Pharmacological and Toxicological Study ofMaytenus ilicifolia Leaf Extract. Part I –Preclinical Studies

Q1 Q2 Ricardo Tabach,1,4* Joaquim M. Duarte-Almeida1,3 and Elisaldo A. Carlini1,21Centro BrasileiroQ3 de Informações sobre Drogas Psicotrópicas (Cebrid), Departamento de Medicina Preventiva, Universidade Federalde São Paulo, UNIFESP, São Paulo, Brazil2Programa de PósGraduação emBiologiaQuímica, Universidade Federal de São Paulo,UNIFESP (DiademaCampus), Diadema, Brazil3Laboratório de Fitoquímica e Produtos Naturais, Universidade Federal de São João del Rei, UFSJ, São João del Rei, Brazil4Universidade de Santo Amaro, UNISA, São Paulo, Brazil

One of the Brazilians medicinal plants most cited in ethnopharmacological surveys for the treatment of ulcersand gastric diseases was evaluated for its efficacy and toxicity.Maytenus ilicifolia leaf extract (MIE) was acutelyand chronically (180 days) administered to rats, mice, and dogs. Acute tests were antiulcer effect andtoxicological trials (observational pharmacological screening, LD50, motor coordination, sleeping time andmotor activity). Chronic tests were the following: weight gain/loss and behavioral parameters in rats and mice;estrus cycle, effects on fertility, and teratogenic studies in rats and mutagenic features in mice, in addition tothe Ames and micronucleus test. The following parameters were assessed in dogs: weight gain/loss, generalphysical conditions, water/food consumption, and anatomopathological examination of the organs subsequentto the 180-day treatment. The results showed a clear antiulcer activity for MIE from 70 mg/kg and an absenceof toxicological effects in the three animal species, even if given in high doses or over a long period. The presentresults confirm the antiulcer property and absence of toxicological effects in three animal species of MIE, whichis in line with its current popular medicinal use. Copyright © 2017 John Wiley & Sons, Ltd.

Keywords: Maytenus; antiulcer; experimental studies; single-dose toxicity; chronic study.

INTRODUCTION

Peptic ulcers are among the commonest complaints byadult populations and are caused by an imbalancebetween factors that produce epithelial and deepermucosal injury and those that protect the mucosa andfacilitate its repair (de Jesus et al., 2012). Peptic ulcerdisease is in general a disease of chronic evolution, withrelapse and activate periods, and results from tissue lossthat occurs in regions of the digestive tract that comesinto contact with stomach secretions (Kumar et al.,2005).The use of plants to treat peptic illness in Brazil is a

common practice by the population. Among the plantsutilized for this purpose, Maytenus ilicifolia Martius exReiss (Celastraceae) known by the folk name of“espinheira santa” is greatly valued (Leite et al., 2010).Previous work in our laboratories with an aqueousextract of this plant has shown a strong protective effectagainst indomethacin and cold–restraint ulcers in rats,as well as a large increase in the volume and pH ofgastric juice (Souza-Formigoni et al., 1991; Tabach &Oliveira, 2003). Although there are several studies thataddress the effectiveness of M. ilicifolia as an antiulceragent, there are few evaluating its safety, possibly

because of its low toxicity in animal studies (Oliveiraet al., 1991).

Despite the great Brazilian biodiversity, the numberof herbal products using native medicinal plants is stillvery low. Evaluating the safety of these plants is alsonot a common practice. The safety and efficacy of thesenative plant products need to be tested in line with theguidelines of national and international regulatoryagencies so that those which might be of benefit to thepopulation become more widely available. This plantwas inserted between the monographs of the mostrecent Brazilian Pharmacopoeia (Brasil, 2010).

This study is part of a broader project aimed atdeveloping an herbal medicine from a standardizedextract of M. ilicifolia. Part I (this study) involved thepreclinical pharmacological and toxicological study,and part II is a clinical study to evaluate the safety andefficacy of this extract in humans.

