Toxicological evaluation of an ethanolic extract from Chiococca alba roots

Journal of Ethnopharmacology 105 (2006) 187–195

Toxicological evaluation of an ethanolic extract from Chiococca alba roots Valeria E. Gazda a,b , Maria Regina Gomes-Carneiro a , Nancy S. Barbi b , Francisco J.R. Paumgartten a,∗ a Laboratory of Environmental Toxicology, Department of Biological Sciences, National School of Public Health, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil b Department of Clinical and Toxicological Analysis, School of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

Received 11 June 2005; received in revised form 28 September 2005; accepted 15 October 2005 Available online 17 November 2005

Abstract The roots of Chiococca alba have been employed to treat rheumatic disorders and for other therapeutic purposes in Brazil and elsewhere. This study was undertaken to evaluate the toxicological properties of an ethanolic extract from Chiococca alba roots (EE), including mutagenicity in the Salmonella assay and acute and subacute toxicity to mice. Single oral doses of EE caused hypoactivity, but no deaths were noted up to the highest dose tested (2000 mg/kg). EE (500 mg/kg p.o.) reduced mouse locomotion in the open field test. EE was markedly more toxic when given by intraperitoneal (i.p.) and subcutaneous (s.c.) routes. Acute approximate lethal doses (ALD) were 125 mg/kg (males) and 250 mg/kg (females) and 250 mg/kg (both sexes) by i.p. and s.c. routes, respectively. Deaths after single doses were preceded by hypoactivity, ataxia and lethargy. Repeated administration of EE by gavage for 14 days caused no deaths. Activity of liver monooxygenases (pentoxy- and ethoxyresorufin-O-dealkylases) was not altered by repeated treatment with EE (2000 mg/kg/day p.o.). Administration of EE by the i.p. route for 14 days decreased weight gain and caused anemia, neutrophilia and deaths. The no-observed-adverse-effect level (NOAEL) for subacute treatment by the i.p. route was as low as 15.6 mg of EE/kg body weight (wt)/day. EE was not mutagenic in the Salmonella/microsome assay with TA100, TA98, TA97a and TA1535 strains. In summary, EE was not mutagenic and presented a low acute and subacute toxicity by the oral route. Toxicities by parenteral routes, however, were more pronounced. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Rubiaceae; Chiococca alba; Mutagenicity; Central nervous system depression; Acute toxicity; Repeated-dose toxicity

1. Introduction Chiococca alba (L.) Hitchc (Rubiaceae) is a tropical plant that has arching branches, dark green foliage, cream-colored blooms and snow-white berries and grows either as a scrambling shrub or as a woody vine that often climbs taller vegetation (Pio-Corrˆea, 1931; Cruz, 1932). It is native to the New World, where it is found in South Florida (USA), the Bahamas, Mexico and through Central and South America up to Paraguay and Southeastern Brazil. Chiococca alba is

Abbreviations: ALD, approximate lethal dose; CNS, central nervous system; EE, ethanolic extract; EROD, ethoxyresorufin-O-deethylase; HCT, hematocrit; HGB, hemoglobin; LD50 , lethal dose 50%; NOAEL, no-observed-adverse-effect level; PROD, pentoxyresorufin-O-depenthylase; RBC, red blood cells; WBC, white blood cells; wt, weight ∗ Corresponding author. Tel.: +55 21 38829044. E-mail address: [email protected] (F.J.R. Paumgartten). 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.10.017

also known by a variety of common names, such as West Indian milkberry, snow-berry, David’s root, ‘bejuco de berac’, ‘buenda’, ‘liane des sorciers’ and in Brazil, as ‘cainca’, ‘cip´ocruz’ ‘raiz-de-frade’ and ‘cruzeirinha’ (Pio-Corrˆea, 1931; Cruz, 1932). The decoction and infusions of roots and other parts of Chiococca alba, alone or mixed with other plant species, have long been used in folk medicine as emetic, purgative, diuretic, antidiarrheic, antipyretic, tonic, aphrodisiac, as well as for a variety of other purposes, such as to treat rheumatism, snakebites, flatulence, delayed menstruation, dementia, alcoholism, nervousness and kidney troubles (Costa, 1932). A decoction of the whole-plant was also reported to be effective as laxative and as a remedy for gonorrhea, skin infections and rheumatism (Cruz, 1932). It is of note that at least until the first half of the 20th century, Chiococca alba was listed in the Brazilian pharmacopeia as well as in those of several European countries (Schapoval et al., 1983). In Brazil, a medicine for rheumatic

