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Toxicological evaluation of 10% Solanum lycocarpum St. Hill fruit consumption in the diet of growing rats: Hematological, biochemical and histopathological effects
Experimental and Toxicologic Pathology 62 (2010) 549–553
Contents lists available at ScienceDirect
Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Toxicological evaluation of 10% Solanum lycocarpum St. Hill fruit consumption in the diet of growing rats: Hematological, biochemical and histopathological effects Milena Rodrigues Soares-Mota a, Aline Schwarz b, Maria Martha Bernardi c, Paulo Cesar Maiorka c, Helenice de Souza Spinosa c, a
~ em Patologia Experimental e Comparada, Brazil Programa de P´ os-Graduac- ao Departamento de Ana lises Cl´ınicas e Toxicol´ ogicas, Universidade Federal do Rio Grande do Norte, RN, Brazil c ~ Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508-270, Departamento de Patologia, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao ~ Paulo, SP, Brazil Sao b
a r t i c l e in fo
abstract
Article history: Received 26 May 2009 Accepted 15 July 2009
Solanum lycocarpum St. Hill (Solanaceae) is a native shrub very common in the Brazilian savanna. The fruit of this plant contains steroidal glycoalkaloids that may disrupt the endocrine system. Because this plant is employed in folk medicine for the management of diabetes, obesity and decreasing cholesterol levels, the present study determined the possible toxic effects of exposure to S. lycocarpum fruit from weaning (21 days old) until adult age (8 weeks of treatment) in male and female rats. In male rats, the plant reduced weight gain, while few significant differences were observed in female animals. Slight significant differences were observed in food and water consumption and in hematological parameters in treated rats. Reductions in adrenal gland, spleen, heart, kidneys and thymus weights of treated males were observed, while increased relative weights were detected in the heart, epididymises, lungs, seminal vesicles, and testicles. In females, no differences were observed in organ weights and few differences were observed in relative weights of some organs. The histopathologic study showed no alteration between groups. Serum biochemical parameters showed triglyceride reductions in treated animals of both sexes; in females, an increase in albumin and alanine aminotransferase levels and a reduction in total protein levels were noted. The present data therefore demonstrate sex-related differences in S. lycocarpum toxicity. & 2009 Elsevier GmbH. All rights reserved.
Keywords: Solanum lycocarpum (St. Hil.) Toxicological study Hematological parameters Biochemical parameters Plant
Introduction Solanum lycocarpum St. Hill (Solanaceae) is a common plant in the Brazilian savanna and, in some regions, people consume its fresh or cooked fruits (Lorenzi, 1991). In Brazil, the effects obtained from S. lycocarpum fruit have been employed in folk medicine for diabetes management, obesity and decreasing cholesterol levels (Dall’Agnol and von Poser, 2000; Oliveira et al., 2003). It has also been reported that this plant possesses diuretic, anti-spasmodic, anti-epileptic, sedative and anti-inflammatory activities (Vieira et al., 2003). Solamargine and solasonine are two major glycoalkaloids found in a large number of Solanum species (Eltayeb et al., 1997) and their steroidal aglycone, solasodine, has been used as a key starting ingredient in the manufacture of steroidal drugs. The compounds 16-dehydropregnenolone acetate and dehydroepian-
Corresponding author. Tel.: +55 11 30917656; fax: +55 11 30917829.
E-mail address: [email protected] (H.d. Spinosa). 0940-2993/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2009.07.006
drosterone acetate, key starting sources for androgen production (Goswani et al., 2003), and the compound 3-B-acetoxypregna5,16-dien-one, a key starting source for progesterone production (Haraguchi et al., 1978), can be obtained from solasodine in laboratory conditions. Recently, we showed that the unripe fruit of this plant contained 1.85% total alkaloids, where 0.06% corresponded to solamargine and 0.09% to solanine (Schwarz et al., 2007). Previously, it has been shown that long-term consumption of S. lycocarpum fruit can produce slight toxic effects in exposed adult rats (Maruo et al., 2003). In this study, male (60 days of administration) and female (37 days) adult rats received a diet containing 3% S. lycocarpum fruit. It was observed that while significant reductions in uterus and liver weight occurred in treated females, no alterations were reported in males. In addition, no significant differences were observed in other organ weights, blood enzymes or proteins. Furthermore, these authors suggested that S. lycocarpum promotes sex-related toxicity. Because of the slight toxic alterations observed following S. lycocarpum fruit ingestion (3% added to diet), the present study
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M.R. Soares-Mota et al. / Experimental and Toxicologic Pathology 62 (2010) 549–553
was undertaken to extend the previous findings, evaluating the possible effects of 10% S. lycocarpum unripe fruit ingestion on hematological, biochemical and histopathological parameters in growing rats.
