EFFECTS OF DETOXIFICATION OF Mucuna pruriens ON THE FEED INTAKE, BEHAVIOR, ORGAN WEIGHTS, BLOOD CELL COUNTS AND METABOLITES OF RATS [EFECTOS DE LA DESINTOXICACIÓN DE LA Mucuna pruriens EN EL CONSUMO, COMPORTAMIENTO, PESO DE LOS ÓRGANOS, RECUENTO DE CÉLULAS SANGUÍNEAS Y LOS METABOLITOS DE LAS RAT

Department of Animal Sciences, Institute of Food and Agricultural Sciences, University of Florida, P.O. Box 110910, Gainesville, FL 32611; Email: huisden@ufl.edu; gisela@ufl.edu; araji@ufl.edu. Phone: +1 (352) 392-7527; Fax: +1 (352) 392 7851; Email: adesogan@ufl.edu; P.O. Box 110910 Gainesville, FL 32608 Department of Pharmaceutics, College of Pharmacy, University of Florida, P.O. Box 100494, Gainesville, FL 32610; Email: butterwk@ufl.edu. Analytical Toxicology Core Laboratory, Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611; Email: nszabo@ufl.edu. Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610; Email: GaskinJ@vetmed.ufl.edu. *Corresponding author


INTRODUCTION
Mucuna pruriens seeds are known for their nutritional value, and also useful in drought stricken areas (Bressani, 2002;Teixeira et al., 2003); in the Ayurveda they are known as an ethnopharmaceutical and its applications in the treatment of Parkinson's disease are well known (Rai et al., 2017).Huisden et al. (2010) reported the anthelmintic properties of the seeds.The high protein content of the seeds contributes to their nutraceutical and nutritional value (Huisden et al., 2012), however, some main antinutrients in Mucuna pruriens have compromised its use as a food and ethnopharmaceutical source.Szabo and Tebbett (2002) and Ukachukwu et al. (2002) noted that 3,4-dihydroxy-L-phenylalanine (L-Dopa) is the most potent toxic compound in the Mucuna bean and the concentrations range between 3 and 7 g/100g in the bean (Daxenbichler et al., 1972).Consequences of increased peripheral dopamine in humans can include nausea, vomiting, and anorexia, orthostatic hypotension, which results in dizziness, staggering, and increased heart rate and Dopamine Dysregulation Syndrome, which causes disabling motor and behavioral features such as pathological gambling, hypersexuality, and mood swings (Grosset, 2008).Tavares et al. (2017) suggest an impact on androgenic and anabolic systems.Psychiatric disturbances have also been reported in patients receiving high doses of L-Dopa in a dose-dependent manner.These can include nervousness, anxiety, agitation, insomnia, vivid dreams, confusion, delirium, depression, and psychotic reactions with hallucinations (Szabo and Tebbett, 2002).Cotzias et al. (1974) reported that feeding rodents high levels of L-Dopa caused motor hyperactivity, muscle jerks, stereotyped movements, jumping, gnawing, corkscrew tails, ataxia, salivation, piloerection, red muzzle, and convulsions.Most side effects arise directly from dopamine's activity as a neurotransmitter involved in the regulation of the heart, vascular system, digestive tract, and excretory system, rather than from its wellknown effect on receptors in the brain (Szabo and Tebbett, 2002).Symptoms of Mucuna intake in broilers and pigs include weight loss, reduced feed intake, and reduced feed conversion efficiency (Del Carmen et al., 2002;Flores et al., 2002).Despite the health hazards caused by L-Dopa, the Mucuna bean's high protein (25-30 g/100g) and starch (39-41 g/100g) concentrations (Ezeagu et al., 2003) make it an important part of the diet in Asia, Africa, and South and Central America.Adebowale et al. (2007) showed that except for methionine and cysteine, concentrations of bioavailable amino acids in Mucuna protein isolates exceeded the recommended levels for human diets by the Food and Agricultural Organization, World Health Organization, and United Nations.The high lysine concentration of Mucuna makes it a suitable supplementary amino acid source to cereal-based diets of livestock that are lysine deficient (Adebowale et al., 2007).Therefore, Mucuna could be used to alleviate malnutrition in developing countries provided its L-Dopa concentration is effectively reduced (Bressani, 2002;Teixeira et al., 2003).
The safety threshold is a bean concentration of less than 0.4 g/100g (Eilitta and Carsky, 2003;Ferreira et al., 2003;Ukachukwu and Szabo, 2003).Processing techniques have been evaluated that reduce the Mucuna L-Dopa concentration to safe levels (Bressani, 2002;Mugendi et al., 2010), but many of these are costly as they rely on fuel to generate heat or require copious amounts of water or are labor intensive and time consuming.Few studies have examined the residual nutritional value of detoxified Mucuna bean and the effects of feeding it to monogastrics.A few promising detoxification methods were identified in preliminary studies (Huisden et al., 2014).Ensiling the Mucuna bean for 28 d reduced the L-Dopa concentration by 54%, preserved the starch and protein concentrations, and resulted in a shelf life (aerobic stability) that exceeded 657 h (Huisden et al., 2014).Acid and alkali extractions for 8 h at pH 3 or 11, respectively almost eliminated (> 90% removal) Mucuna L-Dopa but also reduced the protein concentration by 24-31% (Huisden et al., 2012).The objective of this study was to evaluate the effect of feeding detoxified Mucuna beans on the feed intake, behavior and blood metabolites of rats.

