herbal product antioxidant for mycotoxins

Abstract: The object of this study was to conduct the ability of two medicinal herbs, namely rosemary and parsley, for amelioration of aflatoxicosis in Oreochromis niloticus. Two herbs' extracts at three concentrations of either (0, 2 and 4 g kg^-1 B.W. divided into 2 doses at the start and the 6^th day of the experiment) and three concentrations of aflatoxin B₁, (AFB₁ 0, 9 and 18 mg kg^-1 B.W. as a single intraperitoneal administration) were tested either individually or in combination. The herbs and AFB₁ were dissolved in Dimethylsulphoxide (DMSO 25%) and injected to fish groups. Sixteen groups of fish were investigated in this study, where A group (control) was injected with saline 0.89%, group B injected with DMSO (control solvent), groups F₁ and F₂ were injected with AFB₁ alone (9 and 18 mg kg^-1 B.W. respectively), R₁ and R₂ groups were injected with rosemary alone (2 and 4 g kg^-1 B.W., respectively), groups F₁R₁, F₁R₂, F₂R₁ and F₂R₂ were injected with AFB₁ + rosemary, while groups P₁ and P₂ were injected with parsley alone (2 and 4 g kg^-1 B.W., respectively); however, F₁P₁, F₁P₂, F₂P₁ and F₂P₂ groups were injected with AFB₁ + parsley. At the 12^th day of the experiment, blood and liver samples were taken from each group. The results indicated that the AFB₁ injected groups revealed a significant increase in mortality rate (MR%) compared with AFB₁-not injected, group F₂ was the highest while F₁R₁ and F₁P₁ were the lowest in MR% among all AFB₁ injected fish groups. Also, AFB₁ led to reduction of haemoglobin (Hb), total protein (TP) and globulin (Gl) concentrations and increase in activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). These alterations were significantly ameliorated when fish were injected with herbs' extracts. AFB₁ residues showed that the herbs level of 2g kg^-1 B.W. have higher potency of reducing the AFB₁ residues than the level of 4 g kg^-1 B.W. in case of AFB₁ level 9 mg kg^-1 B.W. While, in case of AFB₁ level 18 mg kg^-1 B.W., the groups F₂ and F₂P₁ showed absence of AFB₁ residues.Microscopically, AFB₁ presented histopathological changes in hepatopancrease which increased in severity with increasing AFB₁ level. These lesions may become less severer in all fish groups injected with AFB₁ combined with herbs' extracts especially with the lowest levels of herbs' extracts and AFB₁. So, this study concluded that either of rosemary or parsley was found to be safe and successful in protection from aflatoxicosis, particulary at the low level.

Key words: Tilapia - Aflatoxicosis - Parsley - Rosemary- Residues.

Aflatoxins are secondary metabolites produced by the ubiquitous fungi Aspergillus flavus and A. parasiticus. Aflatoxin B₁ (AFB₁) has the highest potency as a toxin and is classified as group 1 carcinogen by International Agency for Research on Cancer (IARC, 1993).Aflatoxin has to be activated in order to exert its carcinogenic effect. Also, the free radical and reactive oxygen species (ROS) may, in part, be responsible for the carcinogenic activity of AFB₁(Shen et al., 1996). So, inhibition of cytochromes (CYP₄₅₀) and/or stimulation of the antioxidant defence system, α- tocopherol, ascorbate and reduced glutathione (GSH), may reduce the risk of AFB-mediated carcinogenesis.

Previous studies referred to many medicinal herbs that serve as sources of antioxidant which have antiaflatoxigenic effects such as Thonningia sanguinea (Gyamfi and Ariya, 1998 and Gyamfiet al.,1999), Cymbopogon citratus Staf and Murdannia ioriformis (Vinitketkumnuenet al.,1999), Oldenlandia diffusa and Seutellaria babata (Wong et al., 1993), as well as Ocimum sanctum (Rastogiet al.,2007).

