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Cuban Journal of Agricultural Science

Print version ISSN 0864-0408On-line version ISSN 2079-3480

Cuban J. Agric. Sci. vol.54 no.2 Mayabeque Apr.-June 2020  Epub June 01, 2020



Evaluation of the antioxidant and hepatoprotective activity of Mucuna pruriens (L) cv. utilis forage meal and their polyphenols extract in Sprague Dowley rats

Idania Scull Rodríguez1  *

Lourdes Savón Valdés1

Iraida Spengler Salabarria2

Magaly Herrera Villafranca1

1Dpto. Monogástrico, Instituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba.

2Centro de Estudios de Productos Naturales, Facultad de Química, Universidad de La Habana, Cuidad de La Habana, Cuba.


In order to evaluate the antioxidant and hepatoprotective effect of Mucuna pruriens (L) cv. utilis forage meal and their polyphenol extract, an intoxication model induced with carbon tetrachloride (CCl4) was used. A total of 60 Sprague Dowley rats from both sex of 7-8 weeks of age were used, distributed in three treatments, according to a completely random design. The treatments consisted in a control group with EAO-1004 diet, which contained the premixture with antioxidants, and two experimental groups, which was supplied the Mucuna pruriens forage meal and their polyphenol extract, in a dose of 300 mg/kg body weight respectively, in replacement of the control diet premixture. All groups received the diet of their treatments and water ad libitum. At the end of the experimental period (30d), the animals were slaughtered and biochemical blood indicators were studied, the alanine aminotransferase (ALT) and the aspartate aminotransferase (AST) enzymes were quantified. The activity of the catalase antioxidant enzyme and the antioxidant state of the animals were also determined. The increase of the catalase enzyme concentration in liver (10.42 UI/L) and blood serum (20.50 UI/L) showed that the meal and their extract change the redox state caused by the CCl4.The forage meal and their polyphenols extract showed its hepatoprotective effect against the damage action of the CCl4,evaluated by the alanine aminotransferase and aspartate aminotransferase. The results showed that it is possible to use, in diets for rats the forage meal and their polyphenols extract with antioxidants effects similar to those of the control diet, which used synthetic antioxidants, vitamins and minerals. It is suggested to developed similar studies in productive animals, that includes studies of doses optimization for different animal categories.

Key words: oxidative stress; serum polyphenols; catalase; aspartate aminotransferase

The vegetables have biologically active substances which can exert beneficial effects on the physiology, productivity and health of animals, when acting as antioxidants, antimicrobial and antiparasitics (Salazar et al. 2019).

Scientifics evidences show that the use of some plants, such as legumes or some of their components protects the organism from the oxidative stress damages (Rojas et al. 2015).Tropical legumes, besides constituting an excellent nutrients source, they also supply secondary compounds with certain chemical structures, able to contribute new products with antioxidant activity (García et al. 2014).This is the case of Mucuna pruriens, a legume from the Fabaceae family, with potential to prevent damages derivate from the oxidative stress, an aspect a little known in Cuba until today (Longhi et al. 2011).

The objective of this study was to evaluate the antioxidant and hepatoprotective activity of Mucuna pruriens (L) cv. utilis forage meal and their polyphenols extract, with the use of rats as animal model.

Materials and Methods

Plant material collection. Plants from Mucuna pruriens (L) DC. cv. utilis (Wall. ex Wight) L. H. Bailey, sowing in June, in rainy season, in a brown soil with carbonate were used (Hernández et al. 2019). An area from“Ayala” farm belonging to the Instituto de Ciencia Animal (ICA) was used. The seeds were obtained in the legumes seed bank from the Estación Experimental de Pastos y Forrajes “Miguel Sistach Naya” (ICA). Fertilization and irrigation was not used.

The plants cut was manually carry out, with knife, at 80 d after sowing, at a height of 5cm over soil level, and when 100 % was flowered, according to Díaz et al. (2003). The plot was totally cut during the morning, moment in which samples were selected, and 50cm of border effect was eliminated.

Meals elaboration. The plants (leaves, young stems and flowers) were cut and chopped. Later, were dried at room temperature, in a aired room. Humidity was reduced up 20%, which was monitoring with the dry matter (DM) determination in different moments of the drying process. Samples were milled at 1 mm particle size and stored in paper bags until their use.

