Counteractive Role of White Pepper Extracts for Oxidative Stress and...

[Full title: Counteractive Role of White Pepper Extracts for Oxidative Stress and Hepatotoxicity Induced by Aflatoxin B1 in Rats]

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OPEN ACCESS International Journal of Pharmacology ISSN 1811-7775 DOI: 10.3923/ijp.2019.177.188 Research Article Counteractive Role of White Pepper Extracts for Oxidative Stress and Hepatotoxicity Induced by Aflatoxin B 1 in Rats 1 Ahmed M. S. Hussein, 2 Karem Aly Fouda, 3 Ahmed Noah Badr and 4 Adel G. Abdel-Razek 1 Department of Food Technology, National Research Centre, Cairo, Egypt 2 Department of Nutrition and Food Sciences, National Research Centre, Dokki, Cairo, Egypt 3 Department of Food Toxicology and Contaminants, National Research Centre, Dokki, Cairo, Egypt 4 Department of Fats and Oils, National Research Centre, Dokki, Cairo, Egypt Abstract Background and Objective: Aflatoxin B 1 (AFB 1 ), a pre-carcinogenic and toxic compound which contaminates foodstuffs and edible tissues, is associated with oxidative stress and hepatotoxicity. This investigation aimed to assess the counteractive role of ethanol (EWP), petroleum ether (PWP) and n-hexane (HWP) white pepper extracts for oxidative stress and hepatotoxicity induced by AFB 1 in a rat model Materials and Methods: Concentrated white pepper extracts (WPEs) estimated for total phenolic, total flavonoids, anti-oxidant and anti-fungal activities. Otherwise, the fatty acids composition of white pepper was analyzed. Forty-eight male albino rats were divided into 8 groups, negative and positive AFB 1 groups and the other 6 groups were treated to evaluate the WPEs biological effects either in the AFB 1 presence or absence. Results: The results elucidated that WPEs suppressed both the raising of aminotransferases enzymes (alanine and aspartate) and alkaline phosphatase and the reduction of total protein. The WPEs combat the negative impact of AFB 1 on kidney functions and alleviated AFB 1 mediated oxidative stress either in plasma or liver. Also, it relieved the AFB 1 mediated lipid disturbance and hemoglobin reduction and exhibited antioxidant and antifungal activities. Conclusion: It was concluded that the extracts gave a counteractive role for oxidative stress which support the hepatotoxicity induced by AFB 1 presence Key words: Aflatoxin B 1 , anti-fungal activity, hepatotoxicity, white pepper extracts, oxidative stress, antifungal activities Received: October 25, 2018 Accepted: November 28, 2018 Published: January 15, 2019 Citation: Ahmed M. S. Hussein, Karem Aly Fouda, Ahmed Noah Badr and Adel G. Abdel-Razek, 2019. Counteractive role of white pepper extracts for oxidative stress and hepatotoxicity induced by aflatoxin B 1 in rats. Int. J. Pharmacol., 15: 177-188 Corresponding Author: Karem Aly Fouda, Department of Nutrition and Food Sciences, National Research Centre, El-Buhouth Street, 12622, Dokki, Cairo, Egypt Tel: +201000469262 Copyright: © 2019 Ahmed M. S. Hussein et al. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Competing Interest: The authors have declared that no competing interest exists Data Availability: All relevant data are within the paper and its supporting information files.

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Int. J. Pharmacol., 15 (2): 177-188, 2019 INTRODUCTION Aflatoxins, the secondary metabolites of fungi produced by Aspergillus flavus and A. parasiticus , are considered dangerous and toxic compounds (especially AFB 1 ) which contaminate foodstuffs 1 . Since cereals and food grains included in most human diets are susceptible for the aflatoxins contamination 2,3 . Aflatoxins are not only carcinogenic and teratogenic substances 4 , but also it associated with several health problems such as growth retardation, hematologic disorders, hepato and nephrotoxicity and immunosuppression 5-8 . The metabolism of AFB 1 in the liver results in reactive oxygen species (ROS) production including superoxide anion, hydroxyl radical and hydrogen peroxide (H 2 O 2 ) by cytochrome P 450. The soluble cell compounds and membranes can be attacked by these reactive oxygen species which results in the impairment of cell functions and cytolysis 9 Various phytochemicals compounds are reported to detoxifying aflatoxins 10 . White pepper ( Piper nigrum family Piperaceae) is the sun-dried stony seeds of ripen pepper fruits, it was reported as rich in several phytochemicals compounds 11 . It is utilized as a common spice in food preparation. Otherwise, it was included in several traditional medicine recipes particularly in China and Southeast Asia countries 12 . Several health benefits for white pepper are demonstrated among its anti-oxidant, anti-inflammation, anti-atherogenic as well as anti-platelet aggregation activities 13,14 . Piperine, as a phytochemical component, reported inhibiting aflatoxin production by toxigenic fungi 15 Also a modern study reported the piperine genetic impact on aflatoxigenic fungi strains which leads to suppress aflatoxin production 16 . According to extraction type, piperine reported higher in the non-polar extraction 17 . Despite the microbial load and mycotoxin contamination on pepper fruits (black or white) were dealt with it in previous studies, while aflatoxin reduction by different WPEs types, also the oxidative stress of toxigenic fungi-producing strains in the presence of WPEs has not been evaluated The current research was designed to innovate a novel utilization of WPEs against the hazard biological effects occurred by AFB 1 and to evaluate the counteractive role of WPEs against the oxidative stress and hepatotoxicity induced by AFB 1 in biological systems. Otherwise, the changes in biochemical parameters and tissue enzymes system conjugated to utilize WPEs were also estimated to validate the safety application of WPEs in food products MATERIALS AND METHODS The current study was carried out in the National Research Centre, Cairo, Egypt from January-June, 2018 Materials Plant materials: White pepper was purchased from the local market, sun-dried then ground using an electric grinder and directly extracted Animals: Male albino rats of 125.27±7.52 g as (Mean±SD) were obtained from the animal house of the National Research Centre, Cairo, Egypt. Animals were kept individually in stainless steel cages at room temperature. Water and food had been given ad-libitum Diets: Balanced diet was prepared to contain 10% protein supplemented from casein, 10% corn oil, 10% sucrose, 60.5% maize starch, 5% fiber, 3.5% salt mixture and 1% vitamin mixture provided by the AIN-93 formulation 18 . The oil soluble vitamins had given weekly to rats separately from the diet Methods Extracts preparation: The EWP, PWP and HWP extracts were prepared using ethanol, petroleum ether 40/60 and n-hexane, respectively. The solvent was added to extract the fine powder (4:1 w/v). The mixture was stirred using an overhead ultrasonic probe (30 E C/45 min), filtered through Whatman No. 4 filter paper for separating the extract. The filtrate was concentrated to 25% (v/v) at 40 E C using a rotary evaporator system. Nitrogen gas was utilized to complete solvent removing then stored at -20 E C until further analysis and applications Determination of total phenolic and total flavonoid contents of WPEs: Total phenolic contents (TPC) and total flavonoid contents (TFC) were determined according to Singleton et al 19 and Chang et al 20 , respectively. The TPC was expressed as mg Gallic acid equivalents (GAE) per g extract while the TFC were expressed as mg quercetin equivalent (QE) per g extract DPPH radical scavenging activities of WPEs: The free radical scavenging activity was measured by spectrophotometer using the DPPH method as described by Teke et al 21 178