Corresponding Author: Dr. Manal I. El-Barbary, Aquatic Pathology Lab., National Institute of Oceanography and Fisheries, Egypt.                      E.mail: manal278@yahoo.com

While, parsley (Petroselinum crispum) and rosemary (Rosmarinus officinalis) are medicinal herbs which are widely used around the world that have shown a good antioxidant activity (Lampe et al., 2000; Hinneburg et al., 2006; Sacan Ozsoy et al., 2006 and Caillet et al., 2007). As well as, Lampe et al. (2000) reported that parsley has been shown to inhibit CYP_1A2 in human and it was suggested that the inhibition was possibly related to its phytochemical content. Natural polyphenols found in rosemary have not only potent antioxidant activities but also anticarcinogenic properties. Rosemary components inhibit both the initiation and tumour promotion stages of carcinogenesis in mouse and rat models (Tokuda et al., 1986; Singletary and Nelshoppen, 1991; Yasukawa et al., 1991 and Huang et al., 1994). Also, Offord et al. (1997) reported that rosemary extract strongly inhibits metabolic activation of two important human procarcinogens, AFB₁ and benzo (a) pyrene. Additionally, the medicinal properties of parsley are stimulant, diuretic, carminative, emmenagogue, antipyretic and anti-inflammatory, while the medicinal properties of rosemary are mild irritant, carminative, stimulant and diaphoretic (Peter, 2001). The last author reported that the lethal dose (LD₅₀) of rosemary and parsley essential oils determined in rats are > 5 and 1-5g/kg B.W., respectively. The nutritive values of rosemary and parsley were reported by Farrel (1990), it was higher in parsley than in rosemary as shown from the following:

 

The aim of this study is to investigate the effects of rosemary and parsley at two levels (2 and 4g kg^-1 B.W.) on aflatoxicosis B₁ by fish, Oreochromis niloticus at 0.25 and 0.5 of the LD₅₀ of AFB₁.

Aflatoxin B₁ was produced on liquid medium (Potato dextrose) by Aspergillus parasiticus (NRRL. 2999) according to Ready et al. (1971). Aflatoxin B₁ was dissolved in chloroform and quantitatively estimated by thin layer chromatography, TLC (AOAC, 2000). So, chloroform was evaporated to dryness on a rotary vacuum evaporator at 40ºC and redissolved in Dimethylsulfoxide (DMSO) 25% (1:3 water) to the requirement of each aflatoxin concentration. AFB₁ was freshly dissolved in DMSO before injection.

Fresh rosemary and parsley leaves were obtained from a local farm and carefully washed with tap water then left to dry in the dark at room temperature. Twenty gram of the ground leaves were extracted for 24 h by soaked in 500 ml of methanol (70%). The extract was then filtered and the filtrate was divided into two amounts (one part and its double) before evaporating them till dryness in a rotary evaporator (45ºC). The residues of the two amounts were dissolved in constant volume of 25% DMSO to obtain the two concentrations of herbs extract. The dose levels of 0, 2 and 4 g kg^-1 B.W. were divided into double dose, the first was injected at the start of the experimental period and the second dose was injected one week later.

Two hundred and eighty eight fingerlings of O. niloticus were obtained from El-Serw fish farm, where this study was carried out in summer season 2007. The fish were acclimated to aquaria conditions for two weeks before the experiment was initiated. Six fish (approximately the same size, 20g average) were stocked into each of the 48 aquaria which contained 50 l of water, three glass aquaria (70X40X30 cm) for each treatment, the aquaria were provided with continuous aeration and their water was changed partially daily and totally weekly. All fish were received diet twice daily at a daily feeding rate of 3% of the actual body weight, six days weekly for two weeks. Fish were divided into 16 groups and were administered the test compounds interperitoneally (I.P.). Their effects were studied at the end of the 2^nd week. The experimental setup used is shown in Table (1). AFB₁ was tested at three levels (0, 9 and 18 mg kg^-1 B.W.) being 0, 0.25 and 0.50 the LD₅₀, respectively according to El-Barbary (2008) in a single dose, while both of rosemary and parsley extracts were used at three levels (being 0, 2 and 4 g kg^-1 B.W.) divided into 2 doses (pretreatment at the start of the experiment and one week later). AFB₁ and herbs' extracts were mixed together directly before administration.