Elaboration of polyphenols extract. To evaluate the effect of phenolic compounds on antioxidant activity in vivo, an extract from M. pruriens forage meal was elaborated in triplicate, according to Makkar (2003) methodology. A total of 100 g of meal were weighed and 1000 mL of hydroalcoholic solution (70 %) were added. The extraction was performed for 15 min with the use of a Bandelin Sonorlex Sonicator, serie 2000, made in Germany. The supernatant was separated and the extraction process was repeated three times to the solid waste. The resulting solution was filtered and rotovaporated (volume was reduce to 50%). The obtained extract was keep in amber bottles and refrigerated at 5-8 °C temperature until their use.

The organoleptic characteristics of the hydroalcoholic extract, obtained from M. pruriens forage meal were intense green color, herbarium odor and insipid taste.

Area of research. The study was carry out in the Centro de Toxicología Experimental (CETEX) from the Centro Nacional para la Producción de Animales de Laboratorio (CENPALAB).

The test was performed with the approving of the Comité Institucional para el Cuidado y Uso de los Animales de Laboratorio (CICUAL) from CENPALAB. The research was performed according to the Principles of Good Laboratory Practices (MINSAP 2004), the Operational Procedures of Work for Experimental Toxicology (POT) from CETEX (2012), and by the biosafety considerations expressed in the Biosafety Manual from the CETEX (2012).

Environmental conditions. The environmental conditions were daily recorded by a RH 600(POT meter of temperature and relative humidity. The mean temperature of the room was 23.3 ± 0.3 ºC, humidity 67.7 ± 0.9 % and the photoperiod 12:12 hours light/dark.

Animals and diets. A total of 60 Sprague Dawley young rats of both sex with weight between 125-150 g and average age between 7and 8 weeks were used. They were randomly housed in T3 autoclaveables TECNIPLAST boxes, translucent plastics, individuals, with bottom and stainless metal grid lid.

A total of 20 animals (10/sex) were distributed per treatments. A control with EAO 1004 diet was established, which had the antioxidant premixture (buthylhydroxytoluene BHT, vitamins and minerals) (treatment 1), and other two experimental groups, to which was supplied M. pruriens forage meal (treatment 2) and their polyphenols extract (treatment 3) in replacement of the antioxidant premixture, present in the control diet, that represents 2%. To the experimental groups was supplied a unique dose of forage meal (300 mg/kg live weight) and their polyphenols extract. The diets were formulated in accordance with the nutritional requirements for this species (NRC 1995) (table 1).

Table 1 Chemical composition of the experimental diets 

Indicators (%) Control diet EAO 1004 (M. pruriens) forage meal Polyphenols extract
DM 93.40 88.54 88.91
CP 26.23 26.61 27.90
Ca 0.80 0.56 0.59
P 0.48 0.42 0.43

Diets ingredients. The diets were composed by wheat, barley, soybean, sunflower, fish meal, sugar, calcium carbonate and common salt. The premixture contained retinol, cholecalciferol, tocopherol, phylloquinone, thiamine, riboflavin, pantothenic acid, choline, pyridoxine, cobalamin, folic acid, nicotinic acid, ascorbic acid, antioxidant (BHT), magnesium, potassium, iodine, iron, copper, cobalt, manganese, zinc, selenium and molybdenum.

The adaptation of the animals to the food was carry out when supplying a mixture 50:50 of the EAO 1004 diet and the corresponding diet to each experimental group during three days. Later, in a period of 7d, they intake the diet for each group, to finish the adaptation to the food. Water and food were offered ad libitum.

Experimental procedure. The experiment was developed during thirty days. Twenty hours before finished the experimental period, carbon tetrachloride (CCl4) at 10 % was given to the animals by intraperitoneal way (doses: 1 mL/kg live weight) (POT 2010;, with the objective of inducing the oxidative stress in the organism. Later, the rats were slaughtered by the application of a lethal dose of diethyl ether. With 12h fasting, blood (POT 2010; was extracted to the animals through the orbital sinus (five/sex). The blood was collected in 2 mL vial, where was coagulate. Later, was centrifugated at 12 000 r.p.m. in an Eppendorf centrifuge for 10min. (POT 2010; and the serum was separated. A part was use to perform biochemical determination and the other one was stored at -80ºC to perform other enzymatic determinations. Finally the incision of the abdominal cavity was carried out and the liver and the intestine were extracted for preparing the homogenates.