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Int. J. Pharmacol., 15 (2): 177-188, 2019 ABTS cation decolonization assay of WPEs: The ability to scavenge free ABTS radicals was applied based on the protocol declared by Re et al 22 . Results were expressed as : mol trolox equivalents (TE)/g sample Ferric reducing ability (FRAP) assay of WPEs: The FRAP assay was performed according to Hwang and Thi 23 . The results were expressed as : mol trolox equivalent (TE)/g sample Fatty acid composition evaluation: Fatty acid methyl esters of HWP extract were prepared according to AOAC 24 to be subjected to GLC analysis of fatty acids. Identification of the fatty acid methyl ester was carried out by direct comparison of retention times of each of the separated compounds with authentic standards and the results were reported as weight percentages after integration and calculation using Chem. Station (Agilent Technologies) White pepper extracts effect on fungal growth: Yeast extract sucrose (YES) was utilized to evaluate fungal growth inhibition 25 . The inhibition was represented as a loss in dry weight of fungal growth in the presence of extracts against the control Agar diffusion test: Disk diffusion was evaluated according to the method described by Badr et al 25 . The inhibition effect was determined as a ratio of the calculated as the equation follows: Ac At Growth inhibition (%) = 100 Ac   Where: Ac = Fungal diameter growth in control plate At = Fungal diameter growth in treatment plate Wells diffusion test: Well diffusion was evaluated according to the method described by Badr et al 25 . Each well in a plate media was loaded by 250 µL of WPEs, one plate used for one fungus and one extract type, the plates were incubated at 25 E C/5 days. The inhibition zone was determined as a ratio of decreasing in fungal growth compared to the control Determination of minimal fungicidal concentration of white pepper: The inhibition was used to determine the anti-fungal activity of the extracts against fungi as previously described by Shehata et al 26 . The concentration required to give 50% inhibition of growth IC 50 was calculated from the regression equation. Nystatin was used as a positive control Preparation of standards for aflatoxin: The dry-film standard of aflatoxin was applied to prepare a calculated concentration as ng mL G 1 using a volume of methanol: acetonitrile (9:1) Preparation of dosage form: White pepper extracts were dispersed separately in phosphate buffer saline (pH = 7.2) to be given orally to rats Design of the animal study: Forty-eight rats were used in the present study. After one week of acclimation, rats were divided into 8 groups (n = 6) as follows: Group 1 (G 1) : Normal healthy group (Normal control) Group 2 (G 2) : Rats were treated orally with AFB 1 Group 3 (G 3) : Rats were treated orally with AFB 1 and ethanol extract of white pepper (AFB 1 +EWP) Group 4 (G 4) : Rats were treated orally with ethanol extract of white pepper (EWP) Group 5 (G 5) : Rats were treated orally with AFB 1 and petroleum ether extract of white pepper (AFB 1 +PWP) Group 6 (G 6) : Rats were treated orally with petroleum ether extract of white pepper (PWP) Group 7 (G 7) : Rats were treated orally with AFB 1 and n-hexane extract of white pepper (AFB 1 +HWP) Group 8 (G 8) : Rats were treated orally with n-hexane extract of white pepper (HWP) The daily oral dose of aflatoxin B 1 adjusted at 850 ng kg G 1 b.wt./day. Moreover, the daily oral dose of each extract of white pepper was 250 mg kg G 1 b.wt./day. All rats were fed on a balanced diet all over the study period (4 weeks). During the experiment, body weight and food intake recorded weekly. However, the total food intake, body weight gain and food efficiency ratio calculated at the end of the experiment. Blood samples were collected from all rats after an overnight fast. A portion of the whole blood was analyzed for hemoglobin (Hb) concentration 27 . The remaining blood was centrifuged and the plasma was analyzed for plasma levels of total protein using the method of Rheinhold 28 , the activities of aspartate transaminase (AST) and alanine transaminase (ALT) according to Reitman and Frankel 29 and alkaline phosphatase (ALP) according to Bessey et al 30 . The levels of creatinine, urea, blood urea nitrogen (BUN), albumin and uric acid were determined depending on Larsen 31 , Fawcett and Scott 32 , Christian et al 33 , Doumas et al 34 and Watts 35 in succession as indicators of kidneys function. Total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein 179

