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Title: A toxicological evaluation of the effect of Carapa guianensis Aublet on pregnancy in Wistar rats Authors: J.H. Costa-Silva, M.M.A. Lyra, C.R. Lima, V.M. Arruda, A.V. Ara ujo, A. Ribeiro e Ribeiro, A.C. Arruda, M.C.C.A. Fraga, S.S.L. Lafayette, A.G. Wanderley PII: DOI: Reference: To appear in: Received date: Revised date: Accepted date: S0378-8741(07)00069-4 doi:10.1016/j.jep.2007.02.004 JEP 4575 Journal of Ethnopharmacology 14-8-2006 31-1-2007 7-2-2007

Please cite this article as: Costa-Silva, J.H., Lyra, M.M.A., Lima, C.R., Arruda, V.M., Ara ujo, A.V., Ribeiro e Ribeiro, A., Arruda, A.C., Fraga, M.C.C.A., Lafayette, S.S.L., Wanderley, A.G., A toxicological evaluation of the effect of Carapa guianensis Aublet on pregnancy in Wistar rats, Journal of Ethnopharmacology (2007), doi:10.1016/j.jep.2007.02.004 This is a PDF le of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its nal form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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A toxicological evaluation of the effect of Carapa guianensis Aublet on pregnancy in Wistar rats. J.H. Costa-Silvaa, M.M.A. Lyraa, C.R. Limaa, V.M. Arrudaa; A.V. Arajo b, A. Ribeiro e Ribeiro c, A.C. Arrudac, M.C.C.A. Fragab, S.S.L. Lafayetteb, A.G. Wanderleya,b,*.
a

Department of Pharmaceutical Sciences, Federal University of Pernambuco, Recife,

Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife, Pernambuco, 50760-901, Brazil.
c

Farmacologia, CCB, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, s/n, CEP: 50670-901- Cidade Universitria, Recife, PE, Brazil. Fone: +55-81-21268530; fax: +55-8121268976. E-mail address: almirgw@globo.com Abstract

The effects of the administration of Carapa guianensis Aublet (Meliaceae) seed oil were investigated during pregnancy in female Wistar rats. Five groups of pregnant rats (n=59/group) were treated orally from the 7th to the 14th day of pregnancy (organogenic period), at

sacrificed and laparotomized to evaluate reproductive parameters. The results showed that there was no difference between the control and treated groups in terms of the number of live and dead fetuses, the dam-offspring relationship, the weight of the fetus, the weight of the placentae and ovaries, the number of implantation sites, the number of resorption sites, the number of corpora lutea in the ovaries, and the pre- and post-implantation loss rates. It is therefore concluded that administration of C. guianensis seed oil did not bring about any toxic effect on pregnancy in Wistar rats. 1
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doses of: 0, 0.375, 0.75, 1.5 and 3.0 g.kg -1. On the 20 th day of pregnancy, the animals were

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* Corresponding author. Almir Gonalves Wanderley, Departamento de Fisiologia e

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Department of Chemistry, Federal University of Par, Belm, Par, 66075-110, Brazil.

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Pernambuco, 50740-521, Brazil.

Keywords: Carapa guianensis ; Meliaceae; Toxicology; Pregnancy. 1. Introduction

Carapa guianensis Aublet (Meliaceae) is a popular medicinal plant found in several countries. In Brazil, it is known as Andiroba (Corra, 1984). C. guianesis is an evergreen or

alternate, without stipules, arranged in a helix bunched at the ends of branchlets. Inflorescences are large, many-branched and flowers are white or cream-colored and have a

Fournier, 2003).

repellent and for treatment of arthritis, throat inflammation, ear infections, uterine cancer,

insect repellent has been reported in some studies (Gilbert et al., 1999; Miot et al., 2004). Mendona et al. (2005), using a larvicidal bioassay, observed significant insecticide activity

vector responsible for dengue and yellow fever. Anti-edematogenic and analgesic effects of C. guianensis seed oil (100-400 mg.kg -1) have also been described recently in rats (Penido et al., 2005).

