Journal of Ethnopharmacology, 34 (I 991) 29-41 29

Elsevier Scientific Publishers Ireland Ltd.

Pharmacologic and toxicologic effects of two Maytenus species in

laboratory animals

Maria Gabriela M. Oliveira, Maristela Goldnadel Monteiro, Claudia Macatibas, Valrria

Pereira Barbosa and E.A. Carlini

Department of Psychobiology. Escola Paulista de Medicina. Rua Botucatu 862. I ° andar. C.P. 20399. CEP 04034. Sdo Paulo, SP (Brazil)

(Accepted February 20, 1991)

Leaves of Maytenus species are used in the popular medicine of Brazil for their reported antacid and anfiulcerogenic activity. The
present work examined the effects of a boiling water extract of equal parts of M. aquifolium and M. ilicifolialeaveson acute administration
in rats and mice, in an attempt to detect any general depressant, hypnotic, anticonvulsant and analges!c effects. General depressant
and hypnotic effects were seen only after intraperitoneal administration. After chronic administration, the overall behavior of animals
did not change and they continued to gain weight at the same rate as controls. Several biochemical and hematological parameters
as well as pathological examination of different organs did not show any significant alterations after 3 months of treatment. A search
for the potential effects of the extract on the fertility of female and male rats and on the course of pregnancy as well as a search for
potential teratogenic effects did not reveal any significant differences from controls. Taken together, the results indicate that these
Maytenus species may be safe for human use and deserve further investigation.

Key words: Maytenus species; preclinical pharmacology; toxicology; anticoagulant plant.

Introduction aim of the present study was to evaluate the pre-

Several M a y t e n u s species o f the f a m i l y
Celastracae are commonly used in Brazilian popular
medicine for their antacid and antiulcerogenic ef-
fects (Silva-Araujo and Lucas, 1930). They are
usually used as a tea made by pouring boiling water
on fresh or dried leaves ( " a b a f a d o ' ) (Balbach, 1980;
Cruz, 1982). However, it is k n o w n that plant
preparations can have serious effects both in
laboratory animals and in man (Gunby, 1979; Hux-
table, 1980; Schoental, 1982). Previous work from
our laboratory (Souza-Formigoni et al., 1991) has
indicated the antiulcerogenic effects o f two
Maytenus species against indomethacin- and cold-
restraint-induced ulcers in laboratory animals. The
Correspondence to: M.G.M. Oliveira, Department of
Psychobiology,Escola Paulista de Medicina, Rua Botucatu 862.
I° andar, C.P. 203399. CEP 04034, Silo Paulo, SP (Brazil).
clinical p h a r m a c o l o g y and toxicology o f these
species in rats and mice.
Materials and Methods
Plant material
Leaves o f M a y t e n u s aquifolium Martius and M.
ilicifolia Martius were collected in the state o f
Paranfi and Rio G r a n d e do Sul (South o f Brazil)
and classified by botanists from local universities.
The leaves were dried, ground and kept in dark glass
containers. In all experiments, an equal mixture o f
both species was used. F o r administration to mice
and rats, a tea was prepared by pouring 150 ml o f
boiling water over 3 g o f powdered dried leaves and
the container covered with aluminum foil until it
equilibrated with room temperature. After filtration,
the preparation was then lyophilized giving an
average o f 3.4 mg solid material per ml o f the tea

0378-8741/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd.

