Detoxifying effects of a commercial additive and Phyllanthus amarus extract in pigs fed fumonisins contaminated feed

Nguyen Hieu Phuong, Brian Ogle^*, Hans Petterson^* and Nguyen Quang Thieu

Faculty of Animal Science and Veterinary Medicine

Nong Lam University, Ho Chi Minh City, Vietnam

phuongnguyen180984@yahoo.com

^*Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences,

PO Box 7024, S-750 07, Uppsala, Sweden

Abstract

The effects of a Phyllanthus amarus extract and a commercial detoxifying additive product were evaluated in protecting pigs from fumonisins with respect to growth performance, pathology and blood biochemistry. Forty eight crossbred (Landrace × Yorkshire × Duroc) weanling pigs were randomly assigned in a completely randomized design (CRD) to six diets containing: 1) low fumonisin B₁ and no feed additive (LFNA); 2) low fumonisin B₁ and commercial detoxicant additive at 1g/kg of feed (LFCA); 3) low fumonisin B₁ and Phyllanthus amarus extract at 10g/kg of feed (LFPE); 4) high fumonisin B₁ and no feed additive (HFNA); 5) high fumonisin B₁ and commercial detoxicant additive at 1g/kg of feed (HFCA); 6) high fumonisin B₁ and Phyllanthus amarus extract at 10g/kg of feed (HFPE).

Fumonisin levels, detoxicants and their combination did not have any effect on final weight, average daily weight gain, average daily feed intake, and feed conversion ratio in pigs. Including 10 mg fumonisin B₁ in the diet decreased the total cholesterol significantly compared with the low fumonisin groups (2.19 mmol/L < 2.42 mmol/L) (P<0.05). The aspartate aminotransferase (AST) blood levels of pigs given the commercial additive were higher than in the no feed additive group (110 U/L > 83.1 U/L, P=0.067) and the Phyllanthus amarus group also had a high AST blood level (99.6 U/L). Moreover, fumonisin B₁ thickened the alveolar walls of the lungs, while the commercial and Phyllanthus amarus additives partly reduce the thickened alveolar wall lesions. Liver cells also had more severe fatty degeneration and necrosis in the fumonisin and no additive group than in the commercial and P. amarus groups. However P. amarus extract made the liver tender.  

Key words: Fumonisin B1, blood biochemistry, aspartate aminotransferase, AST, lung, liver

Introduction

Fumonisin is one of the greatest potential mycotoxin risks to human and animal health, as a food and feed contaminant, along with aflatoxins, trichothecenes, zearalenone, ochratoxin A and ergot alkaloids (CAST 2003). There are four main types of fumonisins, B₁, B₂, A₁, A₂ and among these fumonisin B₁ is produced in the largest amount and has highest toxicity. These toxins are produced by Fusarium verticillioides (previously known as F. moniliforme) (Gelderblom et al 1988). Fumonisins cause equine leukoencephalomalacia and pulmonary edema syndrome in pigs and horses (CAST 2003). Raviprakash et al. (1997) revealed that fumonisin B₁ affected kidney weight and induced apoptosis in mouse liver. It is also reported that fumonisins could cause hepatocarcinoma in rats (Gelderblom et al 2001). Fumonisins elevated some serum enzymes, such as AST, ALT, ALP and CREA (Creatinine) and caused the abnormality of liver histophathology (Zomborszky et al 2002; Piva et al 2005). Galvano et al. (2001) indicated that the prolonged exposure of FB₁ at high concentration caused DNA damage of apoptotic type in human fibroblasts. In Vietnam, in earlier studies of Thieu at al. (2008) and Phuong et al. (2010), the occurrence of aflatoxins, fumonisins and zearalenone were widespread and were in high concentrations. Especially, fumonisin incidence was about 70% tested samples and the highest fumonisin B₁ level was around 10.8 ppm in the Southeastern and Highlands provinces (Phuong et al 2010).