Material and methods Plant The S. lycocarpum fruit used in this study was collected in Itajuba City (Minas Gerais – Brazil; July 2003) and were authenticated by the botanist Prof. Dr. J.R. Pirani from Department of Botany, University of Sa~ o Paulo. The voucher specimens are kept in the HSF Herbarium (registration: Aline Schwarz, no. 01). The chemical study of this plant showed that the unripe fruit contained 0.06% solamargine and 0.09% solanine (Schwarz et al., 2007). Animals Twenty-one-day-old genetically similar male and female Wistar rats from our colony were used in this study. The animals were housed in groups of five in plastic cages (40 cm 50 cm 20 cm) at controlled room temperature (2073 1C) with 12 h light– dark cycles (lights on 6:00 am). Water and food were freely available. The animals used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animal, National Research Council, USA (1996) and the experiment was approved by the Ethical Committee, Faculty of Veterinary Medicine (protocol no. 388/2003). Preparing S. lycocarpum fruit to feed rats Unripe S. lycocarpum fruit was sliced and oven-dried at 50 1C, and were then milled and stored at 20 1C in dark plastic bags, protected against humidity and light. Every week, regular chow was mixed with 10% dried and milled S. lycocarpum fruit, pelleted and stored in paper bags in a temperature-controlled room (22–25 1C). The experimental animals were fed this pellet diet, containing 10% dried S. lycocarpum fruit, from 21 days old to adult age (8 weeks of treatment). The control rats received regular chow (Purinas), balanced and specific to lab animals, without the fruit. According to the American Institute of Nutrition (AIN), the chow used in the study, mixed with 10% S. lycocarpum fruit, maintained the minimal amounts of essential nutrients recommended in the AIN-93G diet, formulated for adequate development of rats (Reeves et al., 1993). The animals were weighed daily and the consumption of food and water were also measured daily. Analysis of blood samples At the end of treatment (8 weeks), the animals (n ¼ 15 per group) were submitted to anesthesia (10 mg/kg xylazine+100 mg/kg ketamine, ip) and blood samples were taken from the inferior vena cava for hematological analysis (erythrocyte and leukocyte counts, hemoglobin concentration, mean corpuscular concentration, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and hematocrit) and for determination of the serum contents of albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol, creatinine, glucose, total protein and triglycerides through an automated system, using the apparatus CELM SBA-200 (CELMs, Brazil).
Evaluation of female and male organs After 8 weeks the animals were euthanized, submitted to necropsy, and the weight of organs was recorded. The following organs were fixed in 10% neutral-buffered formalin: adrenal glands, spleen, heart, liver, pancreas, lungs, kidneys, thymus, ovaries, uterus, epididymises, seminal vesicles and testicles. After fixation, the tissues were embedded in paraffin and 5 mm sections were stained with hematoxylin and eosin (HE). Histomorphometric measurements of the ovaries were performed with an image analysis system (Image Pro-Plus Media Cyberneticss). Total area and diameter of the follicles and follicular cysts were measured and compared with the same structures of non-treated animals (control group). The magnification of 10 was used to measure follicular structures and morphometry of 3 consecutive sections was used to evaluate the number/area of follicles in the ovaries.
Experimental design Thirty female and thirty male rats, 21 days old, were randomly assigned to experimental or control groups (n ¼ 15 per group). The rats in the experimental group received 10% S. lycocarpum added to the regular pellet diet, from 21 days old to adult age (8 weeks of treatment), while animals of the control group received only chow during the same period. The body weight was assessed daily and the body weight gain was presented by week. Consumption of food and water were also measured daily and presented as the mean of daily consumption by week. In the eighth week of treatment, all rats were submitted to anesthesia and blood samples were taken for hematological and biochemical examinations, and organs were collected for anatomopathologic study. The relative weights were calculated from the ratio of organ weight to body weight multiplied by 100. In females, the total area and diameter of the follicles and follicular cysts were measured.
Statistical analyses Data are reported as means7SEM and were analyzed statistically by Student’s t test (control group versus experimental group). A value of po0.05 was indicative of a significant difference between groups for all comparisons made.
Results The initial values of body weight (56.471.7 g in control and 56.271.7 g in experimental male; 54.271.6 g in control and 57.470.8 g in experimental female) were similar between groups, while the initial values of food and water intake were only reduced in male and female experimental groups on the first and/ or second days of treatment. Reduced body weight gains were observed in males (1–7 weeks) and females (1, 5 and 6 weeks) treated with 10% S. lycocarpum added to their diet by 8 weeks, but this effect was more evident in male rats (Fig. 1). Few significant differences were observed in the food and water consumption of treated males, while in females prominent increases in food consumption and reductions in water consumption were observed (Fig. 1). In addition, few significant differences were observed in the hematological parameters (Table 1) of male and female rats treated with 10% S. lycocarpum. Serum biochemical parameters showed triglyceride reductions in both male and female treated
M.R. Soares-Mota et al. / Experimental and Toxicologic Pathology 62 (2010) 549–553
551
weight gain Male experimental group
40 30
**
**
**
**
control group
40 weight gain (g)
weight gain (g)
Female
control group
50
**
20
**
10 0
30
experimental group
**
20
* *
10 0
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
week
week food consumption
Female
control group
food consumption (g)
food consumption (g)
Male 30
experimental group
20
*
10 0
1
2
3
4
5
6
7
8
control group
20
experimental group
**
**
** **
10
0 1
2
3
week
4
5
6
7
8
week water consumption Female
control group experimental group
30
**
*
20 10 0 1
2
3
4
5
6
7
8
water consumption (ml)
water consumption (ml)
Male 40
40
control group experimental group
30 *
**
20
**
**
10 0
1
2
3
week
4
5
6
7
8
week
Fig. 1. Weight gain, food and water consumption of male or female rats receiving 10% Solanum lycocarpum fruit added in the diet from weaning (21 days old) until adult age (8 weeks of treatment). Data are presented as the mean7S.E.M of weight gain by week or daily consumption by week; *po0.05 and **po0.01, t test.