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disorders (Rheumoflora® ) that contains an alcoholic extract of Chiococca alba roots, combined with a whole-plant extract of a fern (Polypodium lepidopteris), has been in the phytopharmaceutical market since 1924. Literature on the chemical composition of Chiococca alba is relatively scarce. Two biologically active quinoline alkaloids (El-Abadi et al., 1989), an oleanane-type triterpene (Bhattacharyya and Cunha, 1992), an ent-kaurane (BorgesArg´aez et al., 1997), an iridoid and a seco-iridoid (Carbonezi et al., 1999) and a nor-seco-pimarane known as merilactone (Borges-Arg´aez et al., 2001) have been isolated from Chiococca alba roots, while lignans, coumarins and two new keto alcohols (El-Hafiz et al., 1991) have been found in the leaves. Studies on the pharmacological properties of Chiococca alba and its constituents have revealed anti-inflammatory effects (Schapoval et al., 1983), antimicrobial action against Staphylococcus aureus (Borges-Arg´aez et al., 1997) as well as anticancer activity (Carbonezi et al., 1997). Notwithstanding the widespread medicinal use of Chiococca alba root extracts in Brazil and elsewhere, as far as the authors are aware, there is no published study on their toxicological properties. It should be borne in mind that tradition in use, by no means, warrants that a medicinal plant is safe, particularly with regard to mutagenicity and carcinogenicity because cause–effect relationships in these areas are rather complex and not easily recognized by the population. The present study was undertaken to provide data on the safety of an alcoholic extract of Chiococca alba roots, including its acute and subacute toxicities by different routes of administration as well as an evaluation of its mutagenicity using the Salmonella/microsome assay.

2.3. Chemical characterization of the extract 2.3.1. Gas chromatography coupled to mass spectrometry (GC–MSD) analysis Part of EE (20 g) was fractionated by liquid–liquid partition with hexane (2.1 g), dichloromethane (1.2 g), ethyl acetate (5.7 g), 1-butanol (3.1 g) and water (8 g). The hexane fraction was submitted to silica gel column (80 cm × 1.5 cm) chromatography using hexane/ethyl acetate/methanol gradient to yield 104 fractions. Fractions 39–50 (eluted with hexane/ethyl acetate) were combined, methylated with diazomethane and analysed by using a GC–MSD instrument (Shimadzu model QP5000) equipped with a DB-1 column (30 m × 25 mm, with film of 0.25 ␮m (J&W Scientific, Rancho Cordova, CA, USA). Gas chromatography conditions were as follows: oven temperature initially, 50 ◦ C rising to 270 ◦ C (5 ◦ C/min); split ratio of 30:1; injector temperature 270 ◦ C; interface 230 ◦ C. Mass spectra were recorded at 70 eV. Identification of constituents was made by comparing fragmentation patterns of sample compounds with those found in the literature (McLafferty and Stauffer, 1989). 2.3.2. High performance liquid chromatograph (HPLC) analysis One milligram of EE was dissolved in methanol:water (30:70, v/v) and analysed by using a HPLC instrument (Shimadzu) equipped with ultra violet (SPD-10A) and diode array detection (SPD-M10A). Liquid chromatography conditions were as follows: reverse phase octadecylsilane-functionalized silica gel column, 250 mm × 4.6 mm, particle size of 5 ␮m (LiChrosorb, Merck, Darmstadt, Germany). Mobile phase, linear gradient of methanol:water and flow rate of 1 ml/min. Gradient began with methanol 30% for 10 min (isocratic), 30–40% (10–20 min) and 40–100% methanol (20–60 min).

2. Materials and methods 2.4. Mutagenicity testing 2.1. Plant material Chiococca alba (synonyms Lonicera alba, Chiococca racemosa, Chiococca brachiata) roots were collected in the remains of the Atlantic rain forest located in the municipality of Nova Friburgo, Rio de Janeiro, Brazil, between March and October 2001. Chiococca alba was identified by Dr. Sebasti˜ao Neto, from the Botanical Garden of Rio de Janeiro, Rio de Janeiro, Brazil, and a voucher specimen was deposited (reference number RB 395399) in the Botanical Garden Herbarium. 2.2. Preparation of plant extract Pulverized dried roots of Chiococca alba (2 kg) were initially macerated with 6 l of ethanol for 72 h, and subsequently, Soxhlet-extracted with 2 l of ethanol. The plant ethanol extracts was then concentrated in a rotating evaporator under reduced pressure at 50 ◦ C until all solvent was evaporated. Extraction yield was 86.4 g of ethanol extract residue (EE) per 1000 g of powdered dried roots. EE was kept in a glass dryer with silica gel and protected from light until further use.

The Salmonella typhimurium/microsome assay (tester strains TA97a, TA98, TA100 and TA1535) was performed by the preincubation method, without and with addition of an extrinsic metabolic activation system (S9 mixture), essentially as described by Maron and Ames (1983). All tester strain cultures were prepared from permanents kept in liquid nitrogen. In culture tubes, 100 ␮l of an overnight grown culture (containing approximately, 1 × 109 to 2 × 109 bacteria ml−1 ), 100 ␮l of EE diluted in analytical grade ethanol (70%, v/v), the negative (ethanol 70%) or the positive control substance and 500 ␮l of phosphate buffer or S9 mixture were pre-incubated at 37 ◦ C with shaking for 20 min. Two milliliters of molten top agar was added to each pre-incubation tube the content of which was then poured onto a plate with minimum glucose medium. Sodium azide (SA 1 ␮g/plate), 4-nitroquinoline-Noxide (4-NQNO, 1 ␮g/plate), 2-nitrofiuorene (2-NF, 1 ␮g/plate), 2-aminofluorene (2-AF, 10 ␮g/plate) and 2-aminoanthracene (2-AA, 1 or 0.5 ␮g/plate) were employed as positive control substances. SA was dissolved in distilled water, while dimethylsulfoxide (DMSO) was the solvent employed for the remaining mutagens. The S9 mixture was prepared by using lyophilized