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Int. J. Pharmacol., 15 (2): 177-188, 2019 cholesterol (LDL-C) and triglycerides were determined according to Watson 36 , Burstein et al 37 , Schriewer et al 38 and Megraw et al 39 in succession. Cholesterol/HDL-C ratio and VLDL-C were calculated. Plasma total anti-oxidant capacity was determined according to Koracevic et al 40 . Plasma and liver malondialdehyde (MDA) was determined according to Ohkawa et al 41 . Liver catalase, glutathione-s-transferase (GST) and superoxide dismutase (SOD) activities were determined according to Beers and Sizer 42 , Habig et al 43 and Nishikimi et al 44 in succession. Rats were dissected and liver, kidney were immediately separated from each rat and weighed. This study has been carried out according to the Ethics Committee, National Research Centre, Cairo, Egypt and followed the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985) Statistical analysis: The results were expressed as the Mean±SE. Results of animal experiments were analyzed statistically using the one-way analysis of variance ANOVA followed by Duncanʼs test. In all cases; p<0.05 was used as the criterion of statistical significance RESULTS The represented data in Table 1 declared that the petroleum ether extract of white pepper seeds recorded the highest values of anti-oxidant activities (AA) for the three applied assays (DPPH, ABTS and FRAP). Whereas, n-hexane extract of white pepper showed the lowest values of anti-oxidant activities. The TPC of WPEs was also ordered ascendingly as HWP<EWP<PWP. Moreover, the highest value of total flavonoid content was recorded by petroleum ether extract The fatty acid composition of white pepper (n-hexane extract) was illustrated in Table 2. As shown white pepper n-hexane extract recorded high content of oleic acid (13.22%) and linoleic acid (8.26%). The major saturated fatty acid was luric acid (17.69%). Erucic acid was not detected in the white pepper n-hexane extract Evaluation of minimal fungicidal concentration of white pepper extracts: The results presented in Table 3 showed the minimal fungicidal concentration values of white pepper extracts compared to Nystatin as a standard anti-fungal material. It was obvious that petroleum ether extract of white pepper recorded the best results of minimal fungicidal concentration against the four toxigenic fungi strains Table 1: Total anti-oxidant, total phenolic and total flavonoid of white pepper extracts Parameters EWP extract PWP extract HWP extract DPPH (µmol TE g G 1 ) 5.27±0.02 11.26±0.03 2.17±0.06 ABTS (µmol TE g G 1 ) 42.28±0.62 104.65±1.22 21.37±0.18 FRAP (µmol TE g G 1 ) 9.62±0.41 14.79±0.81 2.56±0.31 TPC (mg GAE g G 1 ) 4.33±0.63 7.57±0.42 2.41±0.12 TFC (mg QE g G 1 ) 12.47±0.74 52.33±1.05 32.66±1.1 TPC: Total phenolic content, TFC: Total flavonoid content Table 2: Fatty acid composition of white pepper n-hexane extract Synonym Fatty acids Percentage C 12:0 Lauric acid 17.69±0.04 C 14:0 Myristic acid 6.82±0.24 C 16:0 Palmitic acid 12.37±0.65 C 16:1 Palmitoleic acid 10.22±0.28 C 17:0 Margaric acid ND C 18:0 Stearic acid 15.72±0.42 C 18:1 Oleic acid 13.22±0.56 C 18:1 trans Vaccenic acid 6.34±0.28 C 18:2 Linoleic acid 8.26±0.48 C 18:3 Linolenic acid 4.84±0.71 C 18:3 n 5 Punicic acid ND C 20:0 Arachidic acid ND C 20:1 Gondoic acid ND C 22:0 Behenic acid ND C 22:1 Erucic acid ND C 24:0 Lignoceric acid 4.52±0.23 C 24:1 Nervonic acid ND SFA Saturated fatty acids 57.12 MUSFA Mono unsaturated fatty acids 29.78 PUSFA Poly unsaturated fatty acids 13.10 USFA/SFA Unsaturated fatty acids/ saturated fatty acids 0.75 ND: Not detected Anti-fungal inhibition of white pepper extracts: The data in Table 4 presented the inhibition percentage of each extract against the control. The highest inhibition (%) recorded by the PWP extract, it was ranged from 39.2-48.6% of the control. The highest inhibition (%) of toxigenic fungi showed for F. oxysporum ITEM 12591 growth (using well diffusion assay), while the lowest inhibition (%) recorded for Aspergillus parasiticus ITEM 11 (on desk diffusion assay). However, the differentiation between the agar desk diffusion and agar well diffusion was not so far which may indicate honesty for the WPEs anti-fungal effect against the toxigenic fungi As shown in Table 4, the highest reduction of mycelial growth was accomplished by PWP. The highest reduction value recorded for F. oxysporium ITEM 12591, while the lowest reduction value was recorded for A. pairasticus ITEM 11 Effect on body weight gain or total food intake: Results (Table 5) revealed that the administration of aflatoxin B 1 to rats lead to reduction in the body weight gain compared to the normal rats. Also orally treated rats with aflatoxin B 1 recorded 180