Table 1 Hematological parameters of male and female rats receiving 10% Solanum lycocarpum fruit in the diet (experimental) or not (control) by 8 weeks. Parameters
Erythrocytes ( 106/mL) Leukocytes ( 103/mm3) Hemoglobin (g/dl) Mean corpuscular volume (fl) Mean corpuscular hemoglobin (pg) Mean corpuscular hemoglobin concentration (%) Hematocrit
Male
Female
Control
Experimental
Control
Experimental
7.0670.2 5.9070.4 15.870.2 79.971.2 22.570.4 28.270.5 56.472.0
7.1370.2 4.8770.3 15.570.3 76.871.0* 21.870.3 28.570.3 54.771.3
6.2170.2 3.9670.2 14.470.4 84.471.1 23.571.2 28.072.0 52.572.0
6.0170.1 4.3970.2 15.170.2 83.271.1 25.170.5 30.371.0 50.171.3
Data are expressed as mean7SEM. (n ¼ 15/group); *po0.05, t test.
rats, while in females we also observed increased albumin and ALT levels, but reduced total protein levels (Table 2). Male treated rats presented a significant decrease in adrenal gland, spleen, heart, kidneys and thymus weights (Table 3). On the other hand, an increase in heart, left and right epididymises, lungs, seminal vesicles, and left and right testicles relative weights
was observed. In females, no differences were detected in organ weights and few significant differences were observed in the relative weights of other organs (reduction in heart, left ovary and lungs) (Table 3). During necropsy, no significant gross lesions were observed in either male or female rats of any of the groups studied. No
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M.R. Soares-Mota et al. / Experimental and Toxicologic Pathology 62 (2010) 549–553
significant histological findings were observed between groups, while the histomorphometry of the ovaries showed no alteration between groups, either (total area: control group 178,613.987 1123.84 mm2 and experimental group 226,347.1071153.83 mm2. Diameter: control group 343.027220.64 mm2 and experimental group 454.347129.31 mm2).
Discussion Our results indicate that exposure to 10% S. lycocarpum in the diet from weaning (21 days old) until adult age (8 weeks of treatment) induced toxicity mainly in growing male rats. In fact, a reduction in weight gain was observed. In addition, the 10% S. lycocarpum mixture was sufficiently palatable to permit normal intake of food for normal growth, because few alterations in female weight gain and increased food consumption were observed. Water consumption between the control and treated groups in both sexes was only slightly affected. Table 2 Serum biochemical parameters of male and female rats receiving 10% Solanum lycocarpum fruit in the diet (experimental) or not (control) by 8 weeks. Parameters
Albumin (g/dl) ALT (U/l) AST (U/l) Cholesterol (mg/dl) Creatinine (mg/dl) Glucose (mg/dl) Total proteins (g/dl) Triglycerides (mg/dl)
Male
Female
Control
Experimental
Control
Experimental
4.1570.10 114.473.01 380.0715.1 168.6714.0 0.7170.02 112.277.0 6.670.1 164.7711.1
4.0270.10 120.675.00 376.50713.6 201.2714.2 0.7070.02 101.176.0 6.470.2 129.575.8**
4.3170.20 63.573.11 318.25729.2 151.7712.6 0.8070.04 94.774.8 6.470.3 189.7725.1
5.0070.15** 68.473.14 427.0732.5* 157.0711.8 0.7670.03 90.074.8 5.670.2* 129.576.0*
Data are expressed as the mean7SEM. (n ¼ 15/group); *po0.05 and **po0.01, t test.
Through phytochemical analysis of medicinal preparation (‘‘povilho-de-lobeira’’) and the fruit of S. lycocarpum Dall’Agnol and von Poser (2000) found complex polysaccharides as the main component. Thus, the weight gain reduction observed in growing male rats is that some polysaccharides slow gastric emptying which, in turn, slow the absorption of glucose and other metabolites into the bloodstream (Spiller, 1994). This enhances satiety because insulin stimulates appetite. Due to the intimate mixing of soluble polysaccharides with food, they could convert some carbohydrates into ‘‘slow-release’’ carbohydrates and act on the endocrine system affecting the liberation of gastrointestinal hormones, lowering blood glucose levels (Dall’Agnol and von Poser, 2000; Spiller, 1994). The adrenal gland, spleen, heart, kidneys and thymus weights were reduced, indicative of decreased growth in male rats. However, increases in the heart, left and right epididymises, lungs, seminal vesicles, and left and right testicle relative weights did occur. This fact is most likely due to a reduction in body weight. Female rats did not present alterations in organ weights and few significant differences in relative weights of some organs (reduction in heart, left ovary and lungs). There were no effects detected on the ovaries as per histomorphometric evaluation between groups. These data suggest that male growing rats were more sensitive to the toxic effects of the plant than female rats. Maruo et al. (2003) showed that 3% S. lycocarpum fruit ingestion in male (60 days of administration) and female (37 days) adult rats caused slight differences in water and food consumption and no changes in male or female weight gain. These authors also reported reductions in uterus and liver weights, without alterations in other organ weights in either sex. However, Maruo et al. (2003) used only adult rats in that study, while we employed not only growing rats, but also a higher plant concentration during testing. Thus, both variables, the growing rats and the higher plant concentration, most likely contributed to a more pronounced endocrine disruptive effect of the steroidal glycoalkaloids of S. lycocarpum.