 

At the end of the 2^nd week of the experiment, blood samples were withdrawn from the fish heart of each group to determinate some blood parameters using commercial colorimetric kits (Diamond, Diagnostic, Egypt), and the obtained data were statistically analyzed by one way analysis of variance using a software (SAS, 1996). Three fish from each group were homogenized and preparated to determinate the residues of AFB₁ in fish by TLC (AOAC, 2000). The histological examination of the fish livers were performed after the preparation of livers which were dissected out from each group and fixed in 10% neutralized formalin solution until use according to the technique of Roberts (2001).

The mortality rate caused by interperitonal injection with AFB₁ alone or in combination with rosemary or parsley extracts was the highest in AFB₁ injected fish as shown in Table (2). The MR% gradually increased by increasing the AFB₁ level in all AFB₁ groups, while this increase in MR% was significantly reduced by using the herbs' extracts against AFB₁. The positive effects of the herbs' extracts on MR% were observed. The level of both extracts 2g kg^-1 B.W. reflected the most significant decrease in MR% with the two levels of AFB₁ comparing with the level 4g kg^-1 B.W. The reduction in MR% ranged from 37.39, 33.30, 37.39 to 42.90% in groups F₁R₁, F₂R₁, F₁P₁ and F₂P₁, respectively as compared with the AFB₁ groups (F₁ and F₂). While, this reduction in MR% ranged from 12.60 to 16.60% in groups F₁R₂ and F₂R₁, respectively and 0 to 33.30% in F₁P₂ and F₂P₂ as compared to F₁ and F₂ groups, respectively.

 

These results agree with the results of AFB₁-residues which confirmed that the level of both herbs' extracts 2g kg^-1 B.W. was more effective in reducing the AFB₁-residues than the level 4g kg^-1 B.W. especially with the AFB₁ level 9 mg kg^-1 B.W.

The significant effect of AFB₁ on MR% was confirmed in previous studies with Nile tilapia (Marzouk et al., 1994; Hussein et al., 2000; Abdelhamid et al., 2002 a; Tuan et al., 2002 and El-Barbary and El-Shaieb, 2006). The positive effect of parsley could be attributed to its medicinal property as anti-inflammation (Peter, 2001). Also, both of rosemary and parsley have antioxidant properties due to polyphenolic compounds  including ferulic acid and syringic acid that are the major phenolic compounds in parsley (El-Barbary, 2008). Ferulic acid exhibit a wide range of pharmacological effects including antiageing, anti-inflammatory, anticancer, antiapoptotic, antidiabetic and neuroprotective (Srinivasan et al., 2006).

                The residual analysis of AFB₁ in the whole body of fish which were injected with 9 mg AFB₁ kg^-1 B.W. with or without herbs' extracts (F₁, F₁P₂, F₁R₂, F₁P₁ and F₁R₁) (Fig. 1) showed that all these groups revealed the presence of AFB₁ residues (1.6 to 7.5 ppb) which consider less than the permissible limit (20 ppb) that was recommended by WHO (Diener et al., 1985). In fish groups injected with AFB₁ with herbs' extracts, traces of AFB₁ were detected in their body at concentration ranged from 1.6 to 3.1 ppb.

Fig. (1): AFB₁ residues in the experimented fish injected with the low dose of AFB₁.

 

Fig. (2) illustrates AFB₁ residues of the fish groups injected with the level of AFB₁ 18 mg kg^-1 B.W. with or without herbs' extracts (F₂, F₂P₁, F₂R₁, F₂P₂ and F₂R₂) which, showed contrast results to those in Fig. (1), since there were no-residues of AFB₁ in either F₂ (18 mg AFB₁ kg^-1 B.W.) or F₂P₁ (18 mg AFB₁ + 2g parsley kg^-1 B.W.). Whereas AFB₁-residues ranged from 2.1, 9.6 to 23.0 ppb in F₂R₁, F₂P₂ and F₂R₂, respectively.

