Antifungal and prophylactic activity of pumpkin ( Cucurbita moschata ) extract against Aspergillus flavus and aflatoxin B 1 Amna

The antifungal effect of pumpkin (Cucurbita moschata) fruit aqueous extract against Aspergillus flavus was investigated in vitro. The result showed that incubation of different pumpkin aqueous extract (0.5 to 2%) with living mass of the fungus has suppressive effect on the fungal growth after 6 days compared with untreated control sample. However 2% of pumpkin aqueous extract was the most effective in inhibiting the fungal growth. The protective efficacy of pumpkin fruit aqueous extract against either A. flavus fungus infection or aflatoxin B1 (AFB1) toxicity induced renal damage in rats was also investigated. A. flavus and AFB1 were administered intraperitoneally (0.1 ml/100 g of body weight) for 15 consecutive days. The result revealed that infection of rats with A. flavus or intoxication with AFB1 significantly induced renal damage as indicated by marked increased levels of serum urea, uric acid and creatinine as well as histopathological pictures compared with normal healthy rats. Oral co-administration of aqueous extract of pumpkin fruits (1.0 mg / kg of body weight) to either rat groups infected with A. flavus or intoxicated with AFB1 for 20 consecutive days effectively normalized the serum kidney function biomarkers and confirmed by histomorphologic pictures which showed normal histological structure.


INTRODUCTION
Fungal infections are mainly caused by opportunistic fungi and are usually associated with immunosuppression (Shoham and Levitz, 2005).Aflatoxins (AFs) are highly toxic secondary fungal metabolites produced by the species of Aspergillus, especially Aspergillus flavus and Aspergillus parasiticus.These fungi can grow on a wide variety of foods and feeds under favorable temperature and humidity (Giray et al., 2007).There are four naturally occurring aflatoxins, the most toxic being aflatoxin B1 (AFB1), and three structurally similar compounds namely aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2).Aflatoxins are not only contaminant our food stuffs, but also are found in edible tissues, milk and eggs after consumption of contaminated feed by farm animals (Fink-Gremmels, 1999;Bennett and Klich, 2003;Aycicek et al., 2005).
Aflatoxins are well known to be potent mutagenic, carcinogenic, teratogenic and immunosuppressive agent; also inhibit several metabolic systems, causing liver, kidney and heart damage (Wogan, 1999;Bintvihok, 2002;Wangikar et al., 2005).These toxins have been incriminated as the cause of high mortality in livestock and some cases of death in human being (Salunkhe et al., 1987).
The mechanism of AFB1 toxic effect has been extensively studied.It has been shown that AFB1 is activated by hepatic cytochrome P450 enzyme system to produce a highly reactive intermediate, AFB1-8,9epoxide, which subsequently binds to nucleophilic sites in DNA, and the major adduct 8,9-dihydro-8-(N7guanyl)-9hydroxy-AFB1 (AFB1 N7-Gua) is formed (Sharma and Farmer, 2004).The formation of AFB1-DNA adducts is regarded as a critical step in the initiation of AFB1induced carcinogenesis (Preston and Williams, 2005).Although the mechanism underlying the toxicity of aflatoxins is not fully understood, several reports suggest that toxicity may ensue through the generation of intracellular reactive oxygen species (ROS) like superoxide anion, hydroxyl radical and hydrogen peroxide (H 2 O 2 ) during the metabolic processing of AFB1 by cytochrome P450 in the liver (Towner et al., 2003;Naaz et al., 2007;Shi et al., 2012).These species may attack soluble cell compounds as well as membranes, eventually leading to the impairment of cell functioning and cytolysis (Berg et al., 2004).
The use of synthetic drugs as antimicrobials was greatly effective, but their application has led to a number of ecological and medical problems due to residual toxicity, carcinogenicity, teratogenicity, hormonal imbalance, spermatotoxicity, etc. (Pandey, 2003).Natural products and their active principles as sources for new drug discovery and treatment of diseases have attracted attention in recent years.Herbs are generally considered safe and proved to be effective against various human ailments.Their medicinal use has been gradually increasing in developed countries.So, natural substances that can prevent AFB1 toxicity would be helpful to human and animal health with minimal cost in foods and feed.Traditional medicinal plants were applied by some authors for their antifungal, anti-aflatoxin and antioxidant activity (Joseph et al., 2005;Kumar et al., 2007).
Pumpkin , belonging to the genus of Cucurbita and family Cucurbitaceae frequently refers to any one of the species Cucurbita pepo, Cucurbita mixta, Cucurbita maxima, and Cucurbita moschata (Itis.gov., 2009).Pumpkin was reported to have medicinal properties.C moschata was reported to have antioxidant components including vitamin A, vitamin E, carotenes, xanthophylls and phenolic compounds which have the principal role in protecting against oxidative tissue damage ( Chanwitheesuk et al., 2005).
Tetrasaccharide glyceroglycolipids were obtained from Cucurbita moschata and showed significant glucoselowering effects in streptozotocin-and high-fat-dietinduced diabetes in mice (Jiang and Du, 2011).watersoluble extract, named PG105, prepared from stem parts of C moschata, contains potent anti-obesity activities in a high fat diet-induced obesity mouse model (Choi et al., 2007).The plant is also used as laxative ,and in the treatment of headaches, heart disease, high blood cholesteroland high blood pressure (AL-Sayed, 2007).
The aim of this study was to evaluate antimicrobial impact of C. moschata fruit aqueous extract against toxigenic A. flavus and also to investigate the in vivo protective effect of the extract against A. flavus infection and AFB1 toxicity induced renal dysfunction and histolopathological structural changes in rats.