MATERIAL AND METHODS

Preparation and evaluation of the extract. Leaves ofM. ilicifolia grown inCentro Pluridisciplinar de PesquisasQuímicas, Biológicas eAgronômicas of theUniversidadede Campinas (Unicamp) were dried and powdered. Avoucher specimen was deposited in UnicampHerbarium(UEC 112745). This material (3 g) was subjected toextraction with 150 mL of water heated between 85°Cand 100°C for 15 min by infusion. The determinations

* Correspondence to: Ricardo Tabach and Joaquim M. Duarte-Almeida,Department of Preventive Medicine, Federal University of São Paulo,UNIFESP, Rua Botucatu, 740, 4th floor, 04023-900 Sao Paulo, Brazil.E-mail: rtabach@unifesp.br; maudall@ufsj.edu.br

PHYTOTHERAPY RESEARCHPhytother. Res. (2017)Published online in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/ptr.5818

Copyright © 2017 John Wiley & Sons, Ltd.

Received 10 October 2016Revised 12 February 2017Accepted 24 March 2017

Journal Code Article ID Dispatch: 08.04.17 CE: Calunod, CherryP T R 5 8 1 8 No. of Pages: 6 ME:

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of total phenolic and tannin in the M. ilicifolia extract(MIE) were performed by Folin–Dennis method aspreviously described by AOAC (1995). The dry extracthad a yield of 17.5%, residual moisture of 3.96%, totalpolyphenol content of 41.57%, and tannin fraction of17.96%. The extract was filtered and spray driedaccording to the method described by Souza et al.(2001). The extraction solution was separated from theplant by pressing material. The dry extract ofM. ilicifoliawas obtained by spray drying tower from the aqueousextraction solution containing 20% of highly dispersedsilicon dioxide (Aerosil 200®/Degussa) under thefollowing conditions: inlet temperature – 146°C; outlettemperature – 97°C; feed stream – 3 mL/min; and spraypressure – 2 bar.

Animals. Female and male Wistar rats, 3–4 months ofage, weighing 300–350 g, and male Swiss albino mice,3–4 months of age, weighing between 30 and 40 g, werebred in the Department of Psychobiology ofUnivesidade Federal de São Paulo (Unifesp) in ananimal facility with air conditioning (23 ± 2°C), light–dark circle (12–12 h) and housed in plastic cages withwater and food provided ad libitum. Wistar rats wereused to perform the toxicological studies: chronic effects,estrus cycle, and teratogenic screening. Swiss albinomice were used to perform the follow pharmacologicaland toxicological studies: antiulcer activity, acidsecretion, observational pharmacological screening,motor coordination, motor activity, sleeping time, andacute and chronic studies. Wistar rats were used toperformer the toxicological studies: chronic effects,estrus cycle, and teratogenic screening. Male and femaleBeagle and mongrel dogs (7–9 kg) fromUniversidade deSão Amaro and Unifesp Canil (respectively), bred inanimal facilities with air conditioning (23 ± 2°C), werehoused with water and food provided ad libitum. Thisresearch was approved by the Unifesp Research EthicsCommittee (CEP 297/00).

Pharmacological studies. The MIE, diluted in water asneeded, was administered either through oral orintraperitoneally (i.p.) routes in constant volume of0.1 mL/10 g of weight in mice and 0.1 mL/100 g ofweight in rats. Different concentrations of the extractswere used in order to obtain the desired posology (mg/kg) for the animals; in all cases, a very fine stablesuspension was obtained. The doses employed in theanimals were based on the human daily dosage of thetraditional extract available in Brazil, approximately7 mg/kg (Oliveira et al., 1991). This dose served as basisof all experiments (varied between 5 and 200 timesconcentrations of lyophilized material).