Mucuna Detoxification
About 370 kg of Mucuna pruriens, cv.preto beans were harvested from a 10-ha field in Sao Paolo, Brazil and used for this study as well as those of Huisden et al. (2010;2014;2019).The beans contained 25 g/100g CP, 4.6 g/100g EE, 17.3 g/100g aNDF, 18.1 g/100g WSC, 38.2 g/100g starch, and 2.8 g/100g L-Dopa.The beans were ground in a Wiley mill to pass through a 1-mm screen (Arthur H. Thomas Company, Philadelphia, PA, USA) and subjected to detoxification by acid or alkaline extraction and ensiling as described by Huisden et al. (2014) and Huisden et al. (2012), respectively.Briefly, for the detoxification by ensiling, air-dry Mucuna beans were crushed in a roller mill (model 10004; Peerless International, Joplin, MO, USA) and weighed into vacuum mini silo bags (26.5 × 38.5 cm; VacLoc Vacuum Packaging Rolls, FoodSaver, Neosho, MO, USA) in quadruplicate.Exactly 900 ml of doubledistilled water were added to the beans in each bag to provide sufficient moisture for the fermentation.A vacuum sealer (V2220, FoodSaver, Neosho, MO, USA) was used to remove residual air and bags were sealed and kept in the dark at room temperature (18-25 °C) for 28 days.

Analysis of L-Dopa
The L-Dopa concentration of undetoxified and detoxified Mucuna beans was measured using the method of Siddhuraju and Becker (2001) and a high performance liquid chromatography system (Hewlett Packard HP1100) comprising a variable wavelength UV detector set at 280 nm, an Apollo C18 (4.6 x 250 mm) column and a mobile phase of 19.5 ml methanol: 1 ml phosphoric acid: 975.5 ml water (pH 2; v/v/v) flowing at 1 ml/min at 25ºC.