Since the occurrence of fumonisins is harmful to animal and human health, there are many methods to reduce their production or their negative impacts, such as using resistant corn varieties, controlling insects, heating, chemical detoxificants like Ca (OH)₂ and ammonia, or degradation of fumonisins by ozone. Nevertheless, some of these methods are not very successful; some are successful but need high temperatures that hydrolyze fumonisins and other toxins (Maja Šegvić and Stjepan Pepeljnjak 2001). Using adsorbents to adsorb mycotoxins, especially aflatoxin, is also one of the methods that can effectively reduce the harmful effects. However, they do not really show any effects on fumonisins, and adsorbants such as activated carbon seem to worsen the toxic of fumonisin B₁ (Piva et al 2005), cholestyramin also did not have a specific effect in adsorption of fumonisins in vivo (Solfrizzo et al 2001). Some commercial products have adsorptive effects on fumonisins, but their costs are too expensive, particularly for small farmers. Besides, fumonisins after being absorbed through the intestine will go to the liver first, and exert effects on it then on it, and then on other organs. According to Taylor (2003), indigenous peoples use plants from Phyllanthus sp., a small annual herb that is distributed throughout the tropical regions all over the world, to cure many diseases including hepatitis and liver diseases. In clinical research, this plant also demonstrated its anti-hepatotoxic function. A study by Huang et al. (2004) showed that the water extract of Phyllanthus urinaria, the same family and the same use as Phyllanthus amarus, could reduce the cell viability of HL-60 cells (human myeloid leukemia), which is additional evidence for its anticancer effect. This plant had the ability to reverse the negative impacts of aflatoxicosis of broiler chickens (Sundaresan et al 2007). Phyllanthin, hypophyllanthin and niranthin are the main substances in P. niruri that have a hepatoprotective effect (Kodakandla et al 1985). Phyllanthus urinaria or P. amarus is a weed that is easy to cultivate and is widespread. In Vietnam, Phyllanthus amarus grows widely in nature and has been used as a medicinal plant for a long time to treat hepatic disease, eye infection, snakebites and acnes (Do Tat Loi 2004). Today, many farmers in the poor Middle provinces of Vietnam are earning money from growing this plant for use as human medicine (Hung Phien 2009). For these reasons, we can hope that adding Phyllanthus amarus or P. urinaria extract to the feed of pigs can reduce the effects of fumonisins on liver, and thus liver, kidney and lung functions will not be affected and the pigs will retain good production performances.

Materials and methods

Site description

The experiment was conducted in the experimental farm of the Animal Husbandry and Veterinary Medicine Faculty of Nong Lam University, Ho Chi Minh City, Vietnam. This area is in Southeastern Vietnam, and has a tropical monsoonal climate, with a rainy season from May to October and a dry season from November to April. The average temperature is 27.5°C with high humidity. The duration of this study was 37 days, from 22 December 2009 to 31 January 2010.

Pigs, treatments and experimental design

The experiment had a completely randomized design (CRD), with a 2 × 3 factorial arrangement and 4 replicates. Each replication had 2 pigs (one male castrate and one female) in one pen. In total 48 crossbred (Landrace × Yorkshire × Duroc) piglets (10.9 ± 1.21 kg initial body weight) were used in the experiment. The pigs had been vaccinated against mycoplasma and swine fever at one week of age and five weeks of age. After 4 days of acclimatization, the pigs were randomly assigned to the treatments and pens according to the design show in Table 1 and Figure 1. The treatments were: 

Fumonisins factor:

HF: 10 mg fermented fumonisin B₁ per kg of feed

LF:  Low fumonisin B₁ in feed

Additive factor:

NA:  No detoxicant additive

CA:  Commercial detoxicant additive (1 g/kg feed)

PE:   Phyllanthus amarus extract (10g extract/kg feed)

Figure 1. Experimental layout

Experimental diets and feeding

Fumonisin B₁ production

Fumonisin B₁ (FB₁) was from two sources: fumonisin B₁ produced by Fusarium proliferatum cultured on maize grains was purchased from the National I-Lan University Laboratory, Taiwan, and production by Fusarium proliferatum isolates that was assessed in cultures grown on autoclaved rice. In brief, 500 g of ground maize was placed in a jar, was moistened for 1 h in distilled water and then autoclaved at 121⁰C for 60 minutes.

The maize medium was inoculated with mycelia from seven-day-old cultures of F. proliferatum grown on PDA (Potato Dextrose Agar and incubated at 26℃ for 15 days. Samples (three replicates) of each isolate were dried in a forced-air draft oven at 55°C for 48 h, crushed with a mortar and pestle, and extracted with 1 ml of CH₃CN:H₂O (1: 1, v ⁄ v) for 1h. The extracts were centrifuged at 1500g for 10 min, and the  supernatants  were cleaned on a Sep-Pak C18 cartridge column (Waters, Milford, MA, USA) with CH₃CN:H₂O (1 : 1, v ⁄ v) as the solvent. The level of FB₁ was determined from the extracts using HPLC. The concentration of fumonisin B₁ in the substrate was 882 ppm. Pure fumonsin B₁ was supported by Biomin company with total 5514 mg fumonisin B₁ in 280.9g powder.

Detoxicant

A commercial detoxicant additive was provided by Biomin Company, Austria. Phyllanthus amarus extract was purchased from Hong Dai Viet Company, Vietnam, and its components (phyllanthin, hypophyllanthin and niranthin) were analyzed at the Institute of Chemical Technology, Vietnam.