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Int. J. Pharmacol., 15 (2): 177-188, 2019 Table 3: Minimal fungicidal concentration of white pepper extracts Minimal fungicidal concentration (MFC) ------------------------------------------------------------------------------------------------------------------------------------------- Parameters EWP (µg mL G 1 ) PWP (µg mL G 1 ) HWP (µg mL G 1 ) Nystatin (ng mL G 1 ) Aspergillus flavus ITEM 698 440 370 1600 180 Aspergillus ochraceus ITEM 5117 400 370 1640 180 Fusarium oxysporum ITEM 12591 380 350 1580 150 Penicillium chrysogenum ATCC 10106 400 370 1600 180 Table 4: Fungal inhibition effect of white pepper extracts on solid and liquid media growth Desk diffusion inhibition zone (5 days incubation) ------------------------------------------------------------------------------------------------------------------------------------------------------------------- Aspergillus flavus Aspergillus parasiticus Fusarium oxysporum Penicillium chrysogenum Parameters ITEM 698 ITEM 11 ITEM 12591 ATCC 10106 Control growth (mm) 77.00±3.17 79.00±2.41 61.00±2.15 72.00±2.90 EWP inhibition (%) 29.13±1.05 25.44±1.71 28.27±2.25 31.28±2.11 PWP inhibition (%) 41.55±1.41 39.24±2.17 47.34±1.12 44.75±1.53 HWP inhibition (%) 12.31±2.16 12.15±1.91 14.94±2.57 11.77±2.89 Well diffusion inhibition zone (5 days incubation) ------------------------------------------------------------------------------------------------------------------------------------------------------------------- Aspergillus flavus Aspergillus parasiticus Fusarium oxysporum Penicillium chrysogenum Parameters ITEM 698 ITEM 11 ITEM 12591 ATCC 10106 Control growth (mm) 85.00±2.31 90.00±1.90 72.00±1.81 81.00±2.45 EWP inhibition (%) 27.55±1.67 26.18±2.11 30.57±3.20 32.37±1.88 PWP inhibition (%) 44.28±2.85 41.73±3.42 48.61±2.48 47.16±2.94 HWP inhibition (%) 14.07±2.74 12.99±2.53 15.87±3.08 13.11±3.47 Mycelial dry weight inhibition (7 days incubation) ------------------------------------------------------------------------------------------------------------------------------------------------------------------- Aspergillus flavus Aspergillus parasiticus Fusarium oxysporum Penicillium chrysogenum Parameters ITEM 698 ITEM 11 ITEM 12591 ATCC 10106 Control growth (g) 12.51±3.42 14.31±3.71 8.57±2.44 12.98±1.99 EWP inhibition (%) 9.43±1.37 11.13±2.41 6.73±1.22 9.88±1.41 PWP inhibition (%) 7.74±1.11 9.21±0.98 5.19±2.05 8.15±1.17 HWP inhibition (%) 10.84±1.23 12.87±1.07 7.13±1.16 11.03±0.61 Table 5: Nutritional parameters of different experimental groups (Mean±SE) Groups Initial body weight (g) Final body weight (g) Body weight gain (g) Total food intake (g) Food intake (g/day) Feed efficiency ratio Normal control 125.17±4.40 a 189.17±5.76 b 64.00±3.10 b 479.17±2.70 b 17.11±0.10 b 0.13±0.01 a AFB 1 125.33±2.98 a 173.67±4.45 a 48.33±5.97 a 455.00±6.78 a 16.25±0.24 a 0.11±0.01 a AFB 1 +EWP 125.17±3.47 a 178.33±4.56 ab 53.17±3.52 ab 466.17±6.06 ab 16.65±0.22 ab 0.11±0.01 a EWP 125.33±2.80 a 187.33±4.03 ab 62.00±5.99 ab 478.33±3.13 b 17.08±0.11 b 0.13±0.01 a AFB 1 +PWP 125.33±2.68 a 180.50±2.92 ab 51.17±2.89 ab 465.33±3.68 ab 16.62±0.13 ab 0.12±0.01 a PWP 125.33±3.54 a 187.17±3.26 ab 61.83±5.69 ab 477.00±2.84 b 17.04±0.10 b 0.13±0.01 a AFB 1 +HWP 125.17±2.41 a 179.50±4.64 ab 54.33±4.38 ab 476.17±3.13 b 17.01±0.11 b 0.13±0.01 a HWP 125.33±3.87 a 188.00±4.08 b 62.67±3.85 ab 477.50±5.22 b 17.05±0.19 b 0.11±0.01 a In each column, same letters means non-significant difference; different letter means the significance among the tested groups at 0.05 probability Table 6: Liver and kidney weight of different experimental groups (Mean±SE) Groups Liver (g) Liver (%) Kidney (g) Kidney (%) Normal control 4.17±0.25 a 2.20 1.28±0.08 a 0.68 AFB 1 5.04±0.07 c 2.92 1.09±0.07 a 0.63 AFB 1 +EWP 4.76±0.13 bc 2.68 1.16±0.06 a 0.66 EWP 4.30±0.19 a 2.30 1.28±0.09 a 0.68 AFB 1 +PWP 4.40±0.15 ab 2.44 1.12±0.05 a 0.62 PWP 4.19±0.19 a 2.24 1.27±0.11 a 0.68 AFB 1 +HWP 4.50±0.17 ab 2.52 1.33±0.12 a 0.74 HWP 4.28±0.20 a 2.28 1.27±0.05 a 0.67 In each column, same letters means non-significant difference, different letter means the significance among the tested groups at 0.05 probability the lowest value of food intake(16.25 g/day) and total food intake (455 g). On the other hand, orally treated rats with ethanol, petroleum ether or n-hexane extracts of white pepper along with aflatoxin B 1 showed improvement in the nutritional parameters either body weight gain or total food intake Effect on liver and kidney weight: With regard to liver and kidney weight of the studied groups, results (Table 6) declared that the liver weight of orally treated rats with aflatoxin B 1 significantly increased in comparison to the normal control rats. Rats administrated with of ethanol, petroleum ether, or n-hexane extracts of white pepper along with aflatoxin B 1 recorded liver weight lower than rats treated with aflatoxin B 1 only. It was obvious that the liver weight 181