Preparation of aflatoxin B1 standard (AFB1)
AFB1 was obtained from Sigma-Aldrich (St. Louis, MO, USA).A concentration of 2 mg/ml of AFB1 was prepared using dimethyl sulphoxide.AFB1 was administered to experimental animals intraperitoneally using a dosage of 0.2 mg/Kg of body weight (Ha et al., 1999) Organism under study A. flavus toxigenic strain was obtained from The MERCIN unit, College of Agriculture, Ein-Shams University-Cairo, Egypt.

Preparation of Aspergillus flavus cultures
Fifty ml of Sabourod dextrose agar were added to sterile Petri dishes and 0.1 ml spore suspension were inoculated.The inoculated media were incubated at 25 ± 2°C for 6 days.

The effect of different concentrations of pumpkin fruit aqueous extract on Aspergillus flavus bio mass
The effect of pumpkin fruit aqueous extract on the mycotic spindle formation of the experimental fungus was studied on the liquid Sabouraud Dextrose media.Exactly 1 ml of different concentrations (0.5, 1.0, 1.5 and 2.0%) of pumpkin fruit aqueous extract was added to a flask contained 50 ml of the liquid media.Control group flasks with no added pumpkin fruit aqueous extract were also prepared.All prepared media were sterilized by bacterial filtration and then fertilized with disks of A. flavus fungus with diameter of 5 mm.Cultures were filtered after incubation at 25 ± 2°C for 3 and 6.

Plant specimens
Pumpkin (Cucurbita moschata) fruits were purchased from the local market in Jeddah, Saudi Arabia during summer 2011.

Preparation of plant aqueous extract
The fruit extract was prepared according to the method of Xia and Wang, (2006b).One hundred grams of dried fruits were mixed with 1000 ml of distilled water and the mixture was boiled at 100°C under reflux for 30 min.The decoction obtained was centrifuged, filtered, frozen at -20°C, and then lyophilized.The lyophilized product of plant extract obtained was either dissolved in water before oral administration to rats or sterilized by bacterial filter for the antifungal studies.