Diets

The diet for pigs was formulated to meet or exceed their nutrient requirement (NRC 1998) (Table 3) and was then mixed with fumonisin B₁ to produce the diet that contained 10 mg FB₁ per kg feed. Finally, detoxicant additives were mixed with feed according to treatments. Feed was analyzed for gross composition, such as crude protein, ether extract, crude fibre, ash, Ca and P at the Department of Animal Nutrition, Nong Lam University (Table 4).

Feed and drinking water were offered ad libitum and hygienic conditions maintained throughout the experiment. Fresh feed was provided at 08:00 h and 15:30 h, when remaining feed was removed and weighed.

Data collection

The animals were weighed at the beginning and the end of the trial to calculate average daily weight gain (ADG) and then feed conversion ratio (FCR) was calculated based on feed consumption. Blood samples of all pigs were collected at the end of the experiment and sent to MEDIC Medical Center, Ho Chi Minh City for analysis of bilirubin T, bilirubin D, bilirubin I, albumin, total cholesterol, glucose, AST (Aspartate Aminotransferase or GOT: Glutamic Oxaloacetic Transaminase), ALT (GPT: Glutamine Pyruvic Transaminase), GGT (Gamma Glutamyl Transpeptidase) and ALP (Alkaline Phosphatase).

At the end of the 37-day experiment, two pigs per treatment were slaughtered and necropsies performed. The lungs, liver and kidneys were sectioned to observe lesions and biopsies carried out to examine the histopathology.

Chemical analysis

The formulated feed was sampled and the composition of the basal diet was determined. The feed also was checked with fumonisins and aflatoxins concentrations.

Serum analysis was done by MEDIC Medical Center, Ho Chi Minh City.

Histopathology examination was carried out in the Veterinary Hospital, Nong Lam University, Ho Chi Minh City.

Statistical analysis

The data were analyzed by analysis of variance (ANOVA) using the CRD procedure of Minitab software (version 13.3). Sources of variance were treatments and error. 

Results

The chemical composition of the basal diet is shown in Table 4.

The concentrations of aflatoxins in the feed were 6.3 and 13.5 µg/kg for LF and HF groups, respectively. Although the expected fumonisin B₁ level in the diet was 10 ppm, the actual level was 3980 µg/kg in the HF group. Moreover, the feed of LF group contained 1132 µg/kg feed.

In order to ensure that the data gave the exact results, the performance data of two pens from two treatments (LFCA and LFNA) in the experiment were not used in data analysis because these pigs were infected by Actinobacillus Pleuropneumonia (APP) and depressed for a long period. Furthermore, the blood profiles of pigs from five pens from four treatments (two from LFPE, three from LFCA, HFNA, HFCA) were also not used in data analysis owing to their identification mistakes in the laboratory. 

High fumonisins and adding detoxicants in the diets did not affect the growth performance (Table 5). Final weight, average daily weight gain, average daily feed intake, and feed conversion ratio were not significantly different among treatments (Table 6).  

Table 7 shows the differences in blood bilirubin T, D, I, albumin and total cholesterol among pigs given diets with different toxin levels and detoxicants. High fumonisins in the diet decreased the total cholesterol significantly (2.19 mmol/L) compared with the low fumonisin groups (2.42 mmol/L) (P<0.05). The differences in these parameters among the no feed additive, commercial additive and Phyllanthus amarus extract treatments were not significant (P>0.05). Moreover, the combination of detoxicant types and fumonisin levels did not significantly affect on bilirubin T, D, I, albumin and total cholesterol levels in pig blood (P>0.05) (Table 8). However, blood albumin in the high fumonisin - no feed additive group tended to be lower than in the other treatments (3.00 g/100mL).  

Serum enzymes levels are presented in Table 9 and Table 10. Although there was no significant difference in blood GGT, ALP, AST and ALT levels between toxin levels and detoxicants and their combination impact, some trends were noted among treatment groups. For example, AST blood levels of pigs given the commercial additive were higher than in the no feed additive group (110 U/L > 83.1 U/L, P=0.067) and the Phyllanthus amarus group also had a high AST blood level (99.6 U/L). Furthermore, pigs given the high fumonisins only diet (HFNA) tended to have lower blood levels of GGT, AST and ALT.

The results of the microscopic and macroscopic pathology of the experimental pigs are summarized in Table 11. The lesions found indicated mycoplasma infection in pigs, and there were some distinct points in the lungs and liver of pigs consuming fumonisin. Microscopic examination showed that fumonisin thickened the alveolar walls of the lungs. However, the groups that were treated with commercial and Phyllanthus amarus additives had less thickened alveolar walls than the no additive group. Moreover, in the pigs given fumonisins fatty degeneration and necrosis of the liver cells was observed and more severe degeneration of the hepatocytes was noted, especially in the groups given diets without additives. In addition, macroscopic observation seemed to show tenderness of the liver including the swollen of liver and easily to be broken by hand in the P. amarus groups.

