatted kernel powder (1.0 g) was extracted with 5.0 ml of methanol: water (55:45) using a mechanical shaker for one hour. Later the samples were centrifuged, the aqueous methanolic phase was transferred into a separating funnel and equal volume of chloroform was added and mixed thoroughly. The chloroform layer containing the toxin was separated and dried using a flash evaporator. The dried samples were stored at 20 C until further analysis by TLC. Aflatoxins were estimated by thin layer chromatography (TLC)/flourodensitometric method 44. The samples were re-dissolved in benzene: acetonitrile (98:2) and were spotted (10 µl) on the activated TLC plates. The TLC plates were developed in toluene:ethyl acetate: formic acid (6:3:1) and visualized under long wave UV light (365 nm). Annotated digital density images of the spots were recorded by the CCD camera (UVItec, Cambridge, UK) and saved in PC-compatible file format (.tif file) on a floppy disk. Later, the digital density images of different toxins were analyzed by the software for determining the net density, which was measured as peak volume. Reference standards AFB 1, AFB 2, AFG 1 and AFG 2 were used for calculating the aflatoxin content in the samples. The aflatoxin content in the samples was expressed as µg/g of defatted powder. Extraction and estimation of ergosterol The extraction procedure followed was a modified method over that of Seitz et al 45. Five grams of the defatted sample was used for extraction. The sample was soaked overnight in 10 ml of ethanol (95%) and kept in dark. After soaking they were mechanically shaken for 1h in a mechanical shaker at room temperature. The extracts were then centrifuged at 900 g in a laboratory centrifuge for 5-10 min. Supernatant (sup-1) was transferred to long screw tubes (18 cm 2 cm, diameter) containing 0.152% butylated hydroxyl toluene and 8% potassium hydroxide. The pellet was re-extracted with 4.0 ml of ethanol using the homogeniser (Polytron PT-MR, Littoue, Switzerland) for 2-3 min at 15,000 rpm. The extract was centrifuged again at 900 g for 5-10 min. The supernatant (sup-2) was transferred in to the screw cap tubes containing sup-1 and the mixture was refluxed at 80 C for 15 min using a heating mantle and later allowed to cool. To this, 15 ml of petroleum ether (60-80%) was added followed by 20 ml of double distilled water for washing. Washing procedure was repeated thrice and both the phases were allowed to separate and the aqueous phase (lower layer) was discarded. The petroleum ether layer was passed through anhydrous sodium sulphate and later evaporated to dryness. The residues were re-dissolved in a known volume of (0.25 ml) of benzene: acetonitrile (98:2). The estimation of ergosterol was done according to the method reported by Sashidhar et al 46. Ergosterol standard in the range of µg / 10 µl was spotted on to an activated silica gel G TLC plate. The plate was developed in toluene: acetone (9:1) solvent system. The plate was air-dried and exposed to iodine vapours in glass chamber pre-saturated with iodine for < 2.0 min. The development of yellow spots indicated the position of ergosterol. The plate was allowed to stand for approximately 10 min at room temperature till the yellow colour, developed due to iodination disappeared. The ergosterol spots were later scanned under UV light (365 nm) to detect the fluorescent spots using TLCdigital image analysis system (UVItec, Cambridge, UK). Calibration curve based on the area under the curve vs concentration of ergosterol (reference standard) was used for calculating the ergosterol content in the infected samples. The ergosterol content was expressed as µg/g of defatted sample.