Antiulcer activity. The ulcers were induced by the cold–restraint stress technique. The animals (male rats) werekept in their home cages for 18 h before this procedure,receiving sweetened milk (diluted in water 1:2) as theironly source of food. They were restrained and kept for2 h in a cold room (4°C), followed by analysis of thegastric lesions (Souza-Formigoni et al., 1991). For theanalysis, each rat was anesthetized by ether, theabdomen opened, and the stomach removed. Thestomach was then cut open along the greater curvature,the mucosa washed under running tap water, and thenumber and severity of lesions determined using a light

microscope. The ulcer index was calculated from thenumber of ulcers according to their size and othersparameters, such as edema and petechiae hemorrhage(Carlini et al., 2010). Tests were included using differentdoses of MIE (70, 140, and 280 mg/kg – equivalent to 10,20, and 40 times the therapeutic dose) i.p. or orally inorder to verify the possible protection against thedevelopment of gastric ulcers induced by the restraintand cold, given 60 min before the cold immobilization.

Effects on pH of acid secretion. Four groups of tenanimals (male rats) were given (i.p.) saline or differentdoses of the extract (70, 140, and 280 mg/kg) and after60 min were euthanized.

Toxicological studies: acute effects. Observationalpharmacological screening. Five groups of three (male)mice each were observed at 30 min intervals for 4 and24 h following the i.p. administration of either saline or1.4, 14, 70, or 140 mg/kg of MIE. The overall behaviorexhibited by the animals was recorded using a checklistincluding urinary frequency, defecation, piloerection,changes in locomotor activity, the presence of tremors,convulsions, muscular tonus, changes in posture, ataxia,ptosis, loss of reflexes, lacrimation, and salivation(Oliveira et al., 1991, Tabach et al., 2009).

Motor coordination (the rota-rod apparatus). Six groupsof ten (male) mice each received saline (control group)or 35, 70, or 140 mg/kg of extract (i.p.) or 700 or1400 mg/kg orally. The mice were first selected on theexperimental day by excluding those animals whichcould not remain on the rota-rod bar for threeconsecutive periods of 60 s each. Immediately, 30 min,1, 2, and 3 h after drug administration, the mice wereagain tested on the rota-rod apparatus and the timespent on the rotating bar was recorded for a maximumof 60 s (Tabach et al., 2009).

Motor activity. Spontaneous locomotor activity wasmeasured by the number of light beam interruptionscumulatively recorded when animals were placed in a40 × 25 × 20 cm cage equipped with three photocells.Groups of ten (male) mice each received either salineor 35, 70, or 140mg/kg of extract (i.p.). Immediately afteradministration, the animals were placed individually inphotocell cages and locomotor activity recorded every10 min over a period of 60 min (Tabach et al., 2009).

Potentiation of pentobarbital sleeping time. Threegroups of ten (male) mice each received saline, 140and 1400 mg/kg of extract orally. After 60 min, theanimals received 50 mg/kg of sodium pentobarbital (i.p.) and were placed in the cage. The duration of the lossof the righting reflex (sleeping time) was recorded.Animals were judged to be awake when they could rightthemselves up to three times (Bezerra et al., 2011).

Acute toxicity assays. Four groups of ten (male) miceeach received either saline or 2.72, 5.44, or 10.88 g/kgof MIE. The animals were kept in wooden cages (tenanimals per cage) and observed for 7 days, during whichthe time and the numbers of deaths was recorded(Carlini et al., 2013).

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Toxicological studies: chronic effects. Mice and rats.Two groups (male) of six mice each and two (male)groups of six rats each received daily water or140 mg/kg orally of MIE for 3 months. During thetreatment, body weight was evaluated weekly. Rota-rodperformance and potentiation of pentobarbital sleepingtime were evaluated in the mice acutely and after 45and 90 days of treatment. Rats were evaluated by openfield test acutely and after 45 and 90 days of treatment.After treatment, the animals (mice and rats) weredecapitated for subsequent pathological examination(Tabach et al., 2009).

Effects on estrus cycle in rats. Four groups of six femalerats were treated orally with water (control) or with 35,70, or 140 mg/kg of MIE over the course of 60 days.From days 1 to 15 (the first 15 days) and from days 45to 60 (the last 15 days), vaginal material was collecteddaily in order to determine the different phases of thecycle (proestrus, estrus, metestrus, and diestrus), inaddition to assessing the number of estrus, the intervalbetween estrus, and total duration of each cycle(Souza-Formigoni et al., 1998; Shivalingappa et al., 2002).