Chemical component analysis
Dried samples of Mucuna were ground to pass through a 1-mm screen in the Wiley mill, and ash was measured by combustion in a muffle furnace at 550 o C overnight.Total N was determined by rapid combustion using a macro elemental N analyzer (Elementar, vario MAX CN, Elementar Americas, Mount Laurel, NJ) and used to calculate CP (CP = N × 6.25).Neutral Detergent Fiber concentration was measured using the method of Van Soest et al. (1991) in an ANKOM 200 Fiber Analyzer (ANKOM Technologies, Macedon, NY).Amylase was used in the analysis and the results were expressed exclusive of residual ash.The anthrone method described by Ministry of Agriculture, Fisheries and Food (1986) was used to quantify water-soluble carbohydrates (WSC).Starch was measured by a modification (Hall, 2001) of the glucose-oxidaseperoxidase method of Holm et al. (1986).Ether extract (EE) was determined using the soxhlet procedure (AOAC, 1984; Method 24.005).
Gross energy was determined with an adiabatic bomb calorimeter (1261 isoperibol bomb calorimeter, Parr Instrument Company, Moline, Illinois, USA), using benzoic acid as a standard.Mucuna silage was made as described by Huisden et al. (2014) and the silage extract was obtained by blending 20 g of the ensiled bean with 200 ml of distilled water for 30s at high speed in a commercial blender (Waring model 31BL91, Dynamics Corporation of America, New Hartford, Connecticut, USA).
The mixture was filtered through two layers of cheesecloth and the pH measured with an electrode (Accumet pH meter, model HP-71, Fischer Scientific, Pittsburg, PA, USA).

Dietary Treatments
The treatments consisted of a control diet (CON) consisting of a commercial rat chow (TD 8604 rodent diet, Harlan Teklad, Madison, WI, USA) and four Mucuna-based diets in which 10 g/100g of the commercial rat chow was replaced with either undetoxified Mucuna (MUC), or Mucuna beans detoxified (Huisden et al., 2019) by acetic acid extraction (ACD), sodium hydroxide extraction (BAS), or ensiling (SIL).The inclusion level of the detoxified and undetoxified Mucuna in the diets was chosen because it had caused hepatotoxic effects on rats in a previous study (Ezeja and Omeh, 2010).The chemical composition of the rat chow and the detoxified and undetoxified Mucuna are shown in Table 1.

Animals and housing
Sixty 6-to 8-week-old male Sprague-Dawley rats with an initial body weight of 203+9g were purchased from Harlan (Indianapolis, IN, USA).Rats were individually housed in 40 x 30 x 20 cm cages and randomly assigned to the five treatments (n=12).All rats were housed for 14 d at 25 + 1 o C in a 12-h light/dark cycle with ad libitum water and feed supply.The rats were weighed daily as change in bodyweight is a sensitive criterion for detecting toxicity in short-term rodent assays (Lyon et al., 1988;Smith et al., 2003).Experiments were performed according to the policies and guidelines of the Institutional Animal Care and Use Committee (IACUC) of the University of Florida, Gainesville, USA.

Open field behavior analysis
Animal behavior was evaluated on d 3 and d 10 using the open field test (Carlini et al., 1986), which consisted of a round grey plastic arena measuring 70 cm in diameter surrounded by a grey plastic wall 34 cm high, lit with three 40W bulbs.The floor of the arena was divided into several concentric units with black lines dividing the arena into 19 fields.Open field activity helps determine locomotion and motor activity as well as speed (Vogel, 2002).On d 3 and 10 the 60 rats were placed one at a time at the center of the arena for 5 minutes.The open field test was videotaped using a high-resolution video camera WV-CP244 (Panasonic, Secaucus, NJ, USA).Video analysis was performed using TopScan, Top View Animal Behavior Analyzing System (version 1.00, Clever Sys Inc., Preston, VA, USA) by an unbiased and treatment-blinded technician.Due to a malfunction of the Top View analyzing system only data from six of the ten and three of the ten rats per treatment were recorded on d 3 and 10, respectively.

Feed intake and organ weights
Rats were individually caged in a laminar flow hood.Feed intake during the first 12 d of the trial was calculated as the difference between the dry weights of the feed offered on one day and the orts remaining the following day.Rats were also weighed daily during the first 12 d of the trial and growth records used to determine total and average daily gain.On d 14 rats were necropsied and the heart, liver, kidneys, spleen, and gonads were weighed.Organ weights were normalized to reflect percent of body weight.