4 398 INDIAN J NAT PROD RESOUR, SEPTEMBER 2012 Estimation of proximate principles and polyphenols The protein, fat and total sugar content was estimated by the methods of Willis et al 47, AOAC 43 and Dubois et al 48, respectively, in neem seed and groundnut kernels. The polyphenol (protein precipitable/bioactive) content in both the substrates was quantitated by the method reported by Ratnavathi and Sashidhar 49. Estimation of azadirachtin content in neem seed kernels Azadirachtin content in neem seed kernels was analyzed by indirect competitive ELISA developed earlier in our laboratory 50. Briefly, the ELISA plates were coated with BSA-azadirachtin (500 ng of BSA containing 76 ng of azadirachtin equivalent) as a coating antigen. Azadirachtin-specific polyclonal antibodies were raised in rabbits against the immunogen, ovalbumin-azadirachtin. The antiserum at a final dilution of 1:30,000 was used in the immunoassay. The following key steps were followed in the ELISA protocol, a) The antisera (25 µl) was pre-incubated with different concentrations of azadirachtin (reference standard) or sample extract in a volume of 25 µl at 37 C for 2 hours. This mixture was added on to ELISA plates coated with 500 ng of BSA-azadirachtin and was further incubated at 37 C for another 2 h; b) Horse-radish peroxidase labeled anti-rabbit IgG (raised in goat) was used as an enzyme label at a dilution of 1:6000 and the plate was incubated at 37 C for 1 h; d) TMB (tetra methyl benzidine) mixed with acetate buffer containing β-cyclodextrin and urea hydrogen peroxidase as substrate was added and after incubation for 15 min, 100 µl of 1.25 M H 2 SO 4 was added as stopping reagent; e) Absorbance at 450 nm was recorded in Micro Scan MS 5608A microplate reader (ECIL, Hyderabad, India) along with reagent blank. All the samples were assayed in triplicate. 1.0 and 6.0 ± 0.4%, respectively. The total toxin elaborated by the infected neem seed and groundnut kernels during various time points of fungal infection was different and statistically significant (P<0.01 and P<0.05) as depicted in Figure 1. When compared to the levels in groundnut (10-12% moisture) the toxin in neem seed kernel samples with varying moisture contents (8-10, and 12-14%) was found to be less and statistically significant (P<0.01 and P<0.05). The amount of total toxin produced was found to increase with increase in the moisture content of the neem seed kernels from 8-10 to 10-12%. However, the total toxin production was not enhanced when the moisture content of the seed was increased to 12-14%. The aflatoxin content was found to be reduced by 38.6, 61.2, 54.4 and 74% on day 3, 6, 9 and 12, respectively in neem seed kernels (10-12%) in comparison to groundnut kernels (Fig. 1). The variation in individual aflatoxins (AFB 1, AFB 2, AFG 1 and AFG 2 ) produced in neem seed kernels with varying moisture levels (8-10, and 12-14%) and in groundnut kernels by A. parasitius is represented in Table 1. The individual aflatoxins were found to increase with time and attained maximum concentration on day 9 in both neem seed and groundnut kernels and their concentration were found to decrease by day 12. Two-way ANOVA showed statistically significant (P<0.01) variation in toxin elaboration between the two substrates as well as during different periods of infection. The interaction between substrates and Statistical analysis Data was analyzed by two way analysis of variance (ANOVA). The software package M. Stat and M.S Excel (Ver 98) was used to analyze the data statistically. The statistical tests, namely paired t-test and correlation were done based on methods reported by Harnett and Murphy 51. Results Moisture content and aflatoxin elaboration The innate moisture content of the semi-dried, dried neem seed and groundnut kernels immediately after procurement was recorded as 14.0 ± 0.6, 7.0 ± Fig. 1 Total toxin content in neem seed kernels at various moisture levels during infestation with Aspergillus parasiticus NRRL 2999 in comparison with groundnut.

5 TANUJA et al: SUITABILITY OF NEEM SEED KERNEL FOR THE GROWTH OF AFLATOXINS 399 Table 1 Production of aflatoxins in neem seed kernels at different moisture levels after infestation with Aspergillus parasiticus NRRL 2999 in comparison with groundnut kernels Substrate Period of Aflatoxin (µg/g) incubation (days) B 1 B 2 G 1 G 2 Groundnut(10-12%) a ± ± ± ± ± ± ±0.10` 2.80± ± ± ± ± ± ± ± ±0.26 Neem (8-10%) a ±0.12** 0.51±0.03** 0.97±0.04** 0.30±0.01** ±0.13** 1.34±0.16** 1.67±0.11** 0.74±0.05** ±0.11** 1.53±0.23** 2.62±0.16** 1.85±0.09** ±0.33** 0.93±0.05** 0.71±0.10** 0.90±0.04** Neem (10-12%) a ±0.05** 0.83±0.02** 1.53±0.11** 1.50±0.02** ±0.23** 1.80±0.12** 2.75±0.06** 0.91±0.03** ±0.13** 2.21±0.13** 3.71±0.21** 2.43±0.02** ±0.15** 0.94±0.04** 2.12±0.19** 0.73±0.06** Neem (12-14%) a ±0.03** 0.83±0.06** 1.57±0.05** 1.54±0.03** ±0.13** 1.73±0.10** 2.77±0.12** 0.87±0.05** ±0.25** 2.23±0.15** 3.50±0.17** 2.41±0.08** ±0.21** 1.01±0.10** 2.10±0.07** 0.71±0.13** Values represented are Mean ± S.D of four replications; a = moisture content; ** =p<0.01 different period of infection for toxin elaboration was also significant (P<0.05) (Fig. 2). Ergosterol content The ergosterol content increased with increase in the period of incubation and reached maximum on day 9 and was found to decrease by day 12 of incubation. The ergosterol content