The characterization of C. guianensis seed oil revealed the presence of miristic, palmitic, oleic, linoleic (Pinto, 1956; Teske and Trentini, 1997), stearic and arachidic fatty acids (Penido et al., 2005). Some tetranortriterpenoids have been isolated from C. guianensis seeds, namely, 6-alfa-acetoxy-epoxyazadiradione, 7-deacetoxy-7-oxogedunin, gedunin, andirobin, methyl angolensate (Penido et al., 2005), 1,3-di-benzene carbon amine-2octadecylic acid-glyceride, hexacosanoic acid-2,3-dihydroxy-glyceride, ursolic acid,

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on the part of C. guianensis seed oil (LC 50 = 57 g.L-1) when used on Aedes aegypti, the

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diarrhea and diabetes (Hammer & Johns, 1993). The potential of C. guianensis seed oil as an

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C. guianensis has been used by indigenous communities in the Amazon as an insect

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delicate musky fragrance. The timber is a valuable product of the species (Ferraz et al., 2002;

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deciduous tree that may grow to 60 m. The trunk is straight and cylindrical and leaves are

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naringenin,

scopoletin,

3,4-dihydroxymethylbenzoate,

2,6-dihydroxymethylbenzoate,

tetratriacontanoic acid, triacontanoic acid (Qi et al., 2004), epoxyazadiradione, 6-alfahydroxygedunin (Lavie et al., 1972). Penido et al. (2006) also observed that the tetranortriterpenoids from C. guianensis seeds exhibit significant anti-allergic activity, due to

pesticide has been established, its toxicological safety needs to be evaluated to establish that it is safe for human use. Studies of the reproductive toxicology of Carapa guianensis need to be

of the Meliaceae family, have demonstrated an antifertility effect in females rats (Sinha et al.,

and Talwar, 1996; Mukherjee et al., 1996). The present study was, therefore, conducted in

female Wistar rats.

2.1. Plant material

Carapa guianensis Aublet (Meliaceae) seeds were collected from the Amazon Rainforest (Par, Brazil). A voucher specimen (MG170789 MA 0404), authenticated by Dra. Ely Simone Cajueiro Gurgel (Museu Paraense Emlio Goedel, Par, Brazil), was deposited in the Joo Mussa Pires herbarium, at the Federal University of Par.

2.2. Extraction

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2. Materials and methods

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order to evaluate the extension of the treatment with C. guianensis seeds on pregnancy in

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1984; Lal et al., 1986; Riar et al., 1988) and an abortive effect in rats and primates (Mukherjee

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carried out, since toxicological findings on Azadirachta indica seed oil, which is also member

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Now the use of C. guianensis seed oil as an anti-inflammatory agent or natural

nuclear factor B inhibition and also IL-5 and CCL11/eotaxin suppression.

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N-hexane was added to 2 kg of previously dried and triturated C. guianensis seeds. The system was allowed to rest for 60 minutes and was then vacuum-filtered. The solvent was replenished until the botanical sample of oil was exhausted and then the solvent was removed under reduced pressure. The yield of extract was 34% v/w. The apparent density of the oil was calculated (0.833 g.mL-1) and this was used to define the exact volume that each animal would

2.3. Animals

standard environmental conditions (25 2 C; 12:12 h dark/light cycle). A water and chow

approved by the Animal Experimentation Ethics Committee of the Biological Science

2.4. Experimental protocol

water (1 mL - control group) orally or Carapa guianensis seed oil at the doses of 0.375, 0.75, 1.5 or 3.0 g.kg-1 from the 7th to the 14th day of pregnancy (organogenic period; Almeida et al., 2000). During pregnancy, the rats were closely observed twice a day for survival, changes in appearance, behavior, and signs of vaginal bleeding and their food and water intake recorded daily. The maternal weight was also recorded on days 1, 7, 14 and 20. The rats were sacrificed by cervical dislocation and, on the 20th day of pregnancy, they were laparotomized and their uterine horns removed. The number of implants, resorptions, live and dead fetuses was then recorded. The fetuses and placentae were weighed and observed for any visible abnormality. The ovaries were weighed and the corpora lutea were

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The animals were randomly divided into five groups (n=5-9/group) and received distilled

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Center, at the Federal University of Pernambuco (Process n 23076.006909/2004-25).