Blood cell counts and metabolite
At the end of the trial (d 14), blood was collected postanesthesia through cardiac puncture and stored in vacutainer tubes containing EDTA anticoagulant (Vacuette, Greiner Bio-One NA, Inc, Monroe, NC, USA).Blood samples were analyzed at the University of Florida veterinary clinical pathology laboratory for complete blood counts (CBC) using a Bayer Advia 120 hematology analyzer (Bayer Diagnostics, Tarrytown, NY, USA) and blood chemistry profile using a Roche/Hitachi 912 Clinical Chemistry System (Roche Diagnostics, Indianapolis, IN).

Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA) with GraphPad Prism software (version 4.00, GraphPad Software Inc., San Diego, CA, USA).The model included treatment effects.When the treatment effect was significant, (P<0.05), the Student-Newman-Keul's multiple comparison test was used to separate means.Tendencies were declared if P>0.05<0.10.

RESULTS
Table 1 shows that ensiling, acid and alkali extraction reduced (P<0.05) the L-Dopa content of the Mucuna bean from 2.8 g/100g in the Control to 1.2, 0.1, and 0.0 g/100g, respectively.Concomitantly, the gross energy value and crude protein and water-soluble carbohydrate concentrations decreased (P<0.05) while the starch and aNDF contents increased (P<0.05).Total DM intake, average daily DM intake, total weight gain, initial and final body weights, feed conversion ratio and average daily weight gain were not different among treatments (P>0.05;Table 2).Dietary treatments did not affect (P>0.05)distance traveled or number of line crossings in the open field test during d 3 or 10 (Figure 1).However, compared to CON, feeding Mucuna diets reduced (P<0.05) the sum of total distance traveled and total number of line crossings on d 3 and 10.The reduction was more statistically significant (P<0.01) in rats fed MUC versus those fed detoxified Mucuna diets (P<0.05).Necropsy revealed that treatments did not affect (P>0.05)liver, kidney, and testicular weights (Table 3).However, rats fed ACD tended to have greater (P=0.07)heart weight (g/100g BW) than other treatments.
Table 2. Effects of feeding diets containing unprocessed or detoxified Mucuna pruriens on DM intake and growth of rats.4).Feeding MUC, CON or BAS also produced greater (P<0.05)hemoglobin concentration distribution width (HDW) than feeding SIL or ACD.

Mucuna
Counts of eosinophils tended (P=0.07) to be greatest in rats fed BAS followed by CON and SIL, and least in those fed ACD.