Published and Printed in Ireland
30
(in human terms, a dose equivalent to about 6.8
mg/kg of starting dry leaf material). This dose served
as the basis for all experiments. In the present study,
the doses used varied between 1 and 50 times that
of 6.8 mg/kg.
Acute effects
Animals. Swiss male mice from our own colony
were used for all acute pharmacological testing.
They were kept in a controlled-temperature (24 +
2°C) room within a 12-h light-dark cycle. At the
time of testing they were 2 - - 3 months old and
weighed 3 0 4 0 g.
Observational screening. Groups of 3 mice were
observed at 30-min intervals for 4 h following the
oral administration of either saline or 1360 mg/kg
or 2720 mg/kg of Maytenus. The overall behavior
exhibited by the animals was recorded using a check
list, which included: 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 (Carlini, 19721.
Rota-rod test. Groups of 10 mice received either
saline or 680 mg/kg or t 360 mg/kg of Maytenus oral-
ly. The apparatus consisted of a bar, with a diameter
of 2.5 cm subdivided into five compartments by
disks 25 cm in diameter (Dunham and Miya, 1957).
The bar rotated at a constant speed of 12 rev/min.
Mice were first selected on the experimental day by
excluding those animals which could not remain on
the rota-rod bar for two consecutive periods of 1
min each.
Thirty minutes, 1 h and 2 h after drug administra-
tion, the mice were again tested on the rota-rod ap-
paratus, and the time spent on the rotating bar was
recorded for a maximum of 60 s.
Locomotor activity. Spontaneous motor activity
was measured by the number of light beam inter-
ruptions cumulatively recorded when animals were
placed in a 40 x 25 x 20 cm Plexiglass cage equip-
ped with three photocells. Groups of 10 mice each
received either saline or 6.8 mg/kg, 170, 340, 680 and
1360 mg/kg of Maytenus intraperitoneally (i.p.). Fif-
teen minutes later they were placed individually in
a photocell cage and the locomotor activity recorded
over a period of 30 min. Activity testing was always
carried out between 13:00 and 16:00 h.
Analgesic testing. Groups of 8 mice each receiv-
ed oral doses of either saline or 680 or 1360 mg/kg
of Maytenus. The animals were placed on a 55°C
hot plate (Gyires and Torma, 1984) before and 30,
60 and 90 min after oral drug administration. The
latency for licking the forepaws was recorded.
In addition, groups of 6 mice each were treated
with oral doses of saline or 680 or 1360 mg/kg of
Maytenus. Thirty minutes later they were injected
i.p. with 0.8% acetic acid. The number of writhing
behaviors was recorded for a period of 5 rain (Gyires
and Torma, 1984).
Potentiation qf penlobarhital sleeping time.
Groups of I0 mice each were treated either with
saline or 6.8, 85.4, 170, 340, 680 or 1360 mg/kg of
Maytenus. Thirty minutes after the i.p. injection or
60 min after the oral (p.o.) administration, the
animals received 30 mg/kg of sodium pentobarbital
i.p., and placed on their backs in a V-shaped trough.
The duration of the loss of the righting reflex (sleep-
ing time) was recorded. Animals were judged to be
awake when they could right themselves three times
in 30 s.
LDso determination. Seven groups of 10 mice
each recieved either saline or 340, 680, 1360, 2720,
5440, or 10880 mg/kg of Mavtenus p.o. The animals
were kept in wooden cages (5 animals per cage) and
observed for seven days, during which time the
number of deaths was recorded.
Anticonvulsant activity. Groups of 10 mice each
received either saline or 1360 mg/kg of Maytenus
p.o. Thirty minutes later, the mice received a
transcorneal electroshock of 8 mA for 2 s. The
number of animals showing tonic convulsions and
the number of deaths were recorded. A convulsion
index was calculated by the ratio between forelimb
extension and flexion times (E/F index). In addition,
two different groups of mice received the same
pretreatment described above, but were injected 30
min later with 100 mg/kg of pentylenetetrazole sub-
cutaneously (s.c.). The latency for tonic convulsions,
the number of convulsive episodes and the number
of deaths were recorded.
Chronic ej]ects
Anima&. Swiss male mice and/or Wistar rats from
our own colony were used for the pharmacological
tests and kept in a controlled-temperature room