in groundnut was found to be 78.0 ±0.2 µg/g while, in the neem seed kernel 24.0±0.15 µg/g accounting to 60% less growth on the neem seeds. The changes in ergosterol content of neem seed and groundnut kernels during infestation was statistically significant (P<0.01) (Fig. 3). The ergosterol content in neem seed kernel as substrate with different moisture contents, (8-10, and 12-14%) was comparatively less and statistically significant (P<0.01) than groundnut (10-12% of moisture content). Ergosterol content remained relatively unchanged in neem seed kernels with increased moisture content from 8-10% to 12-14% and the difference was not statistically significant. The correlation coefficient (r) of ergosterol versus aflatoxin content during different periods of infection, showed a significant and positive correlation for neem seed kernels (r = 0.953) and for groundnut (r = 0.957) (Table 2). The ergosterol content in the infected neem seed and groundnut kernels during various time points of fungal infection was different and statistically significant (P<0.01) as depicted in Figure 3. The two way ANOVA showed a significant difference in ergosterol content among the substrates (P<0.01) and also for various time points of fungal infection (P<0.05) as depicted in Figure 4. Fig. 2 Pie chart of analysis of variance (ANOVA) for total toxin content. Fig. 3 Ergosterol content in neem seed kernels at various moisture levels during infestation with Aspergillus parasiticus NRRL 2999 in comparison with groundnut.

6 400 INDIAN J NAT PROD RESOUR, SEPTEMBER 2012 Table 2 Correlation coefficient values (r) of aflatoxin versus other biochemical parameters Parameter Aflatoxin correlation values (r) Neem Groundnut Ergosterol 0.953** 0.957** Protein content 0.734** 0.756** Fat content ** ** Total sugar content ** ** Polyphenols (-) negative correlation; ** p<0.01 Fig. 4 Pie chart of analysis of variance (ANOVA) for ergosterol content. Proximate analysis The crude protein content in neem and groundnut kernels increased during the course of fungal infection (Fig. 5A). The increase in crude protein content during different periods of fungal infection (P<0.01) and the interaction between substrates at different periods was found to be statistically significant (P<0.05) (Fig. 6A). The crude protein content significantly and positively correlated with total toxin content and the correlation coefficient (r) was and for neem seed and groundnut kernels respectively (Table 2). The fat content in neem seed and groundnut kernels on day 0 was found to be 45.0 ± 2.0 and 48.0 ± 3.4 g%, respectively. It was found to decrease with increased periods of fungal infestation i.e., at end of the day 9 in neem seed kernels (25.0 ± 2.8 g%) and day 6 in groundnut kernels (31.0 ± 1.5 g%) (Fig. 5B). There was no statistically significant difference between the two substrates for the fat content, but the decrease in fat content during various periods of fungal infection was found to be statistically significant (P<0.01). The interaction between substrates and at various time periods of infection for the fat content was also significant (P<0.05) (Fig. 6B). The percent fat was negatively and significantly correlated to aflatoxin produced i.e., the decrease in fat content was associated with the increase in toxin production in both neem seed (r = 0.761) and groundnut kernels (r = 0.940) (Table 2). The total sugar content in neem seed and groundnut kernels on day 0 was found to be 4.6 ± 0.21 and 4.9 ± 0.26 g%, respectively. During fungal colonization, the sugar content was maximally utilized till day 9, in both the substrates and it was found to be reduced from 4.6 to 1.6 g% and 4.9 to 1.8 g% in neem seed and groundnut kernels, respectively (Fig. 5C). There was no statistically significant difference between the two substrates for the sugar content, but a significant difference was observed during various time periods of infection (P<0.01). The interaction between the two substrates during various time periods of infection was not statistically significant (Fig. 6C). The percent sugar content was negatively and significantly correlated to aflatoxin produced, i.e., the decrease in sugar content in both the substrates is associated with the increased toxin production (r = in neem seed and r = in groundnut kernels) (Table 2). Protein precipitable polyphenols The polyphenol (protein precipitable polyphenols or bioactive phenols) content in groundnut kernels (0.81 ± 0.1 g %) was significantly (P<0.01) higher than that of the neem seed kernels (0.45 ± 0.02 g %) on 0 day. In both the substrates the polyphenol content was found to increase in response to fungal infection (Fig. 5D). The maximum increase in polyphenol content up to 0.74 ± 0.02 and 1.3 ± 0.1 g% was recorded for neem seed (day 3) and groundnut kernels (day 6), respectively. The polyphenol content in both the substrates (P<0.01) and during various time points of fungal infestation (P<0.05) was different and was statistically significant. The interaction between the substrates and during different time points of fungal infection for the polyphenol content was also significant (P<0.05) (Fig. 6D). The correlation coefficient of percent polyphenol to aflatoxin content was not significant in both the substrates, with r values being and for neem seed and groundnut kernels, respectively (Table 2).