Fig. (2): AFB₁ residues in the experimented fish injected with the high dose of AFB₁.

 

These results indicate that the herbs' extracts have potency for reducing the AFB₁ residues, particulary in cases of the low levels of AFB₁ and the herbs' extracts. Also, parsley could have higher potency than rosemary.

The absence of AFB₁ residues in F₂ group (18 mg AFB₁ kg^-1 B.W.) could be attributed to the high level of AFB₁ which acts as an acute dose, so its metabolism may be rapid and severely converted into other metabolites which could have more toxicity than AFB₁. The negative results of both MR% and histopathological examination in F₂ group may confirm that. On the other hand, the low level of AFB₁ acts as subacute dose; so, its metabolism may be gradual and slight so its MR% and histopathological manifestation had better results comparing with F₂ group. The mechanism of parsley and rosemary extract may be due to inhibition of the metabolic activation through inhibition of cytochrome P₄₅₀ enzymes, and also through inducation of the detoxifying enzyme glutathione S-transferase.

In this respect, similar results were recorded by Soliman et al. (1998 and 2000). Yet, Abdelhamid et al. (2004 b and 2007) recorded high level of AFB₁ in whole body of Nile tilapia fish. On the other hand, Abdelhamid et al. (2002 a, 2002 b and 2004 a) reported that there were no AFB₁ residues in O. niloticus body. These variable results may be due to AFB₁ level and exposure time as well as to sensitivity variation among fish species to AFB₁. Recently, Oliveira and Furlong (2008) reported that phenolic extracts of different edible plants have antifungal and antimycotoxigenic activity.

There were significant reduction in Hb, TP and Gl levels and increase in the albumin (Al) concentration and activity of hepatic transaminase enzymes (AST and ALT) at all AFB₁ groups, whether injected with AFB₁ alone or AFB₁ with herbs' extracts comparing to the control group (A). Table (3) indicates that the control solvent (B) showed approximately similar values of most blood parameters when compared to the control (A).That indicates the absence of toxicity due to DMSO administration. This alteration in haematology and biochemistry of blood was gradually increased by increasing the level of AFB₁. On contrary, these negative alterations due to AFB₁ were significantly improved by using rosemary and parsley extracts, particulary at the low level of AFB₁.

 

However, these improved values remained lower than the control values. Also, the study indicates that R₁ and P₁ showed significant alterations in some of the studied blood parameters when compared to the control. So, the positive effects of the two levels of these extracts were clearly observed with the low level than the high level of AFB₁.While, the high level of rosemary and parsley was better with the high level of AFB₁.These alterations in blood parameters among fish groups may be due to the alterations in histological structure of livers of AFB₁-injected fish leading to inhibition of blood synthesis, where liver plays an important role in this process. Similarly, Abdelhamid et al. (2002 a) and El-Barbary and El-Shaieb (2006) reported that AFB₁ reduced TP, Al and Gl of O. niloticus. In the same trend, Abdelhamid et al. (2007) found that AFB₁ caused significant decrease in TP, Al and Gl of aflatoxicated O. niloticus fish.

This reduction in TP levels may be due to the hepatotoxic effect of AFB₁. Whereas the reduced Gl levels in AFB₁ injected fish may have been the result of lymphocytolysis (Sahoo et al., 1998). While Youssef and Ashry (1999) attributed the increase in activity of AST and ALT enzymes to the hepatotoxic effect of AFB₁ and consequently hepatic cell damage and liver dysfunction.