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diet (Labina, Purina, Brazil) were available ad libitum. The experimental protocol was

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Pregnant 4-month old Wistar rats, weighing 250 20 g were used. These were kept under

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receive. The oil was stored at -2C until further use.

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counted. From these data, the implantation index (total number of implantation sites/total number of corpora lutea x 100), the resorption index (total number of resorption sites/total number of implantation sites x 100), the number of pre-implantations (number of corpora lutea - number of implantations /number of corpora lutea x 100) and the post- implantation loss rate (number of implantations - number of live fetuses/number of implantations x 100)

2.5. Statistical analysis

differences between treated and control groups. The pre-implantation and post-implantation

and Chi-square tests, respectively. The significance level was set at p < 0.05.

3. Results

C. guianensis seed oil treatment from the 7th to 14th day of pregnancy, at doses of 0.375, 0.75, 1.5 and 3.0 g.kg-1 did not produce any deaths or clinical signs of toxicity (e.g., salivation, piloerection, diarrhea, alteration in locomotor activity, changes in behavior, or signs of vaginal bleeding) in the pregnant rats. Maternal weight gain was unaffected by C. guianensis seed oil administration at all dose levels throughout the study (Table 1). A similar lack of effect was noted in the case of food and water consumption (Fig. 1). Insert table

Insert figure

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loss rates and the implantation and resorption indexes were analyzed using Kruskal-Wallis

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One-way ANOVA followed by Newman-Keuls tests were used to evaluate significant

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were calculated.

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On performing caesarean section, all pregnant females were found to have viable fetuses. No dead fetuses or fetuses with external malformations were observed. There were no statistically significant differences between control and treated dams regarding the number of fetuses in each dam (offspring/dam relationship), fetuses, placentae and ovary masses or the number of corpora lutea (Table 1). Likewise, treatment with C. guianensis did not cause any

(Table 1).

4. Discussion

The present study was designed to evaluate the embryo/fetotoxic and teratogenic potential of C. guianensis seed oil in rats.

C. guianensis (0.375 to 3.0 g.kg-1) did not produce any deaths or toxic clinical signs among the pregnant rats, suggesting absence of maternal toxicity. Maternal parameters such as body weight changes, food and water consumption, and clinical signs of toxicity allowed for a clear evaluation of the integrity of maternal homeostasis (Almeida & Lemonica, 2000). The administration of C. guianensis seed oil did not bring about death or any external malformation in the fetuses and also did not alter fetuses and placentae mass in doses of up to 3.0 g.kg-1 in rats. The ovarian mass and the number of corpora lutea in treated groups were similar to those of the control group. These findings indicate normal development of corpora lutea and suggest that production of progesterone was not influenced by C. guianensis seed oil treatment (Chang et al., 2002).

Both the implantation index and the pre-implantation loss rate evaluate blastocyst implantation in the uterus. These parameters were similar in control and treated groups, suggesting normal reproductive capacity (Chang et al., 2002). 6
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changes in the implantation and resorption index or the pre- and post-implantation loss rates

The resorption index and post-implantation loss rate establishes correlations between the number of implanted blastocysts and those that have not developed (Almeida & Lemnica, 2000). When the implanted blastocysts do not develop, they are known as resorptions and these indicate failure in the development of the embryo. In this study, there were no statistical differences between control and treated groups so far as the resorption

implanted blastocysts.