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rat liver S9 fraction induced by Aroclor 1254 (Moltox® , Molecular Toxicology Inc., Boone, NC, USA) as reported elsewhere (Gomes-Carneiro et al., 1998). Plates were incubated in the dark at 37 ◦ C for 72 h and then scored for his+ (revertant) colonies. Every determination was made in triplicate and experiments were repeated at least once in order to check the reproducibility of results. The tester strains used in the experiments, supplied by Dr. Bruce N. Ames (University of California, Irvine, CA, USA), were checked for their genetic markers at the beginning of the study. 2.5. Animals Male and female Swiss Webster mice (30–35 g), from the Oswaldo Cruz Foundation breeding stock, were used. The animals were separated by sex and housed (four or five per cage) in standard plastic cages with stainless steel coverlids and wood shavings as bedding. Photoperiod (lights on from 06:30 to 18:30 h), room temperature (23 ± 1 ◦ C) and humidity (approximately, 70%) were controlled in the animal facilities. All mice had free access to tap water and were fed ad libitum with a commercial rodent diet (CR1 Nuvital® ; Nuvitab Ltda, Curitiba, PR, Brazil), except for a short fasting period starting 2 h before and lasting until 2 h after the administration of EE or its vehicle (distilled water). Experiments were conducted in accordance with the internationally accepted principles for laboratory animal use and care (EEC Directive of 1986; 86/609/EEC). 2.6. Acute toxicity testing EE was dissolved in distilled water and given as a single dose to mice either by oral (gavage) or parenteral (intraperitoneal and subcutaneous injections) routes. Acute toxicity assays were conducted as suggested by Kennedy et al. (1986), i.e. doses in the tested dose-interval were progressively increased so that each dose was 50% higher than the preceding one (oral: 0, 62.5, 125, 250, 500, 1000 and 2000 mg/kg body weight (wt); parenteral: 0, 62.5, 125, 250 and 500 mg/kg body wt). The animals were observed during 14 days and behavior modifications, deaths or any other sign of toxicity and their respective latencies were recorded. The animals were weighed immediately before treatment and thereafter on post-treatment days 7 and 14. Mice that died during the observation period and all surviving ones (killed on post-treatment day 14 by cervical dislocation) were subject to necropsy. 2.7. Open field test The open field apparatus was a white rectangular (45 cm × 45 cm × 29.5 cm) enclosure with a flat floor divided into nine subdivisions of 225 cm2 each. It was kept in a quiet room and illuminated by a centrally positioned 40 W tungsten lamp pending 150 cm from the floor level. Mice were individually exposed to the open field for 10 min, 15 min after being treated with EE (500 mg/kg body wt) or its vehicle and an observer sat adjacent to the apparatus recorded the following behavioral parameters: ambulation (number of subdivisions tra-

189

versed), rearing (number of rearing episodes), grooming (number of grooming behavior episodes) and defecation (number of fecal boluses left in the arena). 2.8. Repeated-dose (14 days) toxicity testing Groups of 20 mice (10 females and 10 males) were treated with daily doses of EE, either by gavage (0, 500, 1000 and 2000 mg/kg body wt/day) or by intraperitoneal injections (0, 15.6, 31.3, 62.5 and 125 mg/kg body wt/day), for 14 consecutive days. Animals of the vehicle-control group (dose 0) received equal volumes (5 ml/kg body wt/day) of distilled water alone. All mice were daily weighed and examined for signs of toxicity. Animals that died during the treatment period and those killed (by cervical dislocation) 24 h after the last dose of EE or its vehicle were subjected to necropsy. Organs were examined for macroscopic lesions and livers, kidneys and spleen were weighed. Any organ showing macroscopically visible alterations other than those attributable to agony were fixed in a buffered formaldehyde solution (10%) and kept for further histopathological examination. Blood sampling and analysis—blood samples were taken only from animals treated with repeated doses of EE by the intraperitoneal route. Immediately before being killed by cervical dislocation, mice were anesthetized by ethyl ether inhalation and blood (up to 1 ml collected in EDTA-treated centrifuge tubes) was taken by cardiac puncture. Red blood cell and leukocyte counts as well as hemoglobin rate were determined by using an automatic particle counter for veterinary use (DA530 CELM® , Barueri, SP, Brazil). Blood smears for differential leukocyte counting were stained with May-GrunwaldGiemsa. 2.9. Measurement of EROD and PROD activities in the hepatic S9 fraction Immediately after cervical dislocation, livers were quickly removed, weighed and stored at −80 ◦ C. The hepatic S9 fraction was prepared as follows. After thawing in an ice-bath, livers were immersed in a cool 0.25 M sucrose solution and homogenized with a Potter–Elvejhem homogenizer. The homogenate was centrifuged at 9000 × g at 4 ◦ C for 20 min and the supernatant (S9 fraction) was stored in liquid nitrogen until further use. S9 fraction protein levels were determined by the method of Bradford (1976). Pentoxyresorufin-O-depenthylase (PROD) and ethoxyresorufin-O-deethylase (EROD) activities were measured essentially as reported by Burke et al. (1985) with a few adaptations (De-Oliveira et al., 1999). 3. Results 3.1. Chemical analysis of EE A preliminary analysis of EE chemical composition by GC–MS revealed the presence of linear chain fatty acids (hexadecanoic, octadecanoic, eicosanoic, docosanoic and tetracosanoic acids), being the hexadecanoic acid the predominant fatty acid in the mixture, as well as the presence of a triterpenoid