DISCUSSION
At the 10 g/100g inclusion rate, feeding detoxified or undetoxified Mucuna did not affect rat behavior in the open field on d 3 or 10.The open field is an unfamiliar environment to rats, which naturally tend to explore novel situations.A rat that is sedated or ill will travel less when exposed to a new environment such as the open field, which allows for rat locomotion and motor activity (Vogel, 2002).In contrast, an animal that is stimulated and healthy will spend more time exploring the center of the open field and will travel further.
During the open field test on d 10, a software problem prevented computer analysis of data from nine out of twelve animals per treatment.Greater treatment replication may have increased the numerical trends for Mucuna ingestion to reduce line crossings to significant levels and for the latter effect to be less pronounced when Mucuna was detoxified.The fact that detoxification of Mucuna (Huisden et al., 2019) did not affect open field behavior on d 3 or 10 may have been because of the relatively low Mucuna inclusion rate.
Nevertheless, the sum of the total distance traveled and total line crossings on d 3 and 10 were reduced by feeding Mucuna diets, indicating increased abnormal behavior relative to CON, which may be related to side effects caused by Mucuna-derived hallucinogens (Szabo and Tebbett, 2002) or hormonal imbalance (Tavares et al., 2017).Cotzias et al. (1974) reported that feeding rodents high levels of L-Dopa adversely affected behavior and caused motor hyperactivity, muscle jerks, and stereotyped movements.Intake of L-Dopa in humans is also associated with psychiatric disturbances, which can lead to nervousness, anxiety, agitation, insomnia, confusion, delirium, depression, and psychotic reactions with hallucinations and anorexia (Szabo and Tebbett, 2002).The greater statistical significance of the reductions in total line crossing and distance on d 3 and 10 in MUC versus detoxified diets suggests the latter have reduced adverse effects of MUC on behavior in the open field.
The fact that feed intake, weight gain, and feed efficiency did not differ among treatments, indicates that acceptability and nutrient bioavailability of all diets were relatively similar.Mucuna-based diets have reportedly been associated with a decrease in acceptability and intake compared to soybean-based diets (Del Carmen et al., 1999;Flores et al., 2002).The relatively low inclusion rate (10 g/100g of diet DM) in this study may explain why intake and performance was not adversely affected by feeding Mucuna.
Although detoxification strategies.However, longer term feeding trials are needed to validate their effects on the performance of rats and non-ruminant livestock.
It is interesting to note that unlike MUC, feeding detoxified diets did not result in splenomegaly and monocytosis.The splenomegaly caused by MUC agrees with studies where spleen enlargement occurred when poultry were fed Mucuna beans (Iyayi et al., 2005;Pugalenthi et al., 2005;Carew and Gernat, 2006).The spleen is the largest collection of lymphoid tissue in the body and splenomegaly resulting from feeding MUC probably reflects increased workload or hyperfunction of the organ.Splenomegaly is associated with red blood cell destruction in the spleen, congestion due to portal hypertension, infiltration by leukemias and lymphomas, obstruction of blood flow or antigenic stimulation, and infection (Grover et al., 1993).Carew et al. (2003) observed lymphoid necrosis, macrophage proliferation, and lympho-phagocytosis of the spleen when Mucuna was fed at a 12 g/100g inclusion rate in the diet of broilers (Iyayi et al. 2005;Pugalenthi et al., 2005) reported that lymphoid depopulation in the spleen is indicative of the degenerative effects associated with feeding raw Mucuna beans.
Relative to CON, dietary inclusion of MUC also increased the potential for monocytosis, a state of excess monocytes in the peripheral blood indicative of various disease states (Meuten, 2002).Monocytes are leukocytes that replenish macrophages and dendritic cells and elicit an immune response at infection sites.In the tissues, monocytes mature into different types of macrophages that are responsible for phagocytosis of foreign substances in the body.Monocytosis can indicate inflammation, stress due to disease, hyperadrenocorticism, immune-mediated disease, and malignant tumors (Meuten, 2002).
Interestingly, the splengomaly, monocytosis and reduced P concentrations due to feeding MUC did not occur when detoxified diets were fed.Since the detoxified diets contained reduced levels of L-Dopa, the main toxic compound of concern in Mucuna, it is likely that L-Dopa toxicity was at least partially responsible for the adverse clinical conditions in rats fed MUC.
Alkaline phosphatases remove phosphate groups by dephosphorylation, and they are most effective in an alkaline environment (Coleman, 1992).Feeding MUC resulted in lower plasma alkaline phosphatase (hypophosphatasemia) and lower phosphorus concentrations relative to detoxified Mucuna treatments.Phosphatases are involved in signal transduction because they regulate the action of proteins to which they are attached.Therefore, detoxification of Mucuna may have reduced adverse effects of the bean on signal transduction; the modulating effect on transcription factors is in agreement with the findings Ulu et al. (2018).
Elevated MCHC and CHCM are often associated with sickle cell anemia and other diseases (Clark, 1989).Therefore, the higher, MCHC and CHCM in rats fed MUC suggests that they were more anemic and may have had greater red blood cell deformation than Control rats.Whereas, rats fed detoxified diets had similar CHCM and MCHC to Control rats.Hemoglobin distribution width is a measure of the heterogeneity of the red cell hemoglobin concentration and abnormally higher values have been associated with iron deficiency anemia (Lee et al., 1994) and steady state sickle cell anemia (Billet et al., 1988).The HDW values were only reduced by SIL and ACD, suggesting that these treatments were more effective at preventing anemia and red blood cell deformities; this agrees with findings of Mohamed et al. (2018).
. CONCLUSIONS Dietary inclusion of detoxified or undetoxified Mucuna at 10 g/100g of diet DM did not affect any performance measure.Compared to feeding CON, feeding MUC decreased blood phosphorus concentration and caused splenomegaly and monocytosis but feeding detoxified Mucuna diets did not have these effects.Feeding MUC also decreased alkaline phosphatase levels relative to feeding detoxified Mucuna diets.The behavior of rats fed Mucuna diets instead of CON was characterized by decreased total distance travelled and decreased total line crossings in the open field, but the effect was more statistically significant for the undetoxified Mucuna diet.This suggests that detoxifiying Mucuna reduced the severity of the abnormal behavior caused by feeding Mucuna.Therefore, the detoxification processes improved the safety of Mucuna by reducing the toxic effects of the L-Dopa it contained, making it more widely usable as a safe food source and a nutraceutical.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors.
Figure 1.Effect of feeding unprocessed or detoxified Mucuna pruriens on A) distance traveled on d 3; B) open field line crossings on d 3; C) distance traveled on d 10; D) open field line crossings on d 10; E) total distance traveled on both days; and F) total line crossings on both days.Means without a common superscript letter differ (P < 0.05 *, P < 0.01 **).CON = control diet; MUC = untreated Mucuna diet; ACD = Mucuna beans detoxified by acetic acid extraction; BAS = Mucuna beans detoxified by sodium hydroxide extraction; SIL = Mucuna beans detoxified by ensiling.Error bars denote standard error.