Table 3 Weight and relative weight (g) of the organs of male and female rats receiving 10% Solanum lycocarpum fruit in the diet (experimental – E) or not (control – C) by 8 weeks. Sex
Male
Female
Organ
Weight
Relative weight
C
E
C
E
Adrenal glands Spleen Heart Liver Left epididymises Right epididymises Pancreas Lungs Kidneys Thymus Seminal vesicles Left testicle Right testicle
0.0470.003 0.7870.010 1.1570.020 13.370.460 0.5570.020 0.5470.020 1.0570.100 1.7570.100 1.1470.030 0.7070.040 1.7070.030 1.7070.070 0.8570.070
0.0270.002** 0.6570.020** 1.1070.030* 11.970.520 0.5270.010 0.5370.020 1.0070.040 1.6570.100 1.0070.030** 0.5370.030** 1.7070.030 1.7070.030 0.8570.040
0.0170.001 0.2370.010 0.3370.010 4.0070.100 0.1570.004 0.1670.004 0.3170.020 0.5170.014 0.3370.010 0.2070.011 0.2570.020 0.5170.010 0.5170.010
0.0170.001 0.2370.010 0.4070.010* 4.1170.140 0.2070.006** 0.2070.005** 0.3370.013 0.6070.020* 0.3470.010 0.2070.011 0.3070.011* 0.6070.013** 0.6070.014**
Adrenal glands Spleen Heart Liver Left ovary Right ovary Pancreas Lungs Kidneys Thymus Uterine horns
0.0570.002 0.6170.018 0.8370.017 8.5870.190 0.0670.001 0.0670.002 0.9070.030 1.3370.020 0.8470.020 0.4870.020 0.5170.040
0.0470.002 0.5970.022 0.8270.017 8.5470.190 0.0670.003 0.0670.003 0.9170.030 1.3870.060 0.8070.020 0.4470.010 0.4670.030
0.0270.001 0.2770.006 0.3870.007 3.9670.090 0.0370.001 0.0370.002 0.4270.017 0.6370.020 0.3770.009 0.2070.006 0.2170.010
0.0270.010 0.2670.006 0.3670.004* 3.7370.090 0.0270.001* 0.0270.001 0.3970.009 0.5870.010* 0.367v 0.007 0.2070.006 0.2270.020
Data are expressed as the mean7SEM. (n ¼ 15/group) *po0.05 and **po0.01, t test.
M.R. Soares-Mota et al. / Experimental and Toxicologic Pathology 62 (2010) 549–553
Hematological and serum biochemical parameters in both males and females treated with S. lycocarpum were performed to evaluate the possible toxic effects of the plant (Stevens and Gallo, 1989), yet did not reveal toxicity at the levels administered in male and female growing rats. AST and ALT are elevated following tissue damage in which cellular enzymes are released from the cells into the bloodstream (Kaneko, 1989). AST is found in high constitutive levels in the heart and liver, whereas ALT is most active in the liver. Creatinine is found in the heart, skeletal muscle, brains and testes, but not in the liver. Elevated levels are found in muscular disorders, myocardial infarction and pulmonary disorders (Kaneko, 1989). Albumin and total proteins are related with protein metabolism, while cholesterol and triglycerides interfere with lipid metabolism (Kaneko, 1989). In the present work, serum biochemical parameters in females showed more alterations than in male rats. Increased AST and reduction in relative heart weights can indicate light cardiac toxicity in females. On the other hand, reduced triglycerides in male and female rats are similar to those observed in rats fed isoflavone (Demonty et al., 2002; Kawakami et al., 2004), a phytoestrogen. Despite interference with the cholesterol levels was not observed, the decrease in triglyceride levels might represent a reduced lipid metabolism. Thus, Dall’Agnol and von Poser (2000) proposed that hypocholesterolemic activity of this plant could be due the increased fecal bile acid excretion as well as to the action of the short-chain fatty acids (acetate, propionate and butyrate). These short-chain fatty acids derive from the fermentation of some polysaccharides in the colon, on the synthesis of d-aminolevulinate and by the increase of the cholesterol 7-a-hydroxylase and 3-hydroxy-3-metylglutaryl CoA reductase synthesis (Lairon, 1996; Hara et al., 1998; Jimenez et al., 1998). The present data showed sex-related differences in S. lycocarpum toxicity, as male rats presented decreased weight gain as well as female alterations in serum biochemical parameters.
Acknowledgements This research was supported by grants from FAPESP (Fundac- a~ o de Amparo a Pesquisa do Estado de Sa~ o Paulo) and CNPq ´ (Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnologico), and is part of the Master’s dissertation of the first author,
553
´ presented to the Programa de Pos-Graduac - a~ o em Patologia Experimental e Comparada, Faculdade de Medicina Veterina ria e Zootecnia, Universidade de Sa~ o Paulo.