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Int. J. Pharmacol., 15 (2): 177-188, 2019 Table 7: Liver functions of different experimental groups (Mean±SE) Groups ALT (U L G 1 ) AST (U L G 1 ) Alkaline phosphatase (U L G 1 ) Total protein (g dL G 1 ) Normal control 37.90±1.33 a 62.37±2.39 a 52.60±1.42 a 6.37±0.16 d AFB 1 69.70±1.24 d 99.27±2.28 d 89.42±0.61 d 3.06±0.18 a AFB 1 +EWP 54.40±2.42 c 79.25±2.15 c 86.17±0.70 d 3.12±0.15 a EWP 37.60±1.14 a 64.10±1.29 a 54.70±1.40 a 6.27±0.16 d AFB 1 +PWP 42.64±1.17 b 68.90±2.69 ab 67.22±1.38 b 5.31±0.18 c PWP 37.50±1.36 a 62.75±1.28 a 53.20±1.28 a 5.36±0.12 c AFB 1 +HWP 52.40±1.22 c 72.07±2.03 b 77.10±1.74 c 4.88±0.14 b HWP 38.20±1.77 a 63.71±2.29 a 53.70±1.36 a 6.11±0.15 d In each column, same letters means non-significant difference, different letter means the significance among the tested groups at 0.05 probability Table 8: Kidney functions of different experimental groups (Mean±SE) Groups Urea (mg dL G 1 ) BUN (mg dL G 1 ) Uric acid (mg dL G 1 ) Creatinine (mg dL G 1 ) Albumin (g dL G 1 ) Normal control 28.76±0.69 a 13.42±0.32 a 1.17±0.02 a 0.97±0.01 a 4.13±0.12 a AFB 1 33.09±1.35 b 15.44±0.63 b 2.45±0.14 d 1.13±0.02 c 2.28±0.22 e AFB 1 +EWP 31.56±1.59 ab 14.73±0.74 ab 2.06±0.08 c 1.02±0.08 c 2.89±0.27 d EWP 28.91±0.89 a 13.49±0.42 a 1.28±0.04 a 0.99±0.02 b 4.08±0.26 a AFB 1 +PWP 30.26±1.11 ab 14.12±0.52 ab 1.69±0.05 b 0.97±0.07 a 3.73±0.24 c PWP 28.15±0.94 a 13.14±0.44 a 1.22±0.14 a 0.97±0.02 a 4.11±0.12 a AFB 1 +HWP 30.85±1.18 ab 14.40±0.55 ab 2.07±0.13 c 0.99±0.01 b 2.54±0.29 b HWP 28.64±0.77 a 13.37±0.36 a 1.33±0.37 a 0.98±0.14 b 4.07±0.18 a In each column, same letters means non-significant difference; different letter means the significance among the tested groups at 0.05 probability didnʼt affected by the administration of WPEs only without the aflatoxicosis induction. There werenʼt significant differences in kidney weight of the studied groups Effect on liver functions: As illustrated in Table 7, the impact of aflatoxin B 1 on the liver functions was evident. Since the activities of ALT, AST and alkaline phosphatase significantly elevated in orally treated rats with aflatoxin B 1 (69.70, 99.27 and 89.42 U L G 1 in succession) compared to the normal control rats. The total protein value significantly decreased in orally treated rats with aflatoxin B 1 (3.06 g dL G 1 ) The extracts of white pepper suppressed either the raising in ALT, AST and alkaline phosphatase activities or the reduction in total protein. The petroleum ether extract of white pepper exhibit the most promising effect followed by white pepper n-hexane extract. The rats given ethanol, petroleum ether or n-hexane extracts of white pepper only without the aflatoxicosis induction didnʼt show significant differences neither in ALT, AST and alkaline phosphatase activities nor in total protein values in comparison to normal control rats Effect on kidney functions: Kidney functions were not immune to the negative effect of aflatoxin B 1 . Since as noticeable in Table 8, orally treated rats with aflatoxin B 1 exhibited the highest values of urea, uric acid and creatinine (33.09, 2.45 and 1.13 mg dL G 1 in succession) while the albumin value decreased significantly in these rats in comparison to the normal control rats. The extracts of white pepper combat the negative effect of aflatoxin B 1 on kidney functions. The most promising effect on kidney functions was achieved by the petroleum ether extract of white pepper Hemoglobin concentration and oxidative stress impacts: It was manifested from Fig. 1 a that the lowest value of hemoglobin concentration was recorded by the group given aflatoxin B 1 only (10.82 g dL G 1 ). The extracts of white pepper suppressed the reduction of hemoglobin concentration value induced by aflatoxin B 1 Figure 1 b and c declared that the intake of aflatoxin B 1 caused oxidative stress which was clearly demonstrated throughout the elevation of plasma malondialdehyde value as well as the reduction of plasma total antioxidant capacity value for the group given aflatoxin B 1 only in compared to the other groups. On the other hand, the extracts of white pepper reduced the oxidative effect of aflatoxin B 1 when they administrated together As shown in Fig. 2 a-c, no significant differences in the oxidative stress markers (malondialdehyde, catalase, SOD and GST) in liver tissues were observed between the normal control group and the groups treated with each extract of white pepper only. On the other hand, the oxidative stress markers in liver tissues affected by the intake of aflatoxin B 1 While the extracts of white pepper alleviated the raising of liver malondialdehyde and the reduction of liver catalase, SOD as well as GST activities. The petroleum ether extract of white pepper exhibited the most promising effect 182