under the conditions previously described. At the
time of testing, the mice were 2--3 months old and
weighed 30---40 g while the rats were 3 months old
and weighed 250--350 g.
General procedure. Three groups of 12 rats each
received daily oral doses (gavage) of either saline
or 136 or 272 mg/kg of Maytenus for 2 months (one
dose per day). Other groups of rats and mice receiv-
ed, respectively, an aqueous solution containing 17
and 3.4 mg/ml of Maytenus as their only source of
fluids, with free access to food during 3 months.
During the treatment, fluid intake, body weight,
temperature and glycemia (blood was taken from
the tail and blood sugar measured with a Dextro-
meter~ -Ames) were evaluated in both rats and
mice. Rota-rod performance was evaluated every
15th day only in mice. After treatment, half of the
rats were decapitated, with the blood collected for
hematological and biochemical analysis. The other
rats were also decapitated but immersed in 10% for-
maldehyde for subsequent pathological examina-
tion. Pathological examination of organs was also
performed in all mice.
Teratogenic screening
Animals. Wistar male and female rats, 3 months
old and weighing 250--350 g were used for all ex-
periments. They were kept in a controlled room en-
vironment as previously described.
Effects on estrus cycle. Three groups of 5 female
rats received daily oral doses of either saline or 272
or 544 mg/kg of Maytenus for 30 days, during which
the number of estrus cycles and the time between
cycles were determined. For this purpose, a small
volume of saline was introduced into the vagina, im-
mediately aspirated, and examined using a light
microscope.
Treatment prior to mating. Two groups of 10
female rats each received daily oral doses of either
saline or 544 mg/kg of Maytenus for 45 consecutive
days. After treatment, they were mated (5 females:2
males) for 10 days. The number and duration of
pregnancies were recorded and after birth, the
number of pups per litter and the average weight
per litter were also established. The presence or
absence of body malformation was observed.
Treatment during pregnancy. Groups of 10 female
virgin rats received oral doses of saline or 136 and
31
272 mg/kg of Maytenus once a day, beginning at day
1 of pregnancy and extended to the day of delivery.
The number and duration of pregnancies were
recorded, as well as the number of pups per litter.
Six pups per litter were randomly selected for
analysis of the following parameters: (i) external
signs of body malformations and (ii) average weight
per litter on the 1st, 7th, 14th and 21st day of life.
The righting reflex was evaluated on days 3 and 7,
by placing the animals on their backs and recording
the time needed to return to the right posture. Also
recorded was the day of eye opening and ambula-
tion on days 8 and 13. Locomotor activity was
evaluated by placing each animal on a surface divid-
ed in 9 squares of 10 cm each and recording the
number of squares crossed in 2 min.
On day 70, the pups were tested in an open-field
apparatus. The open-field consisted of a circular
white painted plywood arena measuring 60 cm in
diameter, equipped with three 60-watt lamps and
three loudspeakers producing a constant noise of
76 decibels. The floor of the arena was divided into
several units by black painted lines. Each rat was
placed in the center of the arena and during a 3-min
period the number of fecal boluses produced, the
number of rearing episodes (standing up on hind
feet) and the number of square units crossed were
recorded (Masur, 1972).
On day 90, the animals were also tested for lear-
ning abilities in a spatial discrimination task using
a T-maze. The apparatus used was a wooden maze
with a 92 x 12 cm stem, and two 80 x 12 cm arms.
The starting box (10 x 12 cm) was separated from
the stem by a vertical sliding door. Two goal-boxes
(10 x 12 cm) were separated from the respective en-
trance arm by similar sliding doors. Food was
available inside each goal-box. The animals were
deprived of food for 24 h before testing. The experi-
ment lasted 2 days. Day 1 consisted of an habitua-
tion session, where the animal was placed in the
start-box and after 30 s the door to the stem was
opened. When the animal entered one of the arms,
the door of that arm was closed. After a 30-s con-
finement in the chosen arm, the animal was remov-
ed and placing in the starting box for a second
identical trial. The procedure was repeated 10 times.
On day 2, rats were trained for spatial discrimina-
tion learning reinforced ,vith food placed only in
32

TABLE I

EFFECTS OF ORAL ADMINISTRATION O F MA Y T E N U S O N T H E R O T A - R O D PERFORMANCE OF MICE

Treatment Oral dose M e a n time 4- S.D. on rod (s)