References Dall’Agnol R, von Poser GL. The use of complex polysaccharides in the management of metabolic diseases: the case of Solanum lycocarpum fruits. J Ethnopharmacol 2000;71:337–41. Demonty I, Lamarche B, Deshaies Y, Jacques H. Role of soy isoflavones in the hypotriglyceridemic effect of soy protein in the rat. J Nutr Biochem 2002; 13:671–7. Eltayeb EA, Al-Ansari AS, Roddick JG. Changes in the steroidal alkaloid solasodine during development of Solanum nigrum and Solanum incanum. Phytochem 1997; 46:489–94. Goswani A, Kotoky R, Rastogi RC, Ghosh AC. An one-pot efficient process for 16dehydropregnenolone acetate. Org Process Res Dev 2003;7:306–8. Hara H, Haga S, Kasai T, Kiriyama S. Fermentation products of sugar-beet fiber by cacal bacte ria lower plasma cholesterol concentration in rats. J Nutr 1998;128: 688–693. Haraguchi M, Uchimura RT, Motidome M, Gottlieb O. Aproveitamento dos ´ esteroides dos frutos da lobeira. Ciˆen Cult 1978;32:81–2. Institute of Laboratory Animal Research, Commission on Life Sciences, National Research Council, Guide for Care and Use of Laboratory Animals, 1996. Jimenez MV, Conde K, Erickson SK, Fernandez ML. Hypolipidemic mechanisms of pectin and psyllium in guinea pigs fed high fat-sucrose diets: alterations on hepatic cholesterol metabolism. J Lipid Res 1998;39:1455–65. Kaneko JJ. In: Clinical biochemistry of domestic animals, 4th ed. San Diego: Academic Press; 1989. Kawakami Y, Tsurugasaki W, Yoshida Y, Igarashi Y, Nakamura S, Osada K. Regulative actions of dietary soy isoflavone on biological antioxidative system and lipid metabolism in rats. J Agric Food Chem 2004;52:1764–8. Lairon D. Dietary fibres: effects on lipid metabolism and mechanisms of action. Eur J Clin Nutr 1996;50:125–33. Lorenzi H. In: Plantas daninhas do Brasil, 2nd ed. Nova Odessa: Plantarum; 1991 p. 392. Maruo VM, Bernardi MM, Spinosa HS. Toxicological evaluations of long term consumption of Solanum lycocarpum St. Hill fruits in male and female adult rats. Phytomedicine 2003;10:48–52. ´ RJP,, Branda~ o MGL, Coelho MM. The starch from Oliveira ACP, Endringer DC, Araujo Solanum lycocarpum St. Hill. fruit is not a hypoglycemic agent. Brazilian J Med Biol Res 2003;36:525–30. Reeves PG, Nielsen FH, Fahey GC. AIN-93 purified diets for laboratory rodents: final reports of the American Institute of Nutrition Ad Hoc Writing Committee on the reformulation of the AIN-76A Rodent Diet. J Nutr 1993;123:1939–51. Schwarz A, Pinto E, Haraguchi M, Oliveira CA, Bernardi MM, Spinosa HS. Phytochemical study of Solanum lycocarpum (St. Hil.) unripe fruits and its effects in rat gestation. Phytother Res 2007;21:1025–8. Spiller RC. Pharmacology of dietary fiber. Pharmcol Ther 1994;62:407–27. Stevens KR, Gallo MA. Practical considerations in the conduct of chronic toxicity studies. In: Hayes AW, editor. Principles and methods of toxicology, 2nd ed. New York: Raven press; 1989. p. 237–50. Vieira G, Ferreira PM, Matos LG, et al. Antiinflammatory effect of Solanum lycocarpum fruits. Phytother Res 2003;17:892–6.
Contents lists available at ScienceDirect
Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Toxicological evaluation of 10% Solanum lycocarpum St. Hill fruit consumption in the diet of growing rats: Hematological, biochemical and histopathological effects Milena Rodrigues Soares-Mota a, Aline Schwarz b, Maria Martha Bernardi c, Paulo Cesar Maiorka c, Helenice de Souza Spinosa c, a
~ em Patologia Experimental e Comparada, Brazil Programa de P´ os-Graduac- ao Departamento de Ana lises Cl´ınicas e Toxicol´ ogicas, Universidade Federal do Rio Grande do Norte, RN, Brazil c ~ Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508-270, Departamento de Patologia, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao ~ Paulo, SP, Brazil Sao b
a r t i c l e in fo
abstract
Article history: Received 26 May 2009 Accepted 15 July 2009
Solanum lycocarpum St. Hill (Solanaceae) is a native shrub very common in the Brazilian savanna. The fruit of this plant contains steroidal glycoalkaloids that may disrupt the endocrine system. Because this plant is employed in folk medicine for the management of diabetes, obesity and decreasing cholesterol levels, the present study determined the possible toxic effects of exposure to S. lycocarpum fruit from weaning (21 days old) until adult age (8 weeks of treatment) in male and female rats. In male rats, the plant reduced weight gain, while few significant differences were observed in female animals. Slight significant differences were observed in food and water consumption and in hematological parameters in treated rats. Reductions in adrenal gland, spleen, heart, kidneys and thymus weights of treated males were observed, while increased relative weights were detected in the heart, epididymises, lungs, seminal vesicles, and testicles. In females, no differences were observed in organ weights and few differences were observed in relative weights of some organs. The histopathologic study showed no alteration between groups. Serum biochemical parameters showed triglyceride reductions in treated animals of both sexes; in females, an increase in albumin and alanine aminotransferase levels and a reduction in total protein levels were noted. The present data therefore demonstrate sex-related differences in S. lycocarpum toxicity. & 2009 Elsevier GmbH. All rights reserved.
Keywords: Solanum lycocarpum (St. Hil.) Toxicological study Hematological parameters Biochemical parameters Plant
Introduction Solanum lycocarpum St. Hill (Solanaceae) is a common plant in the Brazilian savanna and, in some regions, people consume its fresh or cooked fruits (Lorenzi, 1991). In Brazil, the effects obtained from S. lycocarpum fruit have been employed in folk medicine for diabetes management, obesity and decreasing cholesterol levels (Dall’Agnol and von Poser, 2000; Oliveira et al., 2003). It has also been reported that this plant possesses diuretic, anti-spasmodic, anti-epileptic, sedative and anti-inflammatory activities (Vieira et al., 2003). Solamargine and solasonine are two major glycoalkaloids found in a large number of Solanum species (Eltayeb et al., 1997) and their steroidal aglycone, solasodine, has been used as a key starting ingredient in the manufacture of steroidal drugs. The compounds 16-dehydropregnenolone acetate and dehydroepian-
Corresponding author. Tel.: +55 11 30917656; fax: +55 11 30917829.