Int. J. Pharmacol., 15 (2): 177-188, 2019 6 5 4 3 2 1 0 Pl a sm a MDA ( n m o l m L ) G 1 (b) 3.38 a 4.57 c 4.18 b 3.31 a 3.46 a 3.34 a 4.08 b 3.35 a 16 14 12 10 8 6 4 2 0 Hb ( g d L ) G 1 (a) 13.19 b 10.82 a 11.03 a 12.95 b 12.44 b 13.00 b 12.00 ab 12.90 b 2.5 2.0 1.5 1.0 0.5 0.0 Pl a sm a T A C ( m M L ) G 1 (c) 2.15 bc 1.81 a 1.92 ab 2.19 bc 1.97 abc 2.22 c 1.94 ab 2.19 bc Normal control AFB 1 AFB 1+EWP EWP AFB 1+PWP PWP AFB 1+HWP HWP Experimental groups Fig. 1(a-c): (a) Hemoglobin, (b) Plasma malondialdehyde and (c) Total anti-oxidant capacity of different experimental groups Same letters means non-significant difference, different letter means the significance among the tested groups at 0.05 probability Table 9: Lipid profile of different experimental groups (Mean±SE) Groups T-Ch (mg dL G 1 ) TG (mg dL G 1 ) HDL-Ch (mg dL G 1 ) LDL-Ch (mg dL G 1 ) VLDL-Ch (mg dL G 1 ) T-Ch/ HDL-Ch Normal control 71.60±3.30 a 83.25±3.15 a 33.26±1.37 c 21.69±3.67 a 16.65±0.63 a 2.18±0.16 a AFB 1 98.41±2.69 c 99.53±2.84 b 25.87±1.20 a 52.63±2.09 c 19.91±0.57 b 3.82±0.11 c AFB 1+EWP 85.03±3.77 b 90.28±3.52 a 27.99±1.23 ab 38.99±3.86 b 18.06±0.70 a 3.06±0.17 b EWP 70.20±4.21 a 82.75±3.69 a 33.76±1.44 c 19.89±4.75 a 16.55±0.74 a 2.10±0.16 a AFB 1+PWP 83.31±3.33 b 88.40±3.21 a 31.42±1.16 bc 34.21±3.44 b 17.68±0.64 a 2.66±0.12 b PWP 70.03±3.32 a 82.52±3.39 a 34.38±1.09 c 19.14±4.25 a 16.50±0.68 a 2.05±0.13 a AFB 1+HWP 80.38±3.73 ab 88.07±3.05 a 28.23±1.06 ab 34.54±3.21 b 17.61±0.61 a 2.85±0.13 b HWP 69.92±4.32 a 81.37±2.44 a 35.14±1.28 c 18.51±4.47 a 16.27±0.49 a 2.00±0.15 a In each column, same letters means non-significant difference, different letter means the significance among the tested groups at 0.05 probability Plasma lipid profile: The plasma lipid profile also was affected by the intake of aflatoxin B 1 . Results (Table 9) revealed that the highest values of plasma total cholesterol, triglycerides, LDL-Ch and VLDL-Ch as well as T- Ch/ HDL-Ch ratio were recorded by the group given aflatoxin B 1 only whereas, this group showed the lowest value of HDL-Ch. Significantly the extracts of white pepper improved the lipid profile when they administrated along with aflatoxin B 1 183

[Find the meaning and references behind the names: Natural, Alo, Need]

Int. J. Pharmacol., 15 (2): 177-188, 2019 60 50 40 30 20 10 0 Ca ta la se (µ m g ) G 1 (b) 51.40 a 31.07 d 37.51 c 51.36 a 47.61 a 50.97 a 41.36 b 51.21 a 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 M alo nd ia ld eh y d e ( m mol mg ) G 1 (a) 0.53 a 0.99 d 0.89 c 0.50 a 0.67 b 0.51 a 0.94 d 0.48 a 35 30 25 20 15 10 5 0 SO D ( µ m g ) G 1 (c) 26.10 a 12.34 d 14.57 c 26.16 a 26.70 a 26.20 a 18.99 b 26.4 a 16 14 12 10 8 6 4 2 0 GS T ( µ mg ) G 1 (d) 12.10 a 9.85 c 3.91 c 12.37 a 11.47 a 12.23 a 8.99 b 11.47 a Normal control AFB 1 AFB 1+EWP EWP AFB 1+PWP PWP AFB 1+HWP HWP Experimental groups Fig. 2(a-d): Oxidative stress markers in liver, (a) Malondialdehyde, (b) Catalase, (c) SOD and (d) GST of different experimental groups Same letters means non-significant difference; different letter means the significance among the tested groups at 0.05 probability DISCUSSION Taking into consideration the contamination of food stuffs and edible tissues with mycotoxin, especially aflatoxin B 1 which exhibit several hazards on the health, there is a need to natural products to suppress the negative effects and hazards of aflatoxin B 1 . Anti-oxidant and anti-fungal activities of ethanol and petroleum ether extracts as well as n-hexane 184

[Find the meaning and references behind the names: El Khoury, Change, Purity, Bha, Choi, Galvano, Raman, Mahoney, Red, Khoury, Wahhab, Due, Tac]