Figure 2 .
Figure 2. Effects of feeding detoxified Mucuna pruriens on blood levels of A) alkaline phosphatase; B) phosphorus; and C) monocytes.CON = control diet; MUC = untreated Mucuna diet; ACD = Mucuna beans detoxified by acetic acid extraction; BAS = Mucuna beans detoxified by sodium hydroxide extraction; SIL = Mucuna beans detoxified by ensiling.Means without a common superscript letter differ (P < 0.05); error bars denote standard errors.
the detoxification methods resulted in different L-Dopa and CP concentrations, similar performance and clinical data in rats fed all detoxified diets suggest the CP bioavailability and food safety were comparable among detoxified Mucuna diets.Solvent extraction typically disrupts the protein structure and degrades amino acids in Mucuna(Adebowale et al., 2007), yet, feeding BAS and ACD did not adversely affect performance, indicating the promise of these

Table 1 .
Chemical composition (g/100g DM) and energy value of the Teklaad 8604 rat chow and the undetoxified (Control) and detoxified Mucuna beans.

Table 3 .
Effects of feeding diets containing unprocessed or detoxified Mucuna pruriens on organ weights (g/100 g of body weight) of rats.
Means within a row, means without a common superscripts differ (P<0.05)significantly; a Control diet, standard rat chow without Mucuna; b 10 g/100g of Control diet replaced with untreated Mucuna; c 10 g/100g of Control diet replaced with Mucuna beans detoxified by acetic acid extraction; d 10 g/100g of Control diet replaced with Mucuna beans detoxified by sodium hydroxide extraction; e 10 g/100g of Control diet replaced with Mucuna beans detoxified by ensiling.SEM= Standard error of the mean

Table 4 .
Effects of feeding diets containing unprocessed or detoxified Mucuna pruriens on complete blood count data.
Means within a row without a common superscripts differ (P<0.05); a Control diet, standard rat chow without Mucuna; b 10 g/100g of Control diet replaced with untreated Mucuna; c 10 g/100g of Control diet replaced with Mucuna beans detoxified by acetic acid extraction; d 10 g/100g of Control diet replaced with Mucuna beans detoxified by sodium hydroxide extraction; e 10 g/100g of Control diet replaced with Mucuna beans detoxified by ensiling.SEM= Standard error of the mean