Studies carried out with the oil obtained from Azadirachta indica seeds (Neem),

observed that the administration of A. indica seeds oil during the first days of pregnancy had

purified seed extract of A. indica (Praneem) to pregnant rats, at 0.6 mL/rat, and verified that

seed oil (1-2 g.kg-1) from the 1st to 6th day of pregnancy, showed that their fetuses underwent a reduction in their body mass (Lyra et al., 2005). However, a three-generation study in male

effects on general health or reproductive parameters (Chinnasamy et al., 1993). The structures of more than 100 constituents of A. indica have been elucidated (Siddiqui et al.,1988; Van der

effects on mammals. These include azadirachtin, the best documented pure compound, and nimbidin. The administration of azadirachtin or nimbidin in rats produced no adverse embryo/fetotoxicity or teratogenic effects (Pillai & Santhakumari, 1984; Srivastava & Raizada, 2001). A review of the toxicological data from human and animal studies involving oral administration of different A. indica preparations reveals that most of the pure

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Nat et al., 1991; Siddiqui et al., 1998; Luo et al., 2000) and some have been tested for their

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and female rats fed a diet containing A. indica debitterised oil did not show any adverse

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Praneem also had an abortive effect. Another study of pregnant rats treated with A. indica

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an antifertility effect at a dose of 4-6 mL.kg-1. Mukherjee & Talwar (1996) administered a

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another Meliaceae, have demonstrated an abortive effect in pregnant rats. Lal et al. (1987)

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index and post-implantation loss rate were concerned, indicating normal development of the

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compounds show a relatively low toxicity in comparison with unprocessed seed oil materials (Boeke et al., 2004). Several compounds, such as triterpenes, tetraterpenes, alkaloids and limonoids, which are phytochemically characteristic of all members of Meliaceae family, have been isolated from C. guianensis (Connolly et al., 1966; Banerji & Nigam, 1984; Pereira et al., 1999;

composition (gedunin, epoxyazadiradione and scopoletin) among those species, the results show that C. guianensis did not cause any change in reproductive parameters. These results

in rats are not present in C. guianensis.

According to the results discussed above, treatment with C. guianensis seed oil from the 7th to the 14th day of pregnancy, in doses up to 15 times more than the dose used as an

teratogenic effect in pregnant rats.

C. guianensis oil or tea is administered in indigenous communities at a dose of one

directly applied to troubled joints for arthritis. In cases of uterine cancer, they use the oil to soothe pain in the vaginal region, applying the oil directly to the cervix wall with a finger (Hammer & Johns, 1993). In the present study, doses of up to 20 (three times a day) or 50 times (once a day) that of the dose used by indigenous communities brought about no alteration in the reproductive parameters analyzed in rats, suggesting that C. guianensis is safe for use by pregnant women during organogenic period. Further studies are, however, necessary to confirm this finding.

Acknowledgements

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cup once a day for inflammations, three times a day for diarrhea and diabetes, or it can be

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anti-edematogenic agent (Penido et al., 2005), caused no toxic clinical signs, as well as no

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suggest that the chemical substances of A. indica seed oil responsible for the antifertility effect

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Andrade et al., 2001; Qi et al., 2004). In spite of the similarity in the phytochemical

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The authors would like to thank Rejane de Souza Silva for technical assistance and CAPES and CNPq for financial support.

Almeida, E.R., Melo, A.M., Xavier, H., 2000. Toxicological evaluation of the

Research 14, 99-102.

different periods of prenancy in rats. Journal of Ethnopharmacology 73, 53-60.

Flowers of Carapa guianensis Aubl. J. Essent. Oil Res.13, 436-438 Banerji, B., Nigam, S.K., 1984. Wood constituents of Meliaceae: A review. Fitoterapia 55, 3-36.

Boeke, S.J., Boersma, M.G., Alink, G.M., Van Loon, J.J.A., Van Huis A., Dicke, M., Rietjens, I.M.C.M. 2004. Safety evaluation of neem (Azadirachta indica ) derived pesticides.