Int. J. Pharmacol., 15 (2): 177-188, 2019 extract of white pepper were evaluated also the protective effect of these items against hepatotoxicity induced by aflatoxin B 1 in rats was studied. Logically, the suppression of fungal growth results in prevention of mycotoxin production. The anti-fungal activities of ethanol and petroleum ether extracts as well as n-hexane extract of white pepper, may be attributed to its anti-oxidant activities and contents of phenolic compounds. Since, El Khoury et al 45 reported that; there was a relation between the total phenolic and antioxidant activity of plant extracts and fungal growth degradation. The results reported by Grintzalis et al 46 referred to an inhibition effect of anti-oxidant on mycotoxin production more than its effect on the sclerotial of fungi. Mahoney et al 47 reported that phenolic compounds could inhibit aflatoxin production and fungal growth of A. flavus Moreover, the synthetic anti-oxidant like butylated hydroxyanisole (BHA) also showed a similar impact 48 . It was reported in a previous studies that the materials with a higher minimal inhibitory concentration and minimal fungicidal concentration values represented more potent role in fungal growth inhibition 25,49 . This effect may refer to the change of oxidative stress in the fungal growth media which cause a reduction not only in fungal growth amount, but also in the mycotoxin secreted by the fungi in the media 50 . In addition to anti-oxidant activities and phenolic compounds of ethanol and petroleum ether extracts of white pepper, the antifungal activities of these items may be attributed to the alkaloid compound known as piperine (1-peperoyl piperidine) which possesses anti-fungal and antioxidant effects as mentioned by Gurinderdeep 17 . Although Kanaki et al 51 disclosed that petroleum ether one of the solvents utilized for piperine extraction also ethanol one of the solvents utilized for piperine extraction 52 but Raman and Gaikar 53 found that the highest extraction efficiency of piperine (94% with a purity of 85%) was achieved using petroleum ether as a non-polar solvent while the lowest extraction efficiency of piperine (75-80% with a purity of 72%) was achieved using polar solvent such as ethanol. This finding may interpret the superiority of the effect of white pepper petroleum ether extract as anti-fungal and anti-oxidant agent. In the same pattern, results of the current study demonstrated that the white pepper petroleum ether extract showed the superiority as a hepatic protective agent against aflatoxin B 1 in rats. This superiority may be due to its higher content of piperine which possesses hepatoprotective activity 15 . Aflatoxin B 1 mediated weight gain reduction is related to the anorexia 54 . Choi et al 55 reported that the hepatic damage caused by aflatoxin B 1 is associated with the generation of reactive oxygen species and aflatoxin B 1 - 8,9-epoxide. ALT, AST as well as ALP are considered biomarkers which indicate to the hepatic injury shown to be increased in hepatotoxicity induced by aflatoxin 56-59 B 1 . The elevation of these biomarkers may be due to the disruption of plasma membrane by reactive oxygen species produced during the metabolism of aflatoxin 60 AFB 1 , Galvano et al 61 also mentioned that; the metabolic processing of aflatoxin B 1 by cytochrome P 450 in the liver results in reactive oxygen species. These reactive oxygen species are associated with aflatoxin B 1 mediated oxidative stress which clearly observed in the results of the present study via the elevation of plasma and liver MDA, reduction of plasma TAC and decreasing of liver catalase, SOD as well as GST of rats given aflatoxin B 1 alone. White pepper extracts potentially reversed the oxidative stress (either in plasma or in the liver) with an elevation of elevation of liver functions due to its antioxidant activities. In addition to that, the beneficial effect of white pepper extracts may be attributed not only to piperine which has an important role in free radical damage and can reduce the expressions of MDA and SOD activity 62,63 but also to other bioactive compounds such as flavonoids which exhibit protective effect of cells against aflatoxin 64 AFB 1 and can improve the detoxification of aflatoxin B 1 possibly by enhancing the activities of ROS detoxifying enzymes, thus the redox imbalance caused by aflatoxin B 1 and the cellular macromolecules oxidation and fragmentation can be prevented 65,66 . The results of the current study indicated that rats treated with aflatoxin B 1 alone showed elevation of total cholesterol, triglycerides and low density lipoprotein levels as well as the reduction of high density lipoprotein. Abdel-Wahhab et al 67,68 reported that a disturbance in the lipid metabolism causes by the toxic action of aflatoxin B 1 on liver functions. So, the improvement of lipid profile which accomplished by white pepper extracts may be due to its beneficial effect on liver functions. In addition to the presence of hypolipidemic bioactive compounds such as phenolic compounds and flavonoids in these extracts. Aflatoxin B 1 mediated hemoglobin reduction is related to not only the anemia caused by aflatoxin 54 B 1 but also to the red blood cells haemolysis by elevated lipid peroxides 69 . Thus, white pepper extracts potentially reversed the hemoglobin reduction due to its antioxidant activities CONCLUSION The results of the current investigation indicated to the potency of white pepper extracts as an anti-oxidant and anti-fungal agent. The most promising effect was accomplished by the petroleum ether extract of white pepper, either for its bioactive components contents or its anti-fungal potency. The counteractive role of white pepper extracts for 185

[Find the meaning and references behind the names: Zhang, Lee, Chinese, Ali, New, Vet, Bennett, Wen, Yoshikawa, Khanal, Nielsen, Sharma, Omole, Act, Dong, Brasil, Oswald, Life, Fapohunda, Wang, Hall, Danger, Clin, Han, Wild, Adv, Alabi, Kumar, Sci, Chern, Mahboob, Genet, Lung, Fruit, Egal, Turner, Vivo, Benin, Safe, Yamaguchi, Central, Leaf, Sinha, Huang, Togo, Fahey, Malathi, Nees, Reza, Caceres, Faranak, Ganesh, Smooth, Chioma, Bailly, Gong, Jang, Hoc, Ezekiel, Cortinovis, Chun, Brinda, Dwivedi, Cross, Cardwell, Lett, Uma, Kim, Chemical, Alpha, Med, Klich, Elisa, Chaba, Mice, Bedard, Reeves, Massey, Yasuda, Matsuda, Yang, Young, Marker, Masoud]