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constituent, identified as the ursolic acid. The chromatogram of EE obtained by HPLC with UV detection, on the other hand, suggested the presence of phenolic acids, anthraquinones, iridoid and/or seco-iridoids and phenylpropanoids. 3.2. Mutagenicity testing EE-induced mutagenicity was tested using a wide range of doses the upper limit of which was either an extremely high dose (5000 ␮g/plate) or a dose at which a marked toxicity to the Salmonella typhimurium tester strain was apparent (Table 1). Toxicity to tester strains was noted either as an alteration of the background growth or as a reduction in the number of his+ revertants as compared to solvent control values. In all cases, we tested at least four or five doses of EE that proved to be non-toxic to the tester strain in the presence as well as in the absence of S9 mixture. As shown in Table 1, EE did not cause any increase in the number of his+ revertant colonies over the negative (solvent) control values obtained for the four tester strains, either without or with addition of S9 mixture. The foregoing results indicated that EE was not mutagenic in the Salmonella/microsome assay (Ames test). 3.3. Acute toxicity Except for a reduced locomotion (‘hypoactivity’) in doses equal to or higher than 250 mg/kg body wt, no signs of toxicity were noted in mice treated with a single oral dose of EE (Table 2). Lethality and other signs of toxicity, however, were observed after single doses of EE given by either intraperitoneal or subcutaneous injections. Male mice exposed to EE

by the intraperitoneal route died at doses equal to or higher than 125 mg/kg body wt, while females died at doses equal to or higher than 250 mg/kg body wt. In all cases, the animals died between one and two hours after treatment and death was preceded by symptoms, such as hypoactivity, ataxia and lethargy. Among mice of either sex treated with EE by the subcutaneous route, deaths occurred at doses equal to or higher than 250 mg/kg body weight and in most instances, between 24 and 48 h after treatment. The deaths of mice treated with subcutaneous injections of EE were preceded by symptoms of CNS depression as well. No relevant necropsy finding was recorded either among mice that died after acute treatment with EE or among those that were sacrificed at the end of the 14-day-observation period. 3.4. Behavioral effects in the open field As shown in Table 3, locomotion and frequency of rearing episodes were markedly reduced, while frequency of grooming behavior and defecation remained unaltered, in male as well as in female mice exposed to the open field 15 min after an oral dose of EE as high as 500 mg/kg body wt. The foregoing results indicated that locomotor exploration was decreased by oral treatment with EE, a finding that is consistent with the signs of hypoactivity recorded at the same dose level in the acute toxicity test. 3.5. Repeated-dose (14 days) toxicity No deaths were observed after treatment by the oral route with daily doses of EE as high as 2000 mg/kg body weight for 14 consecutive days. Except for a slightly reduced weight gain

Table 1 Mutagenicity testing of an alcoholic extract from roots of Chiococca alba in the Salmonella/micvosome assay (pre-incubation method) with tester strains TA97a, TA98, TA100 and TA1535 Dose (␮g/plate)

Number of his+ revertants/plate (mean ± S.D.) TA100 −S9

5000 4000 3000 2500 2000 1500 1000 500 250 100 50 25 10 5 0 PC

135 107 100 91 127 134 129 126 114 133 990

– – – – ± 26a ± 38a ± 20a ± 6a ± 24 ± 18 ± 14 ±6 ± 11 – ± 27 ± 32

TA98

TA97a

+S9

−S9

157 ± 12 149 ± 10a 130 ± 1a – 139 ± 13a 134 ± 9 145 ± 10 163 ± 16 169 ± 13 153 ± 24 169 ± 12 – – – 147 ± 12 777 ± 93

± ± 8a ±4 ±7 ±0 ±6 ±9 ±7 ±8 – – – – – 37 ± 4 310 ± 22 14 28 25 28 23 29 29 31 34

+S9 3a

43 ± 3 42 ± 3 47 ± 7 – 48 ± 10 44 ± 9 54 ± 9 36 ± 4 49 ± 5 38 ± 6 43 ± 2 – – – 43 ± 11 236 ± 14