The present study revealed that the toxicity sings began to exist together with mortalities. The most common clinical sings observed were lethargy, loss of appetite, sluggish movement, dark discoloration of the skin and respiratory manifestations. Macroscopically, the common lesions in all necropsied aflatoxicated fish were accumulation of fluids in abdomen, congested gills and dark liver. These symptoms were varied according to the treatment. The photomicrograph of liver showing microscopically the parenchemal architecture of hepatocytes in the control group (A) with central nuclei (Fig. 3A). Fish of the solvent control group (B) presented normal structure of the liver (Fig. 3B). Also, no clear histological changes were observed in any of fish groups which injected with herbs' extracts alone at the different levels (R₁,R₂,P₁,P₂). In contrast, fish injected with AFB₁ presented pathological changes in hepatopancreas which increased in severity with increasing the level (groups F₁, F₂ at 9 and 18 mg kg^-1 B.W respectively). These changes in F₁ group were severe hemolysis in the portal blood vessels (PBV) and presence melanomacrophages (MMC) (Fig. 3C). In addition, lysis of hepatocyte membranes (necrotic cells) besides dilation and congestion in blood sinusoid were noticed (Fig. 3D); while the lesions among F₂ were severe hemolysis, dilation in the portal blood vessels and large area of degenerated hepatocytes (Fig. 3E). Also, liver shows diffuse vaculation and necrosis of hepatocytes and nuclei displaced to the cell periphery; in addition to, congestion and dilation in blood sinusoid (Fig. 3F), thrombosis formation in blood vessels (Fig. 4G) and accumulation of MMC and hemosiderin besides coagulative necrosis in hepatocytes were recorded too (Fig. 4H). These severe pathological alterations in hepatopancrease caused by AFB₁ alone became less severer when these fish were injected with AFB₁ plus either rosemary or parsley at the different levels. The effects of the low level of rosemary on the two levels of AFB₁ were observed in Figs. 4 I and J; whereas, group F₁R₁ showed slight degeneration of hepatocytes (Fig. 4I), while the lesions in group F₂R₁ were necrosis in hepatocytes besides congestion of some pancreatic acini (Fig. 4J). The effects of the high level of rosemary  with AFB₁ were observed, where the hepatocytes showed condensed cytoplasms and loss contact between hepatocytes (Figs.4 K & L), in addition to presence of few numbers of inflammatory cells (Fig. 4L), besides mild congestion and hemolysis in blood vessels and necrosis of hepatocytes (Fig. 4K).Concerning the effect of the low level of parsley with different levels of AFB₁ ,it may have a positive effect in amelioration the toxicity of AFB₁, since the liver of group (F₁P₁)  showed normal  structure of hepatocytes with slight hemolysis of hepatic acini and presence of few numbers of MMC (Fig. 5M).Similar findings were observed in group F₂P₁ but with diffuse of hemosiderin and MMC (Fig. 5N).The effect of the high level of parsley against AFB₁ was presented in groups F₁P₂ and F₂P₂, since the liver of group F₁P₂ showed normal hepatocytes (Fig. 5O).While group F₂P₂ showed slight hemolysis and presence of few inflammatory cells (Fig. 5P). Similar hepatic lesions were reported by El-Banna et al. (1992), Hussein et al. (2000) and Abdelhamid et al. (2002 a) who described congestion, vacuolar degeneration of hepatocytes and activation of melanomacrophages in aflatoxicated tilapia. Also, El-Barbary and El-Shaieb (2006)reported that the liver of aflatoxicated fish showed severe vacuolation of hepatocytes of mainly fatty changes besides focal coagulative necrosis, focal replacements of the hepatic parenchyma with extravagated blood, and severe congestion and hemorrhage. In the same trend, also Mehrim et al., (2006) emphasized similar clinical sings and histopathological lesions in the liver of aflatoxicated Nile tilapia.