E-mail address: [email protected] (H.d. Spinosa). 0940-2993/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2009.07.006
drosterone acetate, key starting sources for androgen production (Goswani et al., 2003), and the compound 3-B-acetoxypregna5,16-dien-one, a key starting source for progesterone production (Haraguchi et al., 1978), can be obtained from solasodine in laboratory conditions. Recently, we showed that the unripe fruit of this plant contained 1.85% total alkaloids, where 0.06% corresponded to solamargine and 0.09% to solanine (Schwarz et al., 2007). Previously, it has been shown that long-term consumption of S. lycocarpum fruit can produce slight toxic effects in exposed adult rats (Maruo et al., 2003). In this study, male (60 days of administration) and female (37 days) adult rats received a diet containing 3% S. lycocarpum fruit. It was observed that while significant reductions in uterus and liver weight occurred in treated females, no alterations were reported in males. In addition, no significant differences were observed in other organ weights, blood enzymes or proteins. Furthermore, these authors suggested that S. lycocarpum promotes sex-related toxicity. Because of the slight toxic alterations observed following S. lycocarpum fruit ingestion (3% added to diet), the present study
550
M.R. Soares-Mota et al. / Experimental and Toxicologic Pathology 62 (2010) 549–553
was undertaken to extend the previous findings, evaluating the possible effects of 10% S. lycocarpum unripe fruit ingestion on hematological, biochemical and histopathological parameters in growing rats.
Material and methods Plant The S. lycocarpum fruit used in this study was collected in Itajuba City (Minas Gerais – Brazil; July 2003) and were authenticated by the botanist Prof. Dr. J.R. Pirani from Department of Botany, University of Sa~ o Paulo. The voucher specimens are kept in the HSF Herbarium (registration: Aline Schwarz, no. 01). The chemical study of this plant showed that the unripe fruit contained 0.06% solamargine and 0.09% solanine (Schwarz et al., 2007). Animals Twenty-one-day-old genetically similar male and female Wistar rats from our colony were used in this study. The animals were housed in groups of five in plastic cages (40 cm 50 cm 20 cm) at controlled room temperature (2073 1C) with 12 h light– dark cycles (lights on 6:00 am). Water and food were freely available. The animals used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animal, National Research Council, USA (1996) and the experiment was approved by the Ethical Committee, Faculty of Veterinary Medicine (protocol no. 388/2003). Preparing S. lycocarpum fruit to feed rats Unripe S. lycocarpum fruit was sliced and oven-dried at 50 1C, and were then milled and stored at 20 1C in dark plastic bags, protected against humidity and light. Every week, regular chow was mixed with 10% dried and milled S. lycocarpum fruit, pelleted and stored in paper bags in a temperature-controlled room (22–25 1C). The experimental animals were fed this pellet diet, containing 10% dried S. lycocarpum fruit, from 21 days old to adult age (8 weeks of treatment). The control rats received regular chow (Purinas), balanced and specific to lab animals, without the fruit. According to the American Institute of Nutrition (AIN), the chow used in the study, mixed with 10% S. lycocarpum fruit, maintained the minimal amounts of essential nutrients recommended in the AIN-93G diet, formulated for adequate development of rats (Reeves et al., 1993). The animals were weighed daily and the consumption of food and water were also measured daily. Analysis of blood samples At the end of treatment (8 weeks), the animals (n ¼ 15 per group) were submitted to anesthesia (10 mg/kg xylazine+100 mg/kg ketamine, ip) and blood samples were taken from the inferior vena cava for hematological analysis (erythrocyte and leukocyte counts, hemoglobin concentration, mean corpuscular concentration, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and hematocrit) and for determination of the serum contents of albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol, creatinine, glucose, total protein and triglycerides through an automated system, using the apparatus CELM SBA-200 (CELMs, Brazil).
Evaluation of female and male organs After 8 weeks the animals were euthanized, submitted to necropsy, and the weight of organs was recorded. The following organs were fixed in 10% neutral-buffered formalin: adrenal glands, spleen, heart, liver, pancreas, lungs, kidneys, thymus, ovaries, uterus, epididymises, seminal vesicles and testicles. After fixation, the tissues were embedded in paraffin and 5 mm sections were stained with hematoxylin and eosin (HE). Histomorphometric measurements of the ovaries were performed with an image analysis system (Image Pro-Plus Media Cyberneticss). Total area and diameter of the follicles and follicular cysts were measured and compared with the same structures of non-treated animals (control group). The magnification of 10 was used to measure follicular structures and morphometry of 3 consecutive sections was used to evaluate the number/area of follicles in the ovaries.
Experimental design Thirty female and thirty male rats, 21 days old, were randomly assigned to experimental or control groups (n ¼ 15 per group). The rats in the experimental group received 10% S. lycocarpum added to the regular pellet diet, from 21 days old to adult age (8 weeks of treatment), while animals of the control group received only chow during the same period. The body weight was assessed daily and the body weight gain was presented by week. Consumption of food and water were also measured daily and presented as the mean of daily consumption by week. In the eighth week of treatment, all rats were submitted to anesthesia and blood samples were taken for hematological and biochemical examinations, and organs were collected for anatomopathologic study. The relative weights were calculated from the ratio of organ weight to body weight multiplied by 100. In females, the total area and diameter of the follicles and follicular cysts were measured.
Statistical analyses Data are reported as means7SEM and were analyzed statistically by Student’s t test (control group versus experimental group). A value of po0.05 was indicative of a significant difference between groups for all comparisons made.