−S9

73 75 87 103 94 115 113 110 128 121 414

– – – – ± 8a – ± 9a ± 17a ±8 ± 11 ± 16 ± 11 ± 16 ±7 ± 12 ± 53

TA1535 −S9

+S9

139 131 147 149 178 191 190 197 193 190 164 785

– – ± 4a – ± 14 ± 23 ± 22 ± 27 ± 19 ± 25 ± 11 ± 10 ± 16 – ± 11 ± 34

10 12 12 16 14 19 26 25 21

23 256

– – – ± 8a ± 1a ±3 ±4 ±4 ±4 ±5 ±4 ±2 – – ±2 ± 27

+S9 8 ± 6a 13 ± 3a 16 ± 2a – 17 ± 6 18 ± 2 15 ± 4 20 ± 2 20 ± 6 18 ± 6 17 ± 3 – – – 20 ± 6 89 ± 10

Values are means ± S.D. of three replicates. Dose 0 (negative control), 100 ␮l of ethanol 70%; PC, positive control; TA100/−S9 and TA1535/−S9, SA (1 ␮g/plate); TA100/+S9 and TA1535/+S9, 2-AA (1 ␮g/plate); TA98/−S9, 2-NF (1 ␮g/plate); TA 98/+S9, 2-AA (0.5 ␮g/plate); TA97a/−S9, 4-NQNO (1 ␮g/plate); TA97a/+S9, 2-AF (10 ␮g/plate). Assays were carried out in the presence (+S9) and in the absence (−S9) of an extrinsic metabolic activation system (S9 mixture). a Toxicity apparent as an alteration of the background lawn.

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Table 2 Toxicity of a single dose of an alcoholic extract (EE) from Chiococca alba roots administered by oral (gavage) or parenteral (intraperitoneal or subcutaneous injections) to male and female mice Dose (mg/kg)

Sex

Route of administration Oral

Intraperitoneal

Subcutaneous

D/T

Symptoms

D/T

Latency

Symptoms

D/T

Latency

Symptoms

0

M F

0/2 0/2

No No

0/2 0/2

– –

No No

0/2 0/2

– –

No No

62.5

M F

– –

– –

0/2 0/2

– –

Piloerection Piloerection

0/2 0/2

– –

No No

125

M

–

–

2/2

Hypoactivity, ataxia, lethargy

0/2

–

No

F

–

–

0/2

>24 h, <48 h >72 h, <96 h –

Lethargy

0/2

–

No

250

M F

0/2 0/2

Hypoactivity Hypoactivity

2/2 1/2

>1 h, <2 h >1 h, <2 h

Hypoactivity, ataxia, lethargy Hypoactivity, ataxia, lethargy

1/2 1/2

>48 h, <72 h >24 h, <48 h

Ataxia Ataxia

500

M F

0/2 0/2

Hypoactivity Hypoactivity

1/2 2/2

>1 h, <2 h >1 h, <2 h

Hypoactivity, ataxia, lethargy Hypoactivity, ataxia, lethargy

2/2 2/2

>24 h, <48 h >24 h, <48 h

Lethargy, hypoactivity Lethargy, hypo activity

1000

M F

0/2 0/2

Hypoactivity Hypoactivity

– –

– –

– –

– –

– –

– –

2000

M F

0/2 0/2

Hypoactivity Hypoactivity

– –

– –

– –

– –

– –

– –

ALD

M F

>2000 mg/kg body wt >2000 mg/kg body wt

125 mg/kg body wt 250 mg/kg body wt

250 mg/kg body wt 250 mg/kg body wt

EE was dissolved in distilled water. All treated animals were carefully examined during 14 days for any sign of toxicity. D/T, dead/treated mice; No, no symptoms; latency, time elapsed between dosing and death; ALD, approximate lethal dose.

(Table 4) and for hypoactivity that appeared within 10–15 min and persisted for a few hours after dosing, no other sign of toxicity was observed in mice treated with repeated oral doses of EE. It is noteworthy that after the 4th or 5th oral dose of EE, hypoactivity was no longer observed. Liver, spleen, heart and kidneys weights did not differ between EE-treated and control mice (Table 4). Necropsy of control and EE-treated mice did not reveal any relevant finding either. Activities of monooxygenases (EROD and PROD) in the liver S9 fraction obtained from mice treated with an oral dose of EE as high as 2000 mg/kg body wt/day for 14 days remained unaltered. In the rodent liver, PROD seems to be predominantly catalysed by cytochrome P450 2B (CYP2B) subfamily isoforms and as expected for mice, activity of this monooxygenase was higher in females than in males, but no difference was found between EEtreated animals and their respective controls (Table 5). EROD, a reaction predominantly catalysed by cytochrome P450 1A

(CYP1A) subfamily isoforms, was not affected by repeated exposure to EE either (Table 5). Results, therefore, suggested that exposure to EE by the oral route did not induce mouse liver cytochrome P450 enzymes, at least as far as CYP1A and 2B subfamilies are concerned. Contrasting to the rather low toxicity by the oral route, repeated administration of EE by intraperitoneal injections caused pronounced lethality at daily doses equal to and higher than 31.3 mg/kg body wt (Table 6). Mice treated with the highest dose (125 mg/kg body wt) died on treatment days 1 and 2, while almost all deaths caused by the two intermediate doses (62.5 and 31.3 mg/kg body wt) occurred at the end of treatment period (days 12–14) (Table 6). Deaths after repeated administration of EE were generally preceded by symptoms, such as piloerection, rapid breathing, hypoactivity and ataxia. Data on EE-induced mortality suggested that males were somewhat more susceptible than females to toxic effects of the plant extract