Animal and treatments
Animal experiment was performed in accordance with legal ethical guidelines of the Medical Ethical Committee of King Abdelaziz University, Jeddah, KSA.Sixty healthy female albino rats (150 to AFB1 was dissolved in dimethyl sulphoxide and administered intraperitoneally (0.1 mg/100 g body weight) to the rats of AFB1 intoxicated groups.A. flavus spore suspension was injected intraperitoneally (0.2 mg/Kg) to A. flavus rat infected groups.AFB1 and A. flavus fungus were administered to rats for 15 consecutive days.pumpkin fruit aqueous extract was administered orally (1.0 mg / kg of body weight) to either rat groups treated with AFB1 or infected with A. flavus fungus for 20 consecutive days.
After the experimental period (20 days), the blood samples were collected from each animal in all groups into sterilized tubes for serum separation.Serum was separated by centrifugation at 3000 rpm for 10 min and used for biochemical serum analysis of kidney function.After blood collection, rats of each group were sacrificed under ether anesthesia and the kidney samples were collected for histopathological examination.

Histopathological examination
A small pieces of kidneys were fixed by 10% formalin and then embedded into paraffin, sectioned for 5 to 6 μm thick, and mounted on the glass microscope slides using standard histopathological techniques.The sections were stained with hematoxylin-eosin and examined by light microscopy.

Statistical analysis
Data were analyzed by comparing values for different treatment groups with the values for individual controls.Results are expressed as mean ± S.D. The significant differences among values were analyzed using analysis of variance (one-way ANOVA) followed by Bonferroni as a post-ANOVA test.Results were considered significant at P < 0.05 (Hill, 1971).

RESULTS
The effect of different concentrations of pumpkin fruit aqueous extract on the vitality of bio mass of A. flavus fungus was illustrated in Table 1.The result showed that incubation of different concentrations of pumpkin aqueous extract (0.5 to 2%) with living mass of the fungus has suppressive effect on the fungus growth after 6 days compared with untreated control sample.2% of the plant extract was the most effective in inhibiting the fungal growth.
The protective role of pumpkin fruit aqueous extract on kidney serum function biomarkers against A. flavus infection and AFB1 toxicity in rats is depicted in Table 2.The result showed that infection of rats with A. flavus (G3) or treated with AFB1 (G5) for 15 consecutive days, dramatically causes renal tissue damage as indicated by marked increases in serum urea, uric acid and creatinine compared with normal rats (P ≤ 0.001).Oral coadministration of pumpkin fruit aqueous extract to either groups (G3 or G5), effectively protected the kidney from the damaging effect of either A. flavus (G4) or AFB1 (G6) as documented by the normalization of the studied kidney function biomarkers.Histopathological observation supported the biochemical result.kidney section of rat infected with A. flavus indicatedits damaging deleterious impact on rat kidney as observed by the damaged architecture of proximal tubular epithelia, such as cell swelling and lysis, cytoplasm vacuolation, nuclear membrane breakdown, cell shrinkage, nuclear condensation, and necrosis of some glomeruli and tubules (Figure 1c).Co-administration of pumpkin fruit aqueous extract to rats infected with A. flavus showed more or less normal histological structure of kidney (Figure 1d).Injection of rats with AFB1 showed sever damage to histomorphological picture of rat kidneys (Figure 1e and f) as observed by marked dilatation and congestion of renal blood vessel (Figure 1e), degeneration of most renal tubules, swelling and lysis of cells, cytoplasm vacuolation and nuclear pyknosis.Coadministration of pumpkin fruit aqueous extract to AFB1 intoxicated rats (Figure 1g) showed more or less normal histological structure of kidney.Administration of pumpkin fruit aqueous extract to normal rats showed no side effects on rat kidneys as indicated by normal kidney function and supported by histomorphologic picture of rat kidneys which showed normal histological structure