Chang, C.V., Felcio, A.C., Reis, J.E.P., Guerra, M.O., Peters, V.M., 2002. Fetal toxicity of Solanum lycocarpum (Solanaceae) in rats. Journal of Ethnopharmacology 81, 265269.

Chinnasamy, N., Harishankar, N.; Kumar, P.U.; Rukmini, C., 1993. Toxicological studies on debitterized neem oil ( Azadirachta indica). Food and Chemical Toxicology 31, 297-301.

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Journal of Ethnopharmacology 94, 25-41.

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Andrade, E.H., Zoghbi, M.G., Maia, J.G., 2001. Volatiles from the Leaves and

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Almeida, F.C.G., Lemonica, I.P., 2000. The toxic effects of Coleus barbatus B. on the

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hydroalcohol extract of the dry leaves of Peumus boldus and boldine in rats. Phytotherapy

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Fournier, L.A., 2003. Carapa guianenses Aublet. Tropical Tree Seed Manual, 360-

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teratogenecity of azadirachtin in rats. Food and Chemical Toxicology 39, 1023-1027.

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Pinto, G.P., 1956. Contribuio ao estudo qumico do leo de Andiroba. Boletim

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Food intake (g/day/rat)

25 20 15 10 5 0 0 2 4 6 8 10 12 14 16 18 20

Control
SO 0.375g.kg-1 SO 0.75g.kg-1 SO 1.5g.kg-1 SO 3.0g.kg-1

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0 2

Water intake (mL/day/rat)

30

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15

10

12 14

16 18

Fig. 1. Daily food and water intake of Wistar rats treated with C. guianensis seed oil (SO, 0.375 to 3.0g.kg-1, p.o.) from the 7th to 14th day of pregnancy.

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Day

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Control
SO 0.375g.kg-1 SO 0.75g.kg-1 SO 1.5g.kg-1 SO 3.0g.kg-1
20

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day of pregnancy (organogenic period). Reproductive parameters Pregnant rats Mass gain in the pregnancy period (g) Number of live fetuses Number of dead fetuses Offspring/dam relationship Fetus mass (g)
a a a a

Control 9 106.7 8.5 27.2 2.8 105 0 11.7 0.5 3.7 0.2 0.49 0.02 17.2 2.8 114 9 105

SO 0.375 g.kg -1 5 109.5 14.0 26.8 4.1 58 0

SO 0.75 g.kg -1

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6 68 0 72 4 68 98.6 5.5 0 0

Table 1 : Reproductive parameters of female rats treated with C. guianensis seed oil (SO, 0.375, 0.75, 1.5 and 3.0g.kg-1, p.o.) from 7 th to the 14th

SO 1.5 g.kg -1 6 110.5 7.1 23.2 3.0 70 0 11.7 0.8 4.2 0.2 0.49 0.01 21.7 4.4 71 1 70 12.2 0.6 97.3 1.4 0 0

SO 3.0 g.kg -1 6 92.8 8,4 17.5 5.1 74 0 12.3 1.1 4.0 0,2 0.48 0.01 21.5 0.9 74 0 74 12.8 0.9 95.8 4.2 0 4.0

Ma
4.0 0.6 0.55 0.05 23.1 8.0 63 5 58 94.0 7.9 7.1 0 13.4 0.9

11.6 1.5

Placentae mass (g)

Ovary mass (mg/100g) a Number of implantation sites Number of resorption sites Number of viable implantation sites Number of corpora lutea a Implantation index (%) Resorption index (%) Pre-implantation loss (%)b
b

13.4 0.7 94.2 7.9 0

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7.1 Post-implantation loss (%) a b The values are expressed as mean S.E.M or median .

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Mass gain in the organogenic period (g)

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100.6 4.2 15.6 4.1

11.3 0.6 3.8 0.1

0.57 0.04 27.6 8.0

12.2 0.7

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