7 TANUJA et al: SUITABILITY OF NEEM SEED KERNEL FOR THE GROWTH OF AFLATOXINS 401 Fig. 5 Changes in proximate composition (crude protein, fat and total sugar) and protein precipitable polyphenol content in neem seed kernels during infestation with Aspergillus parasiticus NRRL 2999 in comparison with groundnut. A. Changes in crude protein content in neem seed kernels during infestation with Aspergillus parasiticus NRRL 2999 in comparison with groundnut. B. Changes in fat content in neem seed kernels during infestation with Aspergillus parasiticus NRRL 2999 in comparison with groundnut. C. Changes in total sugar content in neem seed kernels during infestation with Aspergillus parasiticus NRRL 2999 in comparison with groundnut. D. Changes in protein precipitable polyphenol content in neem seed kernels during infestation with Aspergillus parasiticus NRRL 2999 in comparison with groundnut. Table 3 Changes in azadirachtin content in neem seed kernels during Aspergillus parasiticus NRRL 2999 infestation as analyzed by indirect competitive ELISA Period of infection (days) Azadirachtin (g%) ± ± 0.02* ± 0.01** 9 ND 12 ND Values represented are Mean ± S.D of four replications; ND: not detected; * =p<0.05, ** =p<0.01 Azadirachtin Content Azadirachtin content in neem seed kernels was quantitated by ELISA and its content was found to decrease in response to fungal infection. The azadirachtin content in the neem seed kernels during fungal infection at various time points was found to decrease with increase of time. Its initial concentration in neem seeds was found to be 0.19 ± 0.01 g%, which decreased as the period of infection increased from day 3 (0.14 ± 0.02 g%), to day 6 (0.08 ± 0.01 g%) and in later stages of infection no azadirachtin content was detected (day 9 and 12). The decrease in azadirachtin content was statistically significant on day 3 (P<0.05) and day 6 (P<0.01) when compared to its content on day 0 (Table 3).

8 402 INDIAN J NAT PROD RESOUR, SEPTEMBER 2012 Fig. 6 Pie chart of analysis of variance (ANOVA) of proximate composition (crude protein, fat and total sugar) and protein precipitatable polyphenol content in neem seed kernels during infestation with Aspergillus parasiticus NRRL 2999 in comparison with groundnut. A. Pie chart of analysis of variance of crude protein content in neem seed kernels during infestation with Aspergillus parasiticus NRRL 2999 in comparison with groundnut. B. Pie chart of analysis of variance of fat content in neem seed kernels during infestation with Aspergillus parasiticus NRRL 2999 in comparison with groundnut. C. Pie chart of analysis of variance of total sugar content in neem seed kernels during infestation with Aspergillus parasiticus NRRL 2999 in comparison with groundnut. D. Pie chart of analysis of variance of protein precipitable polyphenol content in neem seed kernels during infestation with Aspergillus parasiticus NRRL 2999 in comparison with groundnut. Discussion Groundnut, a high risk agricultural commodity for aflatoxin elaboration, has relative similarity in proximate composition with neem seed kernel 41,42. Further, the neem seed kernel is the most active part of the neem tree that has the maximum number of bioactive terpenoids 5. The moisture content of the natural plant substrates is an important factor that facilitates the fungal growth and aflatoxin production and it varies with the type of substrate. These values are in agreement with the previous study wherein the moisture content up to 10-15% in semi-dried and 8-9% in dried neem seeds was reported 52. In groundnuts 10-12% was found to be optimum for toxin production 27 and hence was infected at this moisture content. As 10-12% moisture content was found to be optimal for aflatoxin production in neem seed kernels also, the changes in the proximate principles during fungal infestation were carried out at this moisture level. The major target of most of the antifungal compounds was reported to be ergosterol biosynthesis 53. Megan 54, reported that there was a good correlation between the increase in the ergosterol content and fungal growth for a given species of fungi. The present study also showed a positive correlation (r = 0.953) between the ergosterol biosynthesis and aflatoxin production in neem seed kernels by A. parasiticus (NRRL 2999). Though, the ergosterol content in neem seed kernels was enhanced with increase in period of incubation it remained unchanged with augmented moisture content (Fig. 3).