Preparation of the liver and intestine homogenates. A portion of the respective tissues with weight between 1.2 y 1.5 g approximately was cut. It was divided in small fragments and washed twice with 10 mL of sodium chloride solution (NaCl) 0. 9 % at 4°C. A total of 1g of tissue was weighed and it was added 10 mL of trichloroacetic acid solution (5 %) with disodic salt and ethylendiamine tetraacetic acid (EDTA) 1x10-3 M. It was homogenized for 6min. at 10000 rpm, at 4 °C temperature. Later, it was placed in refrigerated centrifuge for 15min, at 5000 rpm and 4 °C. When finished, the precipitate was throwing away and the supernatant was stored at 4°C until performing the conveniently enzymatic determinations.

Clinical observations. The clinical observations were daily performed in the morning during all the experimental period. The general state of animals (POT was evaluated, as well as the changes in skin and coat, mucous membrane and eyes, respiratory and circulatory systems, central nervous system and autonomous, somatosense activity and behavior pattern. A particular attention was taking to the appearance of diarrhea, shaking, convulsions, lethargies, salivations, sleep, and/or possible deaths.

Catalase determination. The activity of this enzyme was determined according to the methodology described in the PNO/TEC/0314, from Centro de Estudios para las Investigaciones y Evaluaciones Biológicas (CEIEB) in Cuba. The method is based on measure the decomposition speed of hydrogen peroxide, catalyzed by the catalase enzyme, at a 240 nm wavelength. For these a spectrophotometer was used (Boehringer 1987). The catalase activity was expressed in international units per liter (IU/L).

Total phenols in serum (TF). To determine the total polyphenols a reference solution of tannic acid (Sigma Aldrich) at 0.5 g/L concentration was used. The analysis was carrying out by using the Folin-Ciocalteau reagent, according to the methodology described by Makkar (2003).

Indicators of the blood biochemical. A total of 10 animals per treatment (5per sex) were used. It was determined the total cholesterol (T- CHOL), triglycerides (TG), uric acid (UA), total proteins (TP), alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). An automatic analyzer Cobas Integra 400 PLUS (Roche Diagnostic Systems) (POT was used for these determinations.

Statistical analysis. The theoretical assumptions of the analysis of variance for the ALT and AST variables for the errors normality were proven. The Shapiro-Wilk (1965) test and the variance homogeneity (Levene 1960) were used. Both variables fulfill with the assumptions that is why a completely random design in 3 x 2 factorial arrangements was used. The factors were the three treatments (control, forage meal and polyphenols extract), and the sex (females and males). The results were analyzed by the Infostat statistical package (Di Rienzo et al. 2012). For means comparison the Duncan (1955) test for P<0.05 was used.

Results and Discussion

During the experiment development there were not changes associated to treatments effects on the animal’s clinical events, whose behavior was normal for the species, except a male animal from treatment 3, which showed crackling noises and apparent increase of the respiratory frequency, between the 9 and 11 study days. The test end with 100% of survival.

The determination of the antioxidant effect of the forage meal and their polyphenols extract was evaluated by the quantification of the enzymatic activity of the catalase in serum, liver and intestine. There were interaction of the factors treatment and sex for this enzyme (table 2).

Table 2 Activity of the catalase antioxidant enzyme in serum, liver and intestine of Sprague Dawley rats, treated with M. pruriens forage meal and their polyphenols extract.  

Catalase activity (UI/L) Sex Treatment (±) SE Signif.
Control Forage meal Polyphenols extract
Serum Male 15.08 a 16.42 a 20.50 b 0.11 P=0.0493
Female 15.08 a 16.00 a 15.33 a
Liver Male 12.17 b 10.42 b 6.67 a 0.76 P=0.0063
Female 6.42 a 6.05 a 6.03 a
Intestine Male 4.33 a 4.33 a 3.92 a 0.53 P=0.0166
Female 8.00 b 5.08 a 4.77 a

a,b Different letters per rows show significant differences for P < 0.05 (Duncan 1955)

The catalase serum activity showed high values (P < 005) in the males from the treatment with the polyphenols extract. While, in the liver there was higher activity of the enzyme in the males from the treatment with forage meal, which was similar to the control. In the case of the intestine, the experimental treatments and the control of the males showed low calatase activity than those found in the females from the control group.