Teratogenic screening. Four groups of female rats(n = 10) received daily oral doses of either saline or70, 140, or 280 mg/kg of MIE. They were mated (threefemales : one male) at the beginning of the treatment.On the 11th day, the male was removed and the extractwas administered during all of the pregnancy. Thenumber and duration of pregnancies were recorded, aswell as the number of pups per litter, the total weightof each litter, and external signs of body malformations.Six pups per litter were randomly selected for furtherevaluation of the following parameters: average weightper litter on the 1st, 7th, 14th, and 21st day of life; dayof eyes and ears openings; the righting reflex wasrecorded on days 3 and 7 by placing the animals on theirbacks and recording the time taken to return to theupright posture; locomotor activity at days 8 and 13was measured by placing each animal on a surfacedivided into nine squares of 10 cm each and recordingthe number of squares crossed in 1 min. On day 70,the pups were tested in an open-field apparatus. Theopen field consisted of a circular white painted plywoodarena measuring 60 cm in diameter, equipped with three60-W lamps and three loudspeakers producing aconstant noise. The floor of the arena was divided intoseveral units by black painted lines. Each rat was placedin the center of the arena, and for 5 min, the number offecal boluses produced, the number of rearing episodes(standing up on hind feet), the time of grooming, thetime of freezing, and the number of square units crossedwere recorded (Carlini et al., 2013; Tabach et al., 2009).

Chronic experiments in dogs. The tests below werecarried out at Unifesp and Unitox – LaboratórioUniversitário de Análises Toxicológicas da Universidadede Santo Amaro (UNISA). Male Beagle dogs weredivided into two groups (two animals each): one controland one experimental, who received either water(control) or 140 mg/kg of MIE orally for 3 months.Female Beagle dogs were divided into two groups (threeanimals each): one control and one experimental, whoreceived either water (control) or 140 mg/kg of MIE(by mouth) for 90 days. The animals were assessed

weekly as to weight gain/loss, general physical condition,and consumption of water and food. Blood and urinewere collected before the start of the treatment (day 0)and on days 45 and 90 of the treatment, and the animalswere then euthanized and sent for anatomopathologicalexamination (Tabach et al., 2009).

Genotoxicity and mutagenicity test. The Ames test.Maytenus ilicifolia was analyzed to study eventualinduce reverse mutation in four lineages of Salmonellatyphimurium in the absence and in the presence ofmetabolic activation of a post-mitochondrial fraction ofrat liver induced by Aroclor 1254 (S-9) according to thatdescribed by Ames et al. (1975). The concentrationsused for the study were 1000, 2000, 3000, 4000, and5000 μg/plate in the absence and presence ofmetabolization. These concentrations were selectedwith the aim of using the highest concentration rangesallowed by solubility in the experiments and with this,to reach possibly mutagenic doses.

The micronucleus test. Maytenus ilicifolia was tested asto its capacity to induce chromosomal mutations in thebone marrow of (male) Swiss mice treated in accordancewith the method described by Schmid (1976). After apreliminary test, a maximum tolerated dose wasdetermined as 2 × 2500 mg/kg, which is the recom-mended dose (DMT) for this test, for little or no toxicproducts. Three test groups were used: 2 × 2500 mg/kg(DMT); 2 × 1250 mg/kg (1/2 DMT); and 2 × 625 mg/kg(1/4 DMT).

Both tests were carried out by Genotox – Laboratóriode Genotoxicidade da Universidade Federal do RioGrande do Sul.

Statistical analysis. Individual data were grouped accor-ding to each treatment, using as a measure of centraltendency for both the ulceration index and, for the pH,respective mean and standard deviation. All resultswere analyzed using analysis of variance, followed byDuncan’s test where appropriate. Results were consi-dered significant when the value was p ≤ 0.05.

RESULTS

Pharmacological effects

Antiulcer activity. The administration, either by i.p. ororally, of the extract at doses of 70, 140, and 280 mg/kgreduced significantly (p ≤ 0.05) the index of ulcerationinduced by restraint and cold, as can be seen inTable T11. Note also that the oral administration of theextract, although it also significantly reduced the rateof ulceration, caused an effect of lesser magnitude whencompared with that produced by the i.p. route. Theseresults show a protective effect of this extract againstthe development of ulcers in rats.