9 TANUJA et al: SUITABILITY OF NEEM SEED KERNEL FOR THE GROWTH OF AFLATOXINS 403 Ergosterol content in the neem seed kernels at various moisture levels was found to be less than groundnut kernels, suggesting it to be comparatively a poor substrate for fungal growth. These results suggest that increase in moisture content perhaps facilitates increase in aflatoxin production by the fungus rather than its growth in neem seed kernels. The fungus infecting the kernels would draw its nourishment from the reserve sources like total sugar, protein and fat present in seeds. Groundnut kernel due to its close similarity in total sugar, protein and fat contents with neem seed kernel was used as a positive control in the present study. There was no statistically significant difference between the two substrates for the crude protein content but showed an increase in the protein content during the period of infection (Fig. 5A). Earlier studies also showed an increase in the protein content during infestation by A. parasiticus in groundnut kernels 55 and sorghum grains 29,56, which could be attributed to the production of non-protein nitrogen by hydrolytic enzymes. Similar observation was made in sorghum grains infected with field fungi, Curvularia lunata and Fusarium moniliforme 57. The decrease in fat content was associated with increase in toxin production in neem seed and groundnut kernels. Earlier studies on alterations in the fat content in stored groundnut seeds infected with Aspergillus species have also shown similar changes 58. During fungal infestation the total sugar content of the substrate decreases due to the utilization of carbohydrates by the fungi through amylolytic activities 57. The present study, also confer a specific correlation between decrease in total sugar content and increase in toxin production in both substrates. Similar observations were made by Ratnavati and Sashidhar 29, with respect to hydrolysis of starch in infected sorghum grains. Plants produce important compounds such as alkaloids, terpenoids, phenolics and a variety of other secondary metabolites in response to fungal infection. These substances have no known function in photosynthesis, growth or other basic aspects of plant physiology and are found to be inhibitory to the growth of insects, nematodes and plant pathogenic fungi 59. The increase in polyphenol content against fungal infection was earlier indicated as a response of plant immune system 60. Resistant cultivators of groundnut were found to respond to fungal infection via increased level of phenolic compounds 61 and pigmentation of spikelet tissues 30. However, the correlation between increase in the polyphenol content and toxin elaboration in the present study was not statistically significant in both the substrates (Table 2). In an earlier study, the free phenolic compounds significantly increased and bound phenolic compounds decreased in viable peanuts 61. The reduction in the aflatoxin biosynthesis by A. flavus due to phenolic compounds was attributed to the inhibition of the synthesis and accumulation of norsolorinic acid 62. The azadirachtin content in the neem seed kernels was found to reduce with increase at different time points of fungal infection. Szeto and Wan 63 reported slow decrease in azadirachtin content with increase of basicity. Presumably, the changes in ph of the substrate during fungal infestation may be attributed to the reduction of azadirachtin content in neem seed kernels in the present study, as the molecule is susceptible to changes in ph. Though the proximate composition of neem seed kernels was similar to that of groundnut, it was found to be relatively poor substrate for fungal growth and aflatoxin elaboration, as evidenced by drastic fall in aflatoxin production by 54% and 74% on day 9 and 12, respectively in comparison to groundnut (Fig. 1). It is pertinent to note that under controlled conditions, the neem seed kernel shows significant resistance to aflatoxin elaboration, when compared to high risk agricultural commodity like groundnut, suggesting that in a natural environment the risk of aflatoxin contamination may be far more less in neem seed kernel. In the present study on day 9 (peak point for aflatoxin elaboration) of fungal infestation the polyphenol content in the neem seed kernels increased while in groundnut it was reduced (Fig. 5D). Thus, the difference in substrate suitability can be attributed to the presence of bioactive compounds in neem seed kernel that might be involved in the inhibition of fungal growth with reduced toxin elaboration. Earlier, studies reported that fungal growth inhibits by 34.9% and 21.4% in neem leaf extract 64. Nimbidin, the bitter principle isolated from the neem seed oil was found to be antifungal. Azadirachtin from neem seeds was shown to significantly inhibit the growth of plant pathogenic fungi such as Fusarium oxysporum, Rhizoctonia solani, Alternaria solani and Sclerotinia sclerotiorum 20. Conclusion The results revealed that, neem seed kernel is a poor substrate to A. parasiticus could perhaps be attributed to the presence of both isoprenoids

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