DISCUSSION
Plants have been used in medicine for a long period of time, since they are easy to obtain and used in treatment of various diseases (Sardi et al., 2011).It contains phosphorus, iron and zinc, vitamin E and vitamin B complex, very poor in sodium (Shaaban, 2005).
Regarding the search for new antifungal ideal agent must have a broad spectrum of fungicidal activity without causing toxicity to the host (Carrillo-Mu˜noz et al., 2006).The treatment of fungal infections is not always effective because of resistance to drugs in addition to presenting high toxicity for human cells.For this reason, there is a continuing search for new drugs which are more potent antifungal, but safer, than existing drugs (Fenner et al., 2006).
The antifungal activity of aqueous extract of pumpkin fruits against A. flavus fungus was studied.The result revealed that different concentrations of pumpkin aqueous extract (0.5 to 2%) showed inhibitory beneficial effect on the growth of fungus living mass after 6 days compared with untreated control.2% of the plant extract was the most effective one in inhibiting the fungal growth.This is consistent with the results of Saddiq, (2010) who reported high ability of the pumpkin fruits and seeds alcohol extract to inhibit the growth of pathogenic bacteria Staphylococcus aureus and Escherichia coli as well as against the toxigenic fungus A. flavus.Also this result is supported by previous study by Wang and Ng. (2003) who isolated antifungal peptide from C. moschata seeds namely cucurmoschin which has abundant arginine, glutamate and glycine residues.Cucurmoschin inhibited mycelial growth in the fungi Botrytis cinerea, Fusarium oxysporum and Mycosphaerella oxysporum.The authors also stated that the cucurmoschin showed a translationinhibitory activity (IC50 = 1.2 µM) which was more effective than some of the antifungal proteins (Lam et al., 2000, Wang and Ng, 2000, 2002) and the antifungal peptides from red bean, pinto bean (Ye and Ng, 2001) and chickpea (Ye et al., 2002).Ribosome-inactivating proteins inhibit translation in rabbit reticulocyte lysate with a much higher potency (IC 50 in pM concentration) (Barbieri et al., 1993) and they also inactivate fungal ribosomes (Roberts and Selitrennikoff, 1986).
The protective role of aqueous extract of pumpkin fruits on kidney serum function biomarkers against A. flavus infection and AFB1 toxicity in rats was investigated.The result showed that either infection of rats with A. flavus or intoxication with AFB1 dramatically induce nephrotoxicity in rats, as demonstrated by the significantly increased levels of serum urea, uric acid and creatinine.The alteration in these kidney function biomarkers was more evidence in rats intoxicated with AFB1.The reno-toxic effect of A. flavus or AFB1 was further confirmed by the severely destructed renal tissue, as shown in the histological analysis.Histomorphological picture of rat kidneys infected with A. flavus showed damaged of renal proximal tubular epithelia, such as cell swelling and lysis, cytoplasm vacuolation, nuclear membrane breakdown, cell shrinkage, nuclear condensation, and necrosis of some glomeruli and tubules.Injections of rats with AFB1 showed sever damage to histomorphological picture of rat kidney indicated by vacuolar degeneration and necrosis of the renal tubular cells.Our results are similar to other studies indicated impairment of kidney functionsand abnormal pathological changes with severe inflammatory response of kidney in animals intoxicated with AFB1 (Valdivia et al., 2001;Salim et al., 2011).Although the mechanism underlying the toxicity of aflatoxins is not fully understood, several reports suggest that toxicity may ensue through the generation of intracellular reactive oxygen species (ROS) like superoxide anion, hydroxyl radical and hydrogen peroxide (H 2 O 2 ) during the metabolic processing of AFB1 by cytochrome P450 in the liver (Towner et al., 2003 ).These species may attack soluble cell compounds as well as membranes, eventually leading to the impairment of cell functioning and cytolysis (Berg et al., 2004).Souza et al. (1999) reported that the oxidative stress is the principle manifestations of AFB1-induced toxicity which could be mitigated by antioxidants.Administration of aqueous extract of pumpkin fruits to rats infected with A. flavus or intoxicated with AFB1 markedly protected the kidney from the damaging deleterious impact on kidney tissue indicated by normalization of kidney function biomarkers in rats administered A. flavus or AFB1 simultaneously with the used plant extract.The beneficial effect of this extract on kidney function biomarkers was supported by histomorphologic picture of kidney which showed more or less normal histological structure.The ameliorative effect of the used plant extract on kidney dysfunction and its histopathological pictures induced by fungal infection or AFB1 may indicate to its antimicrobial effect or antioxidant potential action.Pumpkin was reported to have antioxidant components including vitamin vitamin E, carotenes, xanthophylls and phenolic compounds which have the principle role in protecting against oxidative tissue damage ( Chanwitheesuk et al., 2005;Saddiq, 2010).
Evaluation of the adverse effects of the natural products, accepted as remedies, is important in implementing safety measures for public health.The present work proved that the administered dose of C. moschata fruit aqueous extract (1.0 mg / kg of body weight) to normal healthy rats has no adverse effects which was indicated by the normal levels of the serum renal tested function in comparison to normal untreated rats.This result was confirmed by normal renal histomorphological picture.
In conclusion, aqueous extract of pumpkin fruits has a protective role against A. flavus or AFB1-induced renal damage which may be related to the antioxidant constituents of the plant extract.