Table 3 Effect of a single oral dose of Chiococca alba roots extract on the open field behavior of male and female mice Sex

Dose (mg/kg)

Locomotion (N)

Rearing (N)

Grooming (N)

Defecation (N)

Male

0 500

199 ± 33 89 ± 51*

24 ± 17 19 ± 23*

3±2 4±3

5±1 3±2

Female

0 500

188 ± 25 50 ± 26*

32 ± 10 14 ± 22*

5±3 6±6

4±3 2±1

Locomotion (number of squares traversed), rearing and grooming behaviors and defecation (fecal boluses) were measured during 10 min, starting 15 min after treatment by gavage with Chiococca alba extract (EE, 500 mg/kg) or its vehicle (distilled water) alone. Values are shown as means ± S.D. * P < 0.05, statistically different from the control group (dose 0) of the same sex.

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Table 4 Body weight gain (
g) and organ weights (g) of mice treated by the oral route with an alcoholic extract from roots (EE) of Chiococca alba (0, 500, 1000 and 2000 mg/kg body wt/day) for 14 days Treatment

Chiococca alba EE (mg/kg body wt/day, p.o.)

Males (N = 10) Body weight gain (days 14–1,
g) Organ weight (g) Heart Liver Spleen Kidneys Left Right Females (N = 10) Body weight gain (days 14–1,
g) Organ weight (g) Heart Liver Spleen Kidneys Left Right

0

500

1000

2000

8.87 ± 2.38

7.04 ± 3.82

7.08 ± 2.20

6.20 ± 3.02*

0.16 ± 0.03 1.74 ± 0.63 0.17 ± 0.03

0.18 ± 0.04 1.93 ± 0.38 0.17 ± 0.06

0.18 ± 0.04 2.28 ± 0.25 0.20 ± 0.02

0.17 ± 0.03 2.15 ± 0.05 0.16 ± 0.04

0.22 ± 0.03 0.23 ± 0.02

0.21 ± 0.02 0.21 ± 0.03

0.23 ± 0.04 0.23 ± 0.05

0.22 ± 0.04 0.22 ± 0.04

4.66 ± 1.95

2.27 ± 1.29*

2.32 ± 1.73*

1.56 ± 1.51*

0.16 ± 0.03 1.79 ± 0.19 0.17 ± 0.02

0.18 ± 0.04 1.76 ± 0.28 0.19 ± 0.04

0.15 ± 0.02 1.83 ± 0.17 0.17 ± 0.02

0.14 ± 0.02 1.73 ± 0.21 0.17 ± 0.03

0.19 ± 0.04 0.19 ± 0.04

0.19 ± 0.02 0.19 ± 0.02

0.18 ± 0.02 0.18 ± 0.02

0.18 ± 0.05 0.18 ± 0.02

Values are expressed as mean ± S.D. Data were evaluated by ANOVA and Student’s t-test. * Different (P < 0.05) from control (dose 0). Table 5 Effects of an alcoholic extract (EE) from Chiococca alba roots (2000 mg/kg body wt/day) given by gavage to male and female mice for 14 days on the activities of ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-depentilase (PROD) in the hepatic S9 fraction Enzyme

Activity (pmoles of resorufin/mg protein/min) for male and female sexes Control

EROD PROD

Chiococca alba EE (2000 mg/kg/day)

Male

Female

Male

Female

32.67 ± 1.26 5.79 ± 0.51

27.55 ± 1.10 7.60 ± 0.60*

33.91 ± 1.20 7.48 ± 0.43

27.47 ± 0.84 9.84 ± 0.51*

Values are means ± S.D. of five mice. Within each row (EROD or PROD), means were compared by ANOVA and Student’s t-test. No difference was found between mice treated with Chiococca alba EE and their respective controls of the same sex. * P < 0.05 PROD activity in females was higher than in males of the same treatment group.

(Table 6). Results showed that EE, at all dose levels tested, reduced mouse body wt gain during the 14-day treatment period (Table 7). Decreases of red blood cell count with microcytosis and hypochromia, as well as reductions of hemoglobin concentration and a lower hematocrit indicated that repeated administration of EE by the intraperitoneal (i.p.) route caused a mild microcytic hypochromic anemia (Table 8). No alteration of total white blood cell (WBC) count was detected. WBC differential count, however, revealed that among mice treated with doses of EE equal to or higher than 31.3 mg/kg body wt, the percentage of neutrophils was higher and the proportion of lymphocytes was lower than these percentages in control mice (Table 8). Necropsy of mice that died during the last days of treatment period and also that of those that survived to the scheduled sacrifice, showed sequels of peritoneal membrane irritation, such as an extensive adherence of diaphragm and mesentery to peritoneal cavity organs, as well as granulation tissue on the outer surface of liver and kidneys.