Results The initial values of body weight (56.471.7 g in control and 56.271.7 g in experimental male; 54.271.6 g in control and 57.470.8 g in experimental female) were similar between groups, while the initial values of food and water intake were only reduced in male and female experimental groups on the first and/ or second days of treatment. Reduced body weight gains were observed in males (1–7 weeks) and females (1, 5 and 6 weeks) treated with 10% S. lycocarpum added to their diet by 8 weeks, but this effect was more evident in male rats (Fig. 1). Few significant differences were observed in the food and water consumption of treated males, while in females prominent increases in food consumption and reductions in water consumption were observed (Fig. 1). In addition, few significant differences were observed in the hematological parameters (Table 1) of male and female rats treated with 10% S. lycocarpum. Serum biochemical parameters showed triglyceride reductions in both male and female treated
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weight gain Male experimental group
40 30
**
**
**
**
control group
40 weight gain (g)
weight gain (g)
Female
control group
50
**
20
**
10 0
30
experimental group
**
20
* *
10 0
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
week
week food consumption
Female
control group
food consumption (g)
food consumption (g)
Male 30
experimental group
20
*
10 0
1
2
3
4
5
6
7
8
control group
20
experimental group
**
**
** **
10
0 1
2
3
week
4
5
6
7
8
week water consumption Female
control group experimental group
30
**
*
20 10 0 1
2
3
4
5
6
7
8
water consumption (ml)
water consumption (ml)
Male 40
40
control group experimental group
30 *
**
20
**
**
10 0
1
2
3
week
4
5
6
7
8
week
Fig. 1. Weight gain, food and water consumption of male or female rats receiving 10% Solanum lycocarpum fruit added in the diet from weaning (21 days old) until adult age (8 weeks of treatment). Data are presented as the mean7S.E.M of weight gain by week or daily consumption by week; *po0.05 and **po0.01, t test.
Table 1 Hematological parameters of male and female rats receiving 10% Solanum lycocarpum fruit in the diet (experimental) or not (control) by 8 weeks. Parameters
Erythrocytes ( 106/mL) Leukocytes ( 103/mm3) Hemoglobin (g/dl) Mean corpuscular volume (fl) Mean corpuscular hemoglobin (pg) Mean corpuscular hemoglobin concentration (%) Hematocrit
Male
Female
Control
Experimental
Control
Experimental
7.0670.2 5.9070.4 15.870.2 79.971.2 22.570.4 28.270.5 56.472.0
7.1370.2 4.8770.3 15.570.3 76.871.0* 21.870.3 28.570.3 54.771.3
6.2170.2 3.9670.2 14.470.4 84.471.1 23.571.2 28.072.0 52.572.0
6.0170.1 4.3970.2 15.170.2 83.271.1 25.170.5 30.371.0 50.171.3
Data are expressed as mean7SEM. (n ¼ 15/group); *po0.05, t test.
rats, while in females we also observed increased albumin and ALT levels, but reduced total protein levels (Table 2). Male treated rats presented a significant decrease in adrenal gland, spleen, heart, kidneys and thymus weights (Table 3). On the other hand, an increase in heart, left and right epididymises, lungs, seminal vesicles, and left and right testicles relative weights
was observed. In females, no differences were detected in organ weights and few significant differences were observed in the relative weights of other organs (reduction in heart, left ovary and lungs) (Table 3). During necropsy, no significant gross lesions were observed in either male or female rats of any of the groups studied. No
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significant histological findings were observed between groups, while the histomorphometry of the ovaries showed no alteration between groups, either (total area: control group 178,613.987 1123.84 mm2 and experimental group 226,347.1071153.83 mm2. Diameter: control group 343.027220.64 mm2 and experimental group 454.347129.31 mm2).
Discussion Our results indicate that exposure to 10% S. lycocarpum in the diet from weaning (21 days old) until adult age (8 weeks of treatment) induced toxicity mainly in growing male rats. In fact, a reduction in weight gain was observed. In addition, the 10% S. lycocarpum mixture was sufficiently palatable to permit normal intake of food for normal growth, because few alterations in female weight gain and increased food consumption were observed. Water consumption between the control and treated groups in both sexes was only slightly affected. Table 2 Serum biochemical parameters of male and female rats receiving 10% Solanum lycocarpum fruit in the diet (experimental) or not (control) by 8 weeks. Parameters
Albumin (g/dl) ALT (U/l) AST (U/l) Cholesterol (mg/dl) Creatinine (mg/dl) Glucose (mg/dl) Total proteins (g/dl) Triglycerides (mg/dl)
Male
Female
Control
Experimental
Control
Experimental
4.1570.10 114.473.01 380.0715.1 168.6714.0 0.7170.02 112.277.0 6.670.1 164.7711.1
4.0270.10 120.675.00 376.50713.6 201.2714.2 0.7070.02 101.176.0 6.470.2 129.575.8**
4.3170.20 63.573.11 318.25729.2 151.7712.6 0.8070.04 94.774.8 6.470.3 189.7725.1
5.0070.15** 68.473.14 427.0732.5* 157.0711.8 0.7670.03 90.074.8 5.670.2* 129.576.0*
Data are expressed as the mean7SEM. (n ¼ 15/group); *po0.05 and **po0.01, t test.
Through phytochemical analysis of medicinal preparation (‘‘povilho-de-lobeira’’) and the fruit of S. lycocarpum Dall’Agnol and von Poser (2000) found complex polysaccharides as the main component. Thus, the weight gain reduction observed in growing male rats is that some polysaccharides slow gastric emptying which, in turn, slow the absorption of glucose and other metabolites into the bloodstream (Spiller, 1994). This enhances satiety because insulin stimulates appetite. Due to the intimate mixing of soluble polysaccharides with food, they could convert some carbohydrates into ‘‘slow-release’’ carbohydrates and act on the endocrine system affecting the liberation of gastrointestinal hormones, lowering blood glucose levels (Dall’Agnol and von Poser, 2000; Spiller, 1994). The adrenal gland, spleen, heart, kidneys and thymus weights were reduced, indicative of decreased growth in male rats. However, increases in the heart, left and right epididymises, lungs, seminal vesicles, and left and right testicle relative weights did occur. This fact is most likely due to a reduction in body weight. Female rats did not present alterations in organ weights and few significant differences in relative weights of some organs (reduction in heart, left ovary and lungs). There were no effects detected on the ovaries as per histomorphometric evaluation between groups. These data suggest that male growing rats were more sensitive to the toxic effects of the plant than female rats. Maruo et al. (2003) showed that 3% S. lycocarpum fruit ingestion in male (60 days of administration) and female (37 days) adult rats caused slight differences in water and food consumption and no changes in male or female weight gain. These authors also reported reductions in uterus and liver weights, without alterations in other organ weights in either sex. However, Maruo et al. (2003) used only adult rats in that study, while we employed not only growing rats, but also a higher plant concentration during testing. Thus, both variables, the growing rats and the higher plant concentration, most likely contributed to a more pronounced endocrine disruptive effect of the steroidal glycoalkaloids of S. lycocarpum.