hhggffgFig 1 .Figure 1 .
Fig 1.Sections of rat kidneys of different experimental groups, (a) kidney of normal healthy rats showing normal histological structure of renal parenchyma, (b) kidney section of rat ingested with pumpkin fruit aqueous extract showing normal histological structure of renal parenchyma, (c) kidney section of rat infected with A. flavus showing various degrees of damage to the architecture of proximal tubular epithelia, such as cell swelling and lysis, cytoplasm vacuolation (arrow head), nuclear membrane breakdown, cell shrinkage, nuclear condensation, and necrosis of some glomeruli and tubules (thin arrows), (d) section of rat kidney infected with A. flavus and co-administered with pumpkin fruit aqueous Figure 1.Sections of rat kidneys of different experimental groups, (a) kidney of normal healthy rats showing normal histological structure of renal parenchyma, (b) kidney section of rat ingested with pumpkin fruit aqueous extract showing normal histological structure of renal parenchyma, (c) kidney section of rat infected with A. flavus showing various degrees of damage to the architecture of proximal tubular epithelia, such as cell swelling and lysis, cytoplasm vacuolation (arrow head), nuclear membrane breakdown, cell shrinkage, nuclear condensation, and necrosis of some glomeruli and (thin arrows), (d) section of rat kidney infected with A. flavus and co-administered with pumpkin fruit aqueous extract showing more or less normal histological structure of kidney, (e and f) sections of rat kidney intoxicated with AFB1 showing severe damage to the renal architecture such as marked dilatation and congestion of renal blood vessel (e, arrow), degeneration of most renal tubules (arrow heads), swelling and lysis of cells, cytoplasm vacuolation (stars) and nuclear pyknosis (f, arrows), (g) section of rat kidney intoxicated with AFB1 and co-administered with pumpkin fruit aqueous extract showing more or less normal histological structure of kidney.

Table 1 .
Effect of various concentrations of pumpkin fruit aqueous extract on bio mass of Aspergillus flavus grown on liquid media after 6 days (mg ± SE).
light/dark cycle.The animals were provided with commercial rat pellet diet and deionized water ad libitum.After one week acclimation, the rats were randomly divided into 6 groups (each of 10 rats) as follow: G1: Normal healthy rats, G2: Normal rats ingested with pumpkin fruit aqueous extract, G3: Rats infected with A. flavus, G4: Rats infected with A. flavus and co-administered with pumpkin fruit aqueous extract, G5: Rats intoxicated with AFB1, G6: Rats intoxicated with AFB1 and co-administered with pumpkin fruit aqueous extract.

Table 2 .
Levels of serum kidney function biomarkers (mean ± SE) in different experimental animals.
b Different letters in the same column are significantly different P ≤ 0.001.