4. Discussion EE was toxic to Salmonella typhimurium at doses equal to or higher than 500 ␮g/plate. Toxicity to tester strains was somewhat reduced by addition of S9 mixture, a finding that suggests that it is to some extent detoxified by the action of liver enzymes. Results of the Salmonella assay either in the presence or in the absence of S9 mixture, however, clearly indicated that EE is not genotoxic. As far as the authors are aware, there is no previous study on the genotoxic potential of Chiococca alba crude extracts and their major chemical constituents. Acute toxicity of EE by the oral route was very low. It did not cause deaths at any dose level (approximate lethal doses (ALD) > 2000 mg/kg) and except for a transient hypoactivity at doses equal to or higher than 250 mg/kg, no other sign or symptom of toxicity was observed in the treated mice. The open field test showed that EE, at a single oral dose as high as 500 mg/kg, reduced exploratory behavior of male and female mice, a find-

V.E. Gazda et al. / Journal of Ethnopharmacology 105 (2006) 187–195 Table 6 Cumulative mortality of male and female Swiss Webster mice during treatment with daily doses of an alcoholic extract from Chiococca alba roots (EE; 0, 15.6, 31.3, 62.5 and 125 mg/kg body wt/day) given by intraperitoneal injections for 14 days Days

Chiococca alba EE (mg/kg body wt/day) for male and female sexes 0

1 2 3 4 5 6 7 8 9 10 11 12 13 14

15.6

31.3

62.5

125

M

F

M

F

M

F

M

F

M

F

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 1 1 1 3 4 4

0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 1 1 1 1 1 1 1 1 1 1 1 3 4

0 0 0 0 0 0 0 0 0 0 0 0 0 0

5 9 9 9 9 9 9 9 9 9 9 9 9 9

2 5 7 7 7 7 7 7 7 7 7 7 7 7

No. of treated, N = 10; M, males; F, females; N, number of mice initially treated. Control group (dose 0) received the vehicle (distilled water) only.

ing that is consistent with the hypoactivity noted in the acute and subacute toxicity assays. Single dose toxicity by parenteral routes (i.p. and subcutaneous (s.c.)), nevertheless, was considerably higher. EE-induced deaths at doses equal to or higher than 125 mg/kg (males) and 250 mg/kg (females) after i.p. injections and at doses equal to or higher than 250 mg/kg (both sexes)

193

following s.c. injections. Deaths caused by EE were generally preceded by signs and symptoms of CNS depression, such as hypoactivity, ataxia and lethargy. It should be pointed out that mice that survived to doses equal to or higher than 125 mg/kg body wt, given by i.p. or s.c. injections, also exhibited symptoms of CNS depression but apparently recovered within 12–24 h of treatment without any sequel. The acute approximate lethal doses found in the present study for EE given by i.p. and s.c. injections are very close to the LD50 (134.28 mg/kg i.p., mouse) of an alcoholic extract of Chiococca alba roots reported by Schapoval et al. (1983). A previous study also supports the view that Chiococca alba root extracts have CNS depressant effects. Felix et al. (1999) reported that an aqueous extract of Chiococca alba roots, administered by i.p. injections, markedly depressed the locomotion of rats in the open field arena, as well as impaired the performance of a T maze food rewarded task by mice and the performance of a conditioned active avoidance task by rats. Subacute toxicity of orally administered EE was rather low. Except for a transitory hypoactivity, no indication of EE-induced toxicity was observed during the subacute treatment by the oral route. Moreover, neither induction nor inhibition of liver monooxygenases (EROD and PROD) was detected after a 14day-treatment with oral doses as high as 2000 mg of EE/kg body wt/day. Contrasting to the low toxicity by the oral route, EE proved to be severely toxic to mice when repeatedly administered by the intraperitoneal route. At the highest dose administered by the i.p. route (125 mg/kg body wt/day), most mice were killed during the first 4 or 3 days of treatment being males somewhat more susceptible than females. In this case, deaths were preceded by hypoactivity, ataxia and lethargy and seemed

Table 7 Body weight gain (
g) and organ weights (g) of mice treated by the intraperitoneal route with an ethanolic extract from roots (EE) of Chiococca alba (0, 500, 1000 and 2000 mg/kg body wt/day) for 14 days Treatment

Males (N) Body weight gain(days 14–1,
g) Organ weight (g) Liver Spleen Kidneys Left Right Females (N) Body weight gain(days 14–1,
g) Organ weight (g) Heart Liver Spleen Kidneys Left Right