Int. J. Pharmacol., 15 (2): 177-188, 2019 oxidative stress and hepatotoxicity induced by aflatoxin B 1 in rats was demonstrated. These results directing to recommend implementing WPEs in safe foodstuffs production, mycotoxin health hazard avoidance in food products, besides the possibility to extend food shelf-life SIGNIFICANCE STATEMENT This study confirmed that the white pepper extracts can act as anti-oxidant and antifungal agents. The counteractive role of white pepper extracts for oxidative stress and hepatotoxicity induced by aflatoxin B 1 in rats was demonstrated. The results of the current study directing to recommend implementing WPEs in safe foodstuffs production, mycotoxin health hazard avoidance in food products, besides the possibility to extend food shelf-life REFERENCES 1 Bennett, J.W. and M. Klich, 2003. Mycotoxins. Clin. Microbiol Rev., 16: 497-516 2 Caloni, F. and C. Cortinovis, 2011. Toxicological effects of aflatoxins in horses. Vet. J., 188: 270-273 3 Reza, S.S.M., A. Masoud, T. Ali, G. Faranak and N. Mahboob, 2012. Determination of aflatoxins in nuts of Tabriz confectionaries by ELISA and HPLC methods. Adv Pharmaceut. Bull., 2: 123-126 4 Wangikar, P.B., P. Dwivedi, N. Sinha, A.K. Sharma and A.G. Telang, 2005. Teratogenic effects in rabbits of simultaneous exposure to ochratoxin A and aflatoxin B 1 with special reference to microscopic effects. Toxicololgy, 215: 37-47 5 Bintvihok, A., 2002. New insights to controlling mycotoxin danger in ducks. Feed Technol., 6: 28-29 6 Gong, Y.Y., K. Cardwell, A. Hounsa, S. Egal, P.C. Turner, A.J. Hall and C.P. Wild, 2002. Dietary Aflatoxin exposure and impaired growth in young children from Benin and Togo: Cross sectional study. Br. Med. J., 325: 20-21 7 Guindon, K.A., L.L. Bedard and T.E. Massey, 2007. Elevation of 8-hydroxydeoxyguanosine in DNA from isolated mouse lung cells following In vivo treatment with aflatoxin B 1 Toxicol. Sci., 98: 57-62 8 Fapohunda, S.O., C.N. Ezekiel, O.A. Alabi, A. Omole and S.O. Chioma, 2008. Aflatoxin-mediated sperm and blood cell abnormalities in mice fed with contaminated corn Mycobiology, 36: 255-259 9 Lee, J.K., E.H. Choi, K.G. Lee and H.S. Chun, 2005. Alleviation of aflatoxin B 1 -induced oxidative stress in HepG 2 cells by volatile extract from Allii Fistulosi Bulbus. Life Sci., 77: 2896-2910 10. Brinda, R., S. Vijayanandraj, D. Uma, D. Malathi, V. Paranidharan and R. Velazhahan, 2013. Role of Adhatoda vasica (L.) nees leaf extract in the prevention of aflatoxin induced toxicity in Wistar rats. J. Sci. Food Agric., 93: 2743-2748 11. Ganesh, P., R.S. Kumar and P. Saranraj, 2014. Phytochemical analysis and antibacterial activity of pepper ( Piper nigrum L.) against some human pathogens. Central Eur. J. Exp. Biol., 3: 36-41 12. Wang, B., Y. Zhang, J. Huang, L. Dong, T. Li and X. Fu, 2017 Anti-inflammatory activity and chemical composition of dichloromethane extract from Piper nigrum and P. longum on permanent focal cerebral ischemia injury in rats Rev. Brasil. Farmacogn., 27: 369-374 13. Kim, H.G., E.H. Han, W.S. Jang, J.H. Choi and T. Khanal et al ., 2012. Piperine inhibits PMA-induced cyclooxygenase-2 expression through downregulating NF- 6 B, C/EBP and AP-1 signaling pathways in murine macrophages. Food Chem Toxicol., 50: 2342-2348 14. Son, D.J., S.Y. Kim, S.S. Han, C.W. Kim and S. Kumar et al ., 2012 Piperlongumine inhibits atherosclerotic plaque formation and vascular smooth muscle cell proliferation by suppressing PDGF receptor signaling. Biochem. Biophys. Res. Commun., 427: 349-354 15. Matsuda, H., K. Ninomiya, T. Morikawa, D. Yasuda, I. Yamaguchi and M. Yoshikawa, 2008. Protective effects of amide constituents from the fruit of Piper chaba on D-galactosamine/TNF-alpha-induced cell death in mouse hepatocytes. Bioorg. Med. Chem. Lett., 18: 2038-2042 16. Caceres, I., R. El Khoury, S. Bailly, I.P. Oswald, O. Puel and J.D. Bailly, 2017. Piperine inhibits aflatoxin B 1 production in Aspergillus flavus by modulating fungal oxidative stress response. Fungal Genet. Biol., 107: 77-85 17. Gurinderdeep, S., 2017. Piperine: A remarkable marker with intense biological activity. Int. J. Pharmacogn. Chinese. Med., Vol. 1 18. Reeves, P.G., F.H. Nielsen and G.C. Fahey Jr., 1993. AIN-93 purified diets for laboratory rodents: Final report of the American institute of nutrition Ad hoc writing committee on the reformulation of the AIN-76 A rodent diet. J. Nutr., 123: 1939-1951 19. Singleton, V.L., R. Orthofer and R.M. Lamuela-Raventos, 1999 Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol., 299: 152-178 20. Chang, C.C., M.H. Yang, H.M. Wen and J.C. Chern, 2002 Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J. Food Drug Anal., 10: 178-182 186

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