Discussion

Growth performance

The results in this study are in contrast with the results from Piva et al. (2005), which showed a significant decrease of ADG and a significant increase FCR of piglets fed diets that contained 30 ppm fumonisins given for 42 days. However, there was no difference in the ADFI of these pigs among treatments. Rotter et al. (1996) found out that average daily weight gain of male pigs fed a 10 ppm pure fumonisin B₁ diet through 8 weeks decreased by 11% compared to a 0 ppm diet. Furthermore in the first 4 weeks of the experiment, a general increase of feed consumption was observed. Nevertheless, in agreement with growth performance results in the present study, Osweiler et al. (1992), and Zomborszky et al. (2002) did not find any difference in the feed intake, body weight gain, and feed conversion ratio of pigs fed a 10 ppm fumonisins diet and a 17 ppm fumonisin B₁ diet. Possibly, the concentration of fumonisin B₁ and time on experiment were not high enough and sufficiently long to cause any changes in the growth performance of pigs. In addition, the fumonisin B₁ in this study was a mixture of pure and cultured FB₁, and therefore the results were not as clear as those found by Rotter et al. (1996).

Blood parameters

While high fumonisins in the diets lowered total cholesterol serum of pigs in the present experiment, Rotter et al. (1996) demonstrated an increase in cholesterol serum after 2 weeks. When fumonisins were fed at 30 ppm, cholesterol concentration was significantly higher than in control pigs (Piva et al 2005). On the other hand, the cholesterol and serum enzyme levels were within the normal ranges in the study of Zomborszky et al. (2002), and only some pigs had histopathological changes (fumonisins dose 1 and 5 ppm) and showed an elevation outside the normal ranges of AST, ALT and ALP in serum (ALP >500 U/ l, AST >100 U/ l and/or ALT >70 U/ l). Furthermore, a dose of 17 ppm fumonisin B₁ in the diet did not show clearly any increase in AST, GGT and total bilirubin of pigs (Osweiler et al 1992). High fumonisins concentration in the diet in the present study decreased the total cholesterol concentration (2.19 mmol/L) to values under the normal range of total cholesterol from 2.36 to 3.72 mmol/L (Tumbleson and Kalish 1971). It was reported that the apparent digestibility of the ether extract (EE) of 10 mg FB₁/kg feed was significantly reduced during the weanling phase, and then increased to the level in the control group after this phase (Gbore and Egbunike 2007). Because fumonisins are mentioned in the disruption of lipid metabolism action in animals, this suggests that a 10 ppm mixture of cultured and pure fumonisin B₁ decreased the cholesterol concentration in pig serum, and that this may be related to the lower digestibility of the ether extract over the 37 days of experiment. This mixture level and the length of experiment may not have been enough to increase the total cholesterol serum, as reported in some studies. However, the commercial additive and Phyllanthus amarus extract could have increased the cholesterol level (Table 7 and 8).

In terms of enzyme activity, the commercial additive and Phyllanthus amarus extract elevated the concentration of AST to higher levels than the safe limit (AST >100 U/L) (Zomborszky et al 2002), especially, the commercial additive.

Histopathology

The observed microscopic pathology of lung is in the present study consistent with Osweiler et al. (1992) and Zomborszky et al. (2002), who found thickening of alveolar walls. Moreover, the liver histopathology is also in agreement with Gelderblom et al. (1988) concerning the degeneration of hepatocytes. It can be concluded that the commercial additive and Phyllanthus amarus extract had a mild effect in reducing the negative impacts of fumonisins. However, possibly because of the high dose, P. amarus extract caused tenderness of liver and AST elevation. Furthemore, the commercial additive also showed an AST level increase over the limit which can cause liver damage.

Conclusions

• The concentration of fumonisin and the addition of detoxicants in this study did not cause any changes in the growth performance of pigs

• However, high fumonisin in the diet caused a decrease in serum total cholesterol to under the normal level, and the commercial additive and Phyllanthus amarus extract increased this concentration and reduced the histophathology in lungs and liver, although the reduction was not so clear.

• The Phyllanthus amarus extract concentration in feed may have been too high, and therefore could have induced tenderness of liver including the swollen of liver and easily to be broken by hand.

Acknowledgements

W would like to thank the Department for Research Cooperation with Developing Countries (SAREC), presently a part of the Swedish International Development Cooperation Agency (SIDA) for its financial support for this survey.

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