Table 3 Weight and relative weight (g) of the organs of male and female rats receiving 10% Solanum lycocarpum fruit in the diet (experimental – E) or not (control – C) by 8 weeks. Sex
Male
Female
Organ
Weight
Relative weight
C
E
C
E
Adrenal glands Spleen Heart Liver Left epididymises Right epididymises Pancreas Lungs Kidneys Thymus Seminal vesicles Left testicle Right testicle
0.0470.003 0.7870.010 1.1570.020 13.370.460 0.5570.020 0.5470.020 1.0570.100 1.7570.100 1.1470.030 0.7070.040 1.7070.030 1.7070.070 0.8570.070
0.0270.002** 0.6570.020** 1.1070.030* 11.970.520 0.5270.010 0.5370.020 1.0070.040 1.6570.100 1.0070.030** 0.5370.030** 1.7070.030 1.7070.030 0.8570.040
0.0170.001 0.2370.010 0.3370.010 4.0070.100 0.1570.004 0.1670.004 0.3170.020 0.5170.014 0.3370.010 0.2070.011 0.2570.020 0.5170.010 0.5170.010
0.0170.001 0.2370.010 0.4070.010* 4.1170.140 0.2070.006** 0.2070.005** 0.3370.013 0.6070.020* 0.3470.010 0.2070.011 0.3070.011* 0.6070.013** 0.6070.014**
Adrenal glands Spleen Heart Liver Left ovary Right ovary Pancreas Lungs Kidneys Thymus Uterine horns
0.0570.002 0.6170.018 0.8370.017 8.5870.190 0.0670.001 0.0670.002 0.9070.030 1.3370.020 0.8470.020 0.4870.020 0.5170.040
0.0470.002 0.5970.022 0.8270.017 8.5470.190 0.0670.003 0.0670.003 0.9170.030 1.3870.060 0.8070.020 0.4470.010 0.4670.030
0.0270.001 0.2770.006 0.3870.007 3.9670.090 0.0370.001 0.0370.002 0.4270.017 0.6370.020 0.3770.009 0.2070.006 0.2170.010
0.0270.010 0.2670.006 0.3670.004* 3.7370.090 0.0270.001* 0.0270.001 0.3970.009 0.5870.010* 0.367v 0.007 0.2070.006 0.2270.020
Data are expressed as the mean7SEM. (n ¼ 15/group) *po0.05 and **po0.01, t test.
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Hematological and serum biochemical parameters in both males and females treated with S. lycocarpum were performed to evaluate the possible toxic effects of the plant (Stevens and Gallo, 1989), yet did not reveal toxicity at the levels administered in male and female growing rats. AST and ALT are elevated following tissue damage in which cellular enzymes are released from the cells into the bloodstream (Kaneko, 1989). AST is found in high constitutive levels in the heart and liver, whereas ALT is most active in the liver. Creatinine is found in the heart, skeletal muscle, brains and testes, but not in the liver. Elevated levels are found in muscular disorders, myocardial infarction and pulmonary disorders (Kaneko, 1989). Albumin and total proteins are related with protein metabolism, while cholesterol and triglycerides interfere with lipid metabolism (Kaneko, 1989). In the present work, serum biochemical parameters in females showed more alterations than in male rats. Increased AST and reduction in relative heart weights can indicate light cardiac toxicity in females. On the other hand, reduced triglycerides in male and female rats are similar to those observed in rats fed isoflavone (Demonty et al., 2002; Kawakami et al., 2004), a phytoestrogen. Despite interference with the cholesterol levels was not observed, the decrease in triglyceride levels might represent a reduced lipid metabolism. Thus, Dall’Agnol and von Poser (2000) proposed that hypocholesterolemic activity of this plant could be due the increased fecal bile acid excretion as well as to the action of the short-chain fatty acids (acetate, propionate and butyrate). These short-chain fatty acids derive from the fermentation of some polysaccharides in the colon, on the synthesis of d-aminolevulinate and by the increase of the cholesterol 7-a-hydroxylase and 3-hydroxy-3-metylglutaryl CoA reductase synthesis (Lairon, 1996; Hara et al., 1998; Jimenez et al., 1998). The present data showed sex-related differences in S. lycocarpum toxicity, as male rats presented decreased weight gain as well as female alterations in serum biochemical parameters.
Acknowledgements This research was supported by grants from FAPESP (Fundac- a~ o de Amparo a Pesquisa do Estado de Sa~ o Paulo) and CNPq ´ (Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnologico), and is part of the Master’s dissertation of the first author,
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´ presented to the Programa de Pos-Graduac - a~ o em Patologia Experimental e Comparada, Faculdade de Medicina Veterina ria e Zootecnia, Universidade de Sa~ o Paulo.
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