The phenolic compounds that the polyphenols extract contributes are the main responsible for the increase of the activity of the catalase antioxidant enzyme in the blood serum. The literature reports that the polyphenols which form the different vegetables extracts and foods increased the endogenous antioxidants levels, as the enzymes superoxide dismutase and catalase enzymes (Gupta et al. 2006).

To several flavonoids are known by their ability of inhibit the formation of free radicals, aspect related with the basic chemical structure of this compounds. The hydroxyl groups in the C5 and C7, join by the double bond between the C2 and C3, are essential for its high inhibit activity in the free radicals formation (Spanou et al. 2012). Scull (2018) identified flavone glycosides in M. pruriens forage meal, compounds that corresponds with type of structure, that is why the inhibit activity observed in this study can attributed, in part, to the structural characteristics of these phenolic compounds.

In the liver, the increase of the enzyme activity, in the control group as in the meal treatment, seems to answer to the effect of the high number of bioactive compounds of different chemical structure presents in the diet. Similar results to this study showed Batista et al. (2015), when evaluating the in vivo antioxidant activity of the Buriti fruit (Mauritia flexuosa) in Wistar rats, and no found differences between the catalase values of the experimental group and the control.

When analyzing the intestine, there were significant differences between the treatment and sex. However, the females that intake the control treatment were the ones that have high activity in this organ. This result could be related with the use of one level of doses for the test products (300 mg/kg live weight) was not enough for the meal and their polyphenols extract showed antioxidant defenses in this organ. This aspect become worse, due to the high fiber (NDF) content the meal has could increase this damages.

The CCl4 led the oxidative stress and damages in the intestine, which exceed the antioxidant capacity of the catalase, with the consistent decrease of their activity respect to the female control. These results are similar to those reported by Stojanović et al. (2016), who observed the decrease of the catalase enzyme activity in Wistar rats intestine, when supplying a solution of D.L homocystein tiolactone.

The antioxidant state of animals was determined by the concentration of phenolic compounds in plasma. There was not interaction of the treatments and sex for the serum polyphenols. Figures 1 and 2 shows the results of the total polyphenols in the serum of the Sprague Dawley rats in experimentation, for the components treatments and sex.

P=0.0098; SE (±) 0.67

Figure 1 Concentration of total polyphenols in rats serum treated with M. pruriens forage meal and their polyphenols extracts 

The concentration of total polyphenols in the animals serum that intake the forage meal was similar to that of the control, and differed of the polyphenols content of the group that intake the polyphenols extract. This could be due to chemical structures of the flavonoid glycosides present in the extract, because the aglicone and their glucoside can create derivates, which can be hydrosolubles and more easily to excrete (Kamiloglu et al. 2013).

Several studies coincided in showing that compounds with antioxidants properties as polyphenols, can improve the antioxidant oxidant state in the organism (Quiñones et al. 2012 and Prescha et al. 2018). However, the results are related with the physiology of each animal species, since the action of these compounds directly depend on their bioavailability and intrinsic factors (gastric Ph, digestive enzyme activity and bacterial microflora). These aspects can lead the hydrolysis and/or transformation of polyphenols to biologically active and bioavailability molecules (Lizárraga-Velázquez et al. 2018).

P=0.0008; SE (±) 0.54

Figure 2 Concentration of total polyphenols in the serum of female and male rats, treated with M. pruriens forage meal and their polyphenols extract 

In the sex component there was high (P<005) polyphenol concentration in the serum of the male rats with respect to the males total. The genus is another factor that is associated with the differences in the oxidative stress, probably due to the estrogens with antioxidant properties in females. The estrogens has a hydroxyl group in C3 position, that allows to eliminating reactive species of the oxygen and also showed its protective effect before the cellular damage of the free radicals (Kander et al. 2017).