Effects on pH of acid secretion. There was an increasein pH and a reduction in the acidity of the gastricvolume contents after i.p. administration of differentdoses of MIE as can be seen in Table T22.

3MAYTENUS ILICIFOLIA: PRECLINICAL STUDIES

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Toxicological studies: acute effects

Observational pharmacological screening. The micetreated with 1.4; 14; 70 e 140 mg/kg of MIE (i.p.) didnot show significant differences as compared withcontrols for the parameters observed, that is, urinaryfrequency, defecation (number of fecal bolus), writhes,piloerection, locomotor activity, tremors, convulsions,posture, blepharoptosis, muscular tonus, lacrimation,exoftalmia, salivation, and death (results not shown).

Motor coordination (rota-rod apparatus). No significantdifferences were found between controls and treatedanimals in their performance on the rota-rod apparatus,thus indicating that the acute administration of 70, 140,and 280 mg/kg of MIE (i.p.) or 700 or 1400 mg/kg orallydid not induce gross alterations on the muscular tonusor motor coordination of mice (results not shown).

Motor activity. TableT3 3 shows the average activity inphotocell cages of mice treated with either saline or35, 70, or 140 mg/kg of MIE (i.p.). After 60 min ofadministration, there was a significant decrease(p < 0.05) in locomotor activity.

Potentiation of pentobarbital sleeping time. Theadministration of 140 or 1400 mg/kg of MIE (oraladministration) did not have a significant effect onsleeping time induced by pentobarbital (results notshown).

Acute toxicity assays. It was not possible to calculate theLD50 orally, for even a dose higher than 5000 mg/kg didnot produce deaths up to 7 days following administrationof the extract, suggesting a very low toxicity of thisproduct (Hodgson, 2004) (results not shown).

Toxicological studies: chronic effects

Mice and rats. No significant differences were foundbetween the two groups (water and MIE – 140 mg/kg)in body weight after 90 days of treatment either in ratsor in mice. No differences were observed betweencontrols and treated mice on their performance on therota-rod apparatus or in potentiation of pentobarbitalsleeping time throughout treatment (results not shown).In rats, no changes in the open field test were observedin the experimental groups in all parameters whencompared with the control group.

Chronic administration for 90 days of different dosesof the MIE in both animals (mice and rats) did not alterthe biochemical serum parameters (glucose, cholesterol,amylase, creatinine, uric acid, urea, albumin, totalproteins, GGT Q4, TGP, alkaline phosphatase, and serumglutamic oxalacetic transaminase), hematological redseries (erythrocytes, hemoglobin, hematocrit, mean cellvolume, and mean cell hemoglobin), white series(leukocytes, neutrophils, lymphocytes, monocytes,eosinophils, and basophils), and platelets, whencompared with the control group. No significant resultwas found in the anatomopathological examination ofthe organs that might be attributed to chronic treatmentwith MIE, evidencing absence of toxic effects (resultsnot shown).

Effects on estrus cycle. The treatment with differentdoses of MIE extract (35, 70, and 140 mg/kg) over thecourse of 60 days did not produce any significantalterations in the various parameters of the estrus cyclein rats (number of estrus, interval between estrus, andtotal duration of the cycle both in the first 15 days andin the last 15 days of treatment). At the termination ofthe chronic treatment, the females were killed, andtheir organs subjected to an anatomopathological

Table 1. Effect of i.p. or oral administrations of MIE on theulceration index caused by immobilization and cold (male rats,n = 10)

Treatment Dose (mg/kg) Ulceration index % reduction

i.p. Control 22.6 ± 1.8 —

70 11.7 ± 1.1* 48MIE 140 9.7 ± 0.6* 57

280 10.6 ± 1.2* 53

Oral Control 20.7 ± 2.2 —

70 13.5 ± 1.8* 35MIE 140 13.2 ± 1.7* 36

280 13.1 ± 1.3* 37

MIE, Maytenus ilicifolia extract.Values are means ± standard deviation,*p ≤ 0.05 compared with control group (ANOVA, followed by theDuncan test).