The positive effects of both of the parsley and rosemary on overcoming the toxic effects of AFB₁ could be attributed to the antioxidative and nutritive properties of these herbs. These results showed that the ability of parsley extract to counteract the toxic effects of AFB₁ on the fish could be better than rosemary extract. That may be attributed to the high nutritive value of parsley that included too high percent of vitamins (A, C, riboflavin and niacin) and minerals (Fe, Mg, P, K, Ca, Na and Zn) compared to rosemary. Vitamin C is related to the immunological system performance, and has antioxidant properties. This antioxidant activity of Vit. C makes it as a hunter of free radicals, thus preventing the autointoxication of immunological cells, such as macrophages which are the first processors of the information about the alien bodies and maximizing the defense of fish (Brake, 1997). Also, metal ions such as Se, Zn, Cu, Mn and Fe are essential for most organisms. Essential trace elements are important parts of antioxidant enzymes as superoxide dismutase and glutathione peroxidase and may affect the antioxidant defense system (Hung et al., 2007).

Fig. 3: Histopathological changes in liver of O niloticus injected with different levels of AFB₁ as compared to control (stained with H&E). (A&B): The control and control solvent fish groups showing normal structure of the liver (X600&250, respectively). (C&D); fish injected with AFB₁ (9 mg kg^-1 B.W., F₁ group) showing hemolysis and dilation in pbv (C, X200) besides congestion and dilation in sinusoid with necrosis (D, X600). (E&F); fish injected with  AFB_I (18 mg kg^-1 B.W., F₂ group) showing dilation and hemolysis in pbv, degeneration in hepatocytes (E, X300), vaculation and necrosis in hepatocytes and congestion with dilation in sinusoid (F, X450). h; hepatocyte. s; sinusoid. pbv;  portal blood vessel.

 

 

Fig. 4: Histopathological changes in liver of O niloticus injected with AFB₁ with or without rosemary at the different levels (stained with H&E). (G, H); fish injected with (18 mg kg^-1 B.W., F₂ group) showing thrombosis formation in bv (G, X250), coagulative necrosis, besides diffusion of MMC and hemosiderin (H, X250). (I); fish injected with AFB₁ + rosemary (9 mg and 2g kg^-1 B.W., F₁R₁ group) showing slight necrosis (X250). (J); fish injected with AFB₁ + rosemary (18 mg and 2g kg^-1 B.W., F₂R₁ group) showing congestion and necrosis in pi (X400). (K); fish injected with AFB₁ + rosemary (9 mg and 4g kg^-1 B.W., F₁R₂ group) showing necrosis and congestion (X400). (L); fish injected with AFB₁ + rosemary (18 mg and 4g kg^-1 B.W., F₂R₂ group) showing necrosis and congestion (X400). bv; blood vessel. pi; pancreatic acini.

 

 

Fig. 5: Histopathological changes in liver of O niloticus injected with AFB₁ + parsley at the different levels (stained with H&E). (M); fish injected with AFB₁+ parsley (9 mg and 2g kg^-1 B.W., F₁P₁ group) showing hemolysis in pi and presence of MMC (X600). (N); fish injected with AFB₁+ parsley (18 mg and 2g kg^-1 B.W., F₂P₁ group) showing diffusion of MMC and hemosiderin (X600).(O); fish injected with AFB₁+ parsley (9 mg and 4g kg^-1 B.W., F₁P₂ group) showing normal structure (X250). (P); fish injected with AFB₁+ parsley (18 mg and 4g kg^-1 B.W., F₂P₂ group) showing slight hemolysis and infiltration (X400).

From previous results, it could be recommended the useful using of both of medicinal herbs, namely rosemary and parsley, at low level (2 g kg^-1 B.W.) to eliminate the drastic effects of aflatoxin B₁ on O. niloticus. Yet, it must be exist more scientific efforts to use the medicinal herbs and other natural materials against the contamination with the mycotoxins. But, it will be still the prevention from toxic effects of aflatoxin B₁ and other toxins are more useful usually.

The authors would like to thank Dr. Abdelhamid M. Abdelhamid, Prof. of Animal Nutrition, Fac., of Agriculture, Mansoura Univ., Egypt for his critical reading of the manuscript and generous assistance.     

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