Chiococca alba EE (mg/kg body wt/day, i.p.) 0

15.6

31.3

62.5

125

10 4.12 ± 2.51

10 −3.53 ± 3.11*

6 −3.51 ± 2.51*

6 −2.80 ± 1.62*

1 −4.36

2.30 ± 0.22 0.19 ± 0.06

2.48 ± 0.14 0.24 ± 0.08

2.28 ± 0.25 0.20 ± 0.07

2.51 ± 0.22 0.29 ± 0.11

2.18 0.15

0.30 ± 0.03 0.30 ± 0.04

0.30 ± 0.04 0.29 ± 0.03

0.23 ± 0.04 0.23 ± 0.04

0.27 ± 0.02 0.27 ± 0.02

0.28 0.28

10 2.90 ± 2.45

10 −4.44 ± 2.69*

10 −6.09 ± 2.28*

10 −8.28 ± 4.54*

3 −7.43 ± 4.53

1.77 ± 0.17 0.18 ± 0.04

1.74 ± 0.24 0.21 ± 0.05

1.84 ± 0.25 0.22 ± 0.03

1.90 ± 0.31 0.20 ± 0.09

2.07 ± 0.10 0.27 ± 0.12

0.19 ± 0.03 0.19 ± 0.02

0.20 ± 0.03 0.21 ± 0.03

0.21 ± 0.03 0.21 ± 0.03

0.20 ± 0.02 0.20 ± 0.02

0.30 ± 0.08 0.30 ± 0.10

Values are expressed as mean ± S.D. Data were evaluated by ANOVA and Student’s t-test. * Different (P < 0.05) from control (dose 0).

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Table 8 Hematological parameters of male and female Swiss Webster mice treated with daily doses of an alcoholic extract from Chiococca alba roots (EE; 0, 15.6, 31.3, 62.5 and 125 mg/kg body wt/day) given by intraperitoneal injections for 14 days Treatment parameters

Males (N) RBC (106 ml−1 ) HGB (g dl−1 ) HCT (%) WBC (103 ml−1 ) Differential count (%) Eosinophils Neutrophils Lymphocytes Monocytes Females (N) RBC (106 ml−1 ) HGB (g dl−1 ) HCT (%) WBC (103 ml−1 ) Differential count (%) Eosinophils Neutrophils Lymphocytes Monocytes

Chiococca alba EE (mg/kg body wt/day i.p.) 0

15.6

31.3

62.5

125

10 9.90 ± 1.2 17.7 ± 2.2 47 ± 3

10 10.10 ± 2.5 17.1 ± 4.9 41 ± 4

6 8.07 ± 1.00* 14.0 ± 1.9* 43 ± 4*

6 7.61 ± 0.60* 13.4 ± 1.0* 42 ± 2*

1 6.35 11.3 38

3.8 ± 1.1

5.3 ± 2.1

4.2 ± 0.7

5.1 ± 2.5

4.1

1±1 23 ± 9 73 ± 9 2±1

2±2 25 ± 13 71 ± 11 3±2

3 ± 1* 28 ± 14 66 ± 17 4±3

4±4 43 ± 16* 52 ± 12* 2±1

2 32 65 1

10 9.49 ± 0.8 17.0 ± 1.7 47 ± 2

10 11.10 ± 1.2* 20.2 ± 2.6* 47 ± 2

10 7.58 ± 1.20* 14.5 ± 0.9* 44 ± 3*

10 8.86 ± 1.70* 14.4 ± 1.6* 40 ± 3*

3 9.18 ± 2.10 13.0 ± 2.1* 39.3 ± 3.0*

3.0 ± l.l

4.4 ± 1.6*

3.5 ± 1.4

4.3 ± 2.1

2.3 ± 0.2*

1±1 13 ± 4 84 ± 5 3±1

2 ± 1* 14 ± 7 83 ± 6 1±2

4 ± 2* 21 ± 9* 74 ± 9* 2±1

4 ± 2* 32 ± 16* 62 ± 16* 2±2

2±1 32 ± 7 47 ± 31 1±1

RBC, red blood cells; HGB, hemoglobin concentration; HCT, hematocrit; WBC, white blood cells. Values are means ± S.D. Data were evaluated by ANOVA and Student’s t-test. * Different (P < 0.05) from dose 0 (control group).

to have resulted mainly from an EE-induced CNS depression. At lower doses (31.3 and 62.2 mg/kg body wt/day), nevertheless, deaths occurred predominantly at the end of treatment period (days 12–14) and were probably related to other effects of EE, such as anemia and peritonitis. Mice treated for 14 days with doses of EE equal to or higher than 31.3 mg/kg body wt/day showed, at the end of the treatment period, a marked weight loss and a mild anemia. Since neither evidence of bleeding nor signs of hemolysis were found and red blood cells were small and hypochromic, anemia probably arose from an inadequate production of cells. The mechanism by which repeated parenteral doses of EE-induced anemia, however, is not clear. Necropsy of treated mice that survived to the scheduled sacrifice indicated that EE, injected directly into the peritoneal cavity, produced a chemical peritonitis. Therefore, peritonitis was possibly a contributory cause or even the main cause of death of animals treated with EE doses as high as 31.3 and 62.2 mg/kg body wt. WBC changes (neutrophilia) also seem to be consistent with this interpretation. It should be pointed out that the presence of irritating substances, such as methyl salicilate in Chiococca alba ethanolic extracts was previously reported by Schapoval et al. (1983). In summary, results from this study showed that Chiococca alba root ethanolic extract is not mutagenic in the Salmonella assay. Data presented here also demonstrated that acute and subacute oral toxicity of EE is very low when it is administered by the oral route. The Chiococca alba root ethanolic extract, on the other hand, proved to be considerably more toxic by parenteral (i.p. and s.c.) administration. The acute approximate lethal dose

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