The evaluation of the antioxidant and hepatoprotective effect of the forage meal and their polyphenols extract before the hepatic intoxication, lead by the carbon tetrachloride (CCl4), was performed by the transaminase activity. In the serum levels of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) there was interaction of the factors treatment and sex for both enzymes (table 3).

Table 3 Transaminase enzymes in blood serum of rats treated with M. pruriens forage meal and their polyphenols extract.  

Catalase activity (UI/L) Sex Treatment (±) SE Signif.
Control Forage meal Polyphenols extract
ALT (U/L) Male 67.00 a 126.67 ab 72.00 a 28.62 P=0.0385
Female 187.00 b 80.67 a 133.33 ab
AST (U/L) Male 187.00 a 349.67 bc 202.67 ab 50.11 P=0.0366
Female 488.00 c 389.33 c 496.67 c

a,b,c Different letters per rows show significant differences for P < 0.05 (Duncan 1955)

The serum activity of the ALT enzyme differed (P<0.05) between females and males that received the control treatment. The groups that intake the resting treatments showed similar performance in both sex. The ALT enzymatic activity in the male control group did not have significant differences between the groups that were added the polyphenols extract and mucuna forage for females and males, respectively. In the serum levels of the ATS enzyme there were not differences between the female groups that intake these treatments, which showed the highest activity values. With respect to males, there were not significant differences in the control groups and those that intake the meal treatment.

When analyzing these results, it was observed that the CCL4 supply caused in the male rats from the control treatment increase of the ALT serum levels over the reference values (32,2-80,9 UI/L), reported by Alemán et al. (1998) and León et al. (2011) for Sprague Dawley species. This observation was provided by several authors during the evaluation of the antioxidant activity of different substances, in which a model of hepatic intoxication with carbon tetrachloride was used (Gupta et al. 2006 and Bhoomannavar et al. 2011). In comparison, the males showed an activity that is considered in the referenced limits for this sex, according the previous mentioned authors.

An aspect to highlighted in female rats is the decrease of the ALT serum levels in the group corresponding to the mucuna forage meal related to the control group (P<005). This show the forage meal efficiency as natural antioxidant for counteracts the CCl4 toxic effects.

The ALT is an enzyme that is in the hepatocytes mitochondria, whose crossing to the surrounding environment occurs due to damage in the hepatic functioning. The CCl4 is a chemical inductor of the hepatic damage that is why the supplying causes increase of the serum levels of this enzyme as a secondary effect (Weber et al. 2003). In this sense, the supplying of substances with antioxidant properties could avoid or decrease the toxic effect of this substance.

In the case of the AST enzyme, the protective effect is better for the males from the treatment where the polyphenols extract was supplied. In females, although the serum concentrations of this enzyme did not differ between treatments, the enzymatic activity was superior to the referenced levels (253 UI/L) (León et al. 2011). The previous could be due to the high dispersion of data (SE= 50.11 %).

Likewise, the AST is in the cytosol and mitochondria of hepatic cells, heart, skeletal muscle, brain, kidney, pancreas, lung, erythrocytes and lymphocytes, that is why the AST high levels are not specific of hepatic damage (Grattagliano et al. 2009). The results with the ALT enzyme are similar to those showed by Yang et al. (2018) in the evaluation of the hepatoprotective effect of the methyl ferulic acid before the oxidative stress induced by CCl4 in rats.

Substances as total proteins and the uric acid acted as defense mechanism against the oxidative damage of body fluids. In this experiment, where the effect of the replacement of the antioxidant premixture from the control group by the forage meal and their polyphenols extract in the blood biochemical indicators was analyzed, there were not significant interactions between sex and treatments. Tables 4 and 5 show the determinations for the main effects.

Table 4 Performance of the blood biochemical indicators in rats treated with M. pruriens forage meal and their polyphenols extract.  

Blood biochemical Control Forage meal Polyphenols extract (±) SE Signif. Refernce values1
ALP (U/L) 110.33 119.67 120.00 11.66 P=0.8047 82.8-311.7
PT (g/L) 68.00 66.48 67.93 1.49 P=0.7246 56.69-73.5
AU (mg/dL) 0.89 1.01 0.84 0.12 P=0.5957 0.87-4.72
CHOL-T (mmol/L) 1.95 1.91 1.84 0.15 P=0.8670 2.23-4.70
TG (mmol/L) 0.66 0.82 0.60 0.12 P=0.4242 2.49-6.65

1References values, according to León et al. (2011).