Table 2. pH of gastric volume contents of rats pretreated (i.p.)with extract of MIE (male rats, n = 10)

Treatment Dose (mg/kg) pH % elevation

Control —

2.4 ± 0.1—

704.1 ± 0.4*

71

MIE 1404.7 ± 0.4*

96

2803.3 ± 0.3*

38

MIE, Maytenus ilicifolia extract.Values are means ± standard deviation,*p ≤ 0.05 compared with control group (ANOVA, followed by theDuncan test).

Table 3. Motor activity during the 60-min after administration (i.p.) of MIE (male mice per dose, n = 10)

Treatment Dose (mg/kg) Motor activity % reduction

Control — 2000 ± 194 —

35 1001 ± 88* 50MIE 70 957 ± 148* 48

140 871 ± 149* 44

MIE, Maytenus ilicifolia extract.Values are means ± standard deviation,*p ≤ 0.05 compared with control group (ANOVA, followed by theDuncan test).

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examination. No alteration worthy of note was observedin the macroscopic and microscopic examination of theorgans (results not shown).

Teratogenic screening. Maytenus ilicifolia extracttreatment did not cause significant differences inparameters such as the number and duration ofpregnancies, as well as the number of pups per litter,the total weight of each litter, and external signs of bodymalformations. Finally, no significant differences werefound among the groups for all other parameters suchas eye-opening or ambulation of the pups on the 8thand 13th day of life indicating no teratogenic effect(results not shown).

Chronic experiments in dogs. The administration of upto 140 mg/kg of MIE orally did not cause significantchanges in both males and females in all parametersevaluated during treatment (data not shown). Thehematological and biochemical alterations were fewand were within the reference standards for the breedand age. Some of these parameters observed in thefemales are presented in TableT4 4.

Genotoxicity and mutagenicity tests

Ames test. The results indicate that MIE did not inducemutations in the four lineages of S. typhimurium,according to the report supplied by the UFRGSLaboratory of Genotoxicity (results not shown).

Micronucleus test. The maximum tolerated dose(2 × 2500 mg/kg) of MIE did not produce signs oftoxicity, nor any significant reduction in the productionof erythrocytes, and was considered negative forchromosomic mutagenicity, according to the reportsupplied by the UFRGS Laboratory of Genotoxicity(results not shown).

DISCUSSION

In Brazil, espinheira santa is popularly regarded as amedicament for the treatment of tumors, the prevention

of fermentation and formation of intestinal gas, andcombating diseases of the liver and kidneys, as a healingagent, a diuretic, an aid to lactation, and an analgesic,with anti-asthmatic, contraceptive, antiulcer, tonic, andantiseptic properties (Almeida, 1993; Niero et al.,2011). However, more emphasis has been given to itsuse in gastrointestinal disorders such as hyperacidity,gastric and duodenal ulcers, gastralgia, and functionaldyspepsia (Simões et al., 1995). In the case of gastritisand gastric ulcers, previous data obtained in ourlaboratory has shown that freeze-dried extracts ofM. ilicifolia leaves and Mahonia Q6aquifolium were ableto protect the gastric mucosa against ulcer inducingagents in experimental rats (Souza-Formigoni et al.,1991). In addition, no toxic effect was observed, evenafter chronic administration (3 months) of Maytenus inlaboratory animals (Oliveira et al., 1991).

The activity of M. ilicifolia on peptic ulcers andgastritis seems to involve multiple mechanisms of action,not yet fully elucidated, which may be associated withmore than one group of active ingredients (Santos-Oliveira et al., 2009). Tannins (epigallocatechin, forexample) and terpenes (fridenelol, for example) areamong the active ingredients that can protect the gastricmucosa by regulating the secretion of hydrochloric acidin the stomach and also through bacteriostatic actionagainst the Helicobacter pylori, a bacteria usuallyassociated with gastritis and ulcers (Santos-Oliveiraet al., 2009).