Table 5 Sex component for the bichemical blood indicators in rats treated with M. pruriens forage meal and their polyphenols extract.  

Blood biochemical Female Male (±) SE Signif. Reference values1
ALP (U/L) 86.33 147.00

  • 9.52

  • P=0.0007

  • F: 82.8- 297.3

  • M: 85.4- 311.7

PT (g/L) 69.30 65.64

  • 1.22

  • P=0,0550

  • F: 56.69-73.5

  • M: 59.9- 73.5

AU (mg/dL) 0.89 0.94

  • 0.10

  • P=0.7289

  • F: 2.22- 4.72

  • M: 0.97- 4.72

CHOL-T (mmol/L) 2.14 1.66

  • 0.12

  • P=0.0188

  • F: 2.28- 3.28

  • M: 2.23 - 4.70

TG (mmol/L) 0.52 0.86

  • 0.10

  • P=0.0313

  • F: 4.24-7.37

  • M: 2.49-6.65

1Reference values, according to León et al. (2011).

The applying of CCl4 to the rats did not caused differences for the treatment factor (table 5). All the analyzed indicators were similar to the reference values for the species (León et al. 2011).

Al Said et al. (2011) when using the CCl4 model in rats found that the Grevistenax ethanolic extract, in 250 mg/kg doses, significantly prevent the alkaline phosphatase increase.

In several researches of natural products with hepatoprotective and antioxidant activity is referred, generally, decrease of the protein synthesis, caused by the carbon tetrachloride (Bhoomannavar et al. 2011 and Heba et al. 2011).These reports are not corresponding with the results found in this experiment, which could be mean that the mucuna forage meal and their polyphenols extract exerted their antioxidant action. This not allowed that the liver biosynthetic capacity could be affected.

The serum levels of the cholesterol and triglycerides of the experimental treatments were similar to the control. It is known that the CCl4 cause hepatic damage through peroxidation process of the lipids that are in the membrane of the hepatic tissue, or through oxidant mechanisms (Surendra and Bodakhe 2007, Bhoomannavar et al. 2011 and Heba et al. 2011). In addition, the carbon tetrachloride is a very lipophilic compound, that could also acting increasing fat solubility and making easier its passing to the bloodstream.

Related to sex factor (table 5), the males differed to the females in all the analyzed indicators, except the AU levels. Females, in general, were superior to males for total proteins and cholesterol, while the males exceed the females in the serum values of phosphatase and triglycerides.

The fact that the males have serum concentrations of alkaline phosphatase higher than the females could be due to their fasting growing and this enzyme reached higher values in fast growing animals (Wolford et al. 1986). Similar results were fund by Alemán et al. (1998) and Hatayama et al. (2003), when they analyzed biochemical blood indicators in laboratory rats from the Sprague Dawley species.

León et al. (2011), when reporting the common performance ranges for hematochemical indicators in rats, explained that the differences between genus can be due to the organism physical size and the hormonal changes of each sex. Kander et al. (2017) showed the relation between the genus and the oxidative stress, since stress is involved in many diseases that are in different ways between males and females. For the oxidative stress occurred, should exits imbalance between the ERO production and the antioxidant defense system. That is why, it is considered that there is difference in the expression and/or activities of the antioxidant enzymes between man and women.

In spite of the results showed until today, a great number of researchers to better explore the function that the genus developed in the gene expression of the antioxidants and other genes associated to the oxidative stress are needed.

The results of this experiment showed that the M. pruriens forage meal and their poliphenos extract have antioxidant activity and hepatoprotective functions against the oxidative stress damage in Sprague Dawley rats. It suggested to evaluate their antioxidant potential in productive animals and to include studies of doses optimization for different animal categories.

The performed study should contribute and stimulate the use of the aggregate value of this legume as local resource for animal feeding, because the use of plants in productive systems is still poor.


Thanks to the researches, specialists and technicians from the Centro de Toxicología Experimental (CETEX), del Centro Nacional para la Producción de Animales de Laboratorio (CENPALAB), for carry out part of this experiment.


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Received: June 24, 2019; Accepted: February 21, 2020

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