Ferreira et al. (2004) found that freeze-dried aqueousextract of M. ilicifolia reduced, in a dose dependentform, acid secretion of the isolated gastric mucosa offrogs through antagonism of the histamine H2 receptor.These results were similar to those obtained usingcimetidine as a positive control. Although other mecha-nisms cannot be ruled out (inhibition of the protonpump and inhibiting the biosynthesis of histamineand/or its release), the H2 receptor blockage appearsto play an important role in the action mechanism ofM. ilicifolia.

Cipriani et al. (2009), working with female rats, foundthat polygalacturonic acid, obtained from the aqueousfractionation of M. Maytenus leaves, significantlyinhibited the gastric lesions induced by ethanol.According to the authors, the mechanism involved isrelated to the ability of this substance to act as aprotective layer of the gastric mucosa, reducing acidsecretion and pepsin and increasing mucus synthesisand/or scavenging free radicals.

Some species of the genus Maytenus have been theobject of toxicological evaluation, for example, Maytenusheterophylla senegalensis (da Silva et al., 2011) orgenotoxicity as with Maytenus robusta (Raymundoet al., 2012). Niero et al. (2011), a review of studies of theMaytenus genus, highlighted M. ilicifolia as the moststudied species, with numerous studies on its potentialantiulcerogenic properties, either as a crude extract oras a fraction or even as an isolated component, makingthis of great interest as a medicinal plant. However,unlike the preclinical pharmacological studies, thetoxicological studies involving M. ilicifolia are scarceand show a gap in the study of this plant.

This study evaluated both the antiulcer activity andthe preclinical toxicology of this species with the resultsobtained from this extract showing that it protected thegastric mucosa through a significant reduction in the

Table 4. Effect of 90-day oral administration of 140 mg/kg of MIEon hematological parameters in female Beagle dogs (n = 6)

Control MIE

Erythrocytes (mm3) 7.3 ± 0.7 7.6 ± 0.5Hemoglobin (%) 16.0 ± 1.5 16. 3 ± 1.5Platelets (mm3) 375.0 ± 49.3 390.2 ± 78.4Total leukocytes (mm3) 10.6 ± 1.3 11.9 ± 2.4Uric acid (mg/dL) 0.4 ± 0.1 0.7 ± 0.4Creatinina (U/L) 0.7 ± 0.1 0.7 ± 0.1GOT (U/L) 48.0 ± 7.4 44.0 ± 5.6GPTQ5 (U/L) 40.8 ± 5.9 55.3 ± 16.9Glucose (mEq/L) 97.3 ± 2.2 101.3 ± 2.5

MIE, Maytenus ilicifolia extract.Values are means ± standard deviation, (p > 0.05, NS, analysis ofvariance).

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ulceration index, both intraperitoneally as well as orally,in addition to causing an increase in pH and gastric juicevolume. One of the few observed effects was asignificant reduction in locomotor activity in animalstreated with MIE. This was probably the result of anunspecific effect of the preparations, becauseadministration of i.p. injections, regardless of thesubstance or extract, often causes abdominal discomfortand irritation, hindering the motor activity of animalssubjected to this route of administration.Prolonged administration did not cause toxic or

teratogenic effects, even when tested in three differentanimal species. The results of genotoxicity tests werealso negative, indicating that this extract may be an

important therapeutic tool for the treatment of gastritisand gastric ulcers.

All these results indicate therapeutic potential and alack of toxicity that allow the continuation of studies,making it possible to start the clinical toxicological studydescribed in the second part of this project (Tabachet al., 2017; PTR-16-1270).

A Q7cknowledgement

The authors would like to thank Dr Marna Eliana Sakalen forreviewing the text and José Roberto Lazarini (Laboratório Aché).This work has financial support from the CEBRID and AFIP.

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Q8Tabach R, Duarte-Almeida JM, Carlini EA. 2017. Pharmacologicaland toxicological study of Maytenus ilicifolia leaf extract. PartII - Clinical Study (Phase I). Phytother Res PTR-16-1270.

6 R. TABACH ET AL.

Copyright © 2017 John Wiley & Sons, Ltd. Phytother. Res. (2017)

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