(mg/kg)
+30 +60 +120 min

Saline -- 46.4 ± 22.5 53.8 4- 9.5 48.3 4- 18.6

Maytenus 680 39.5 4- 18.7 41.1 4- 21.3 48.0 4- 21.0
1260 46.8 4- 12.1 37.8 4- 21.4 37.3 4- 19.7

the same goal-box first chosen. Criterion for learn- Results

ing the task consisted of 5 consecutive correct runs
to the same reinforced goal-box (Bueno et al., 1984). Acute effects
Treatment of male rats before mating. Three Observational screening. The mice treated with
groups of 5 male rats each received daily oral doses 1360 and 2720 mg/kg of Maytenus did not show
of either saline or 136 or 272 mg/kg of Maytenus significant differences as compared to controls for
for 60 days. After treatment, each rat was placed the parameters observed, i.e., urinary frequency,
in a mating cage with two females for 6 days. When defecation (number of fecal bolus), polierection,
pregnancy was detected, the females were separated locomotor activity, tremors, convulsions, muscular
and the number of pregnancies, number of pups per tonus, posture, ataxia, blepharoptosis, loss of
litter and the average weight per litter at birth were reflexes, lacrimation or salivation (data not shown).
recorded. Rota-rod test. The results obtained are presented
in Table 1. No significant differences were found
between controls and treated animals on their per-
formance on the rota-rod apparatus, thus indicating
TABLE 2
that the acute administration of 680 and 1360 mg/kg
of Maytenus did not induce gross alterations on the
LOCOMOTOR ACTIVITY OF MICE TREATED WITH muscular tonus or m o t o r coordination of mice.
S A L I N E O R MA Y T E N U S Locomotor activity. Table 2 shows the average ac-
tivity in photocell cages of mice treated p.o. or i.p.
Treatment Dose Route M e a n 4- S.D.
(mg/kg) Locomotor count with saline or different doses of Maytenus. No dif-
(n) ferences were found after oral administration of
Maytenus," however, after i.p. administration there
Saline i.p. 243 4- 121
was a significant decrease in locomotor activity. This
Maytenus a 6.8 i.p. 212 ± 86
170 i.p. 117 4- 51" effect occurred with all but the lowest dose of
340 i.p. 85 4- 45* Maytenus (i.e., 6.8 mg/kg) as compared to controls.
680 i.p. 31 4- 24* Analgesic testing. As can be seen in Table 3, treat-
1360 i.p. 53 4- 25* ment with the two doses of Maytenus caused slight
increases in the reaction time at 30, 60 and 90 min,
Saline p.o. 216 ± 101
Maytenus b 340 p.o. 296 4- 62 but these responses were not statistically different
680 p.o. 301 4- 103 from baseline values.
1360 p.o. 243 ± 98 Table 4 shows that oral administration of 680 and
a A N O V A FI5,54~ = 16.073, followed by D u n n e t t ' s t-test: * P <
1360 mg/kg of Maytenus did not alter significantly
0.05. the number of abdominal contractions induced by
b A N O V A F(3,361 = 2.136: not significant. acetic acid administered i.p. as compared to con-
 33

TABLE 3

REACTION TIME OF MICE SUBMITTED TO THE HOT PLATE (55°C) TEST BEFORE A N D AFTER ORAL
A D M I N I S T R A T I O N OF MA YTENUS OR SALINE

Treatment Dose Mean 4. S.D. reaction time (s)

(mg/kg)
-5 +30 +60 +90 min

Saline -- 13.1 4. 2.6 12.3 4- 4.6 12.3 ~- 5.5 10.1 4. 2

Maytenus 680 I 1.6 4. 1.8 15.0 4. 4.5 13.1 + 4.8 12,3 4. 8
1360 10.3 4. 1.9 14.9 4. 9.7 16.9 4. 9.8 15.4 4. 7

TABLE 4
trois. Taken together with the above findings, it ap-
EFFECTS OF ORAL ADMINISTRATION OF pears that Maytenus does not have a significant
MAYTENUS OR SALINE ON THE NUMBER OF analgesic effect.
W R I T H I N G MOVEMENTS (ABDOMINAL CONTRAC-
Potentition of pentobarbital sleeping time. As can
TIONS) I N D U C E D BY ACETIC A C I D IN MICE
be seen in Table 5, there was an increase in the sleep-
Treatment Dose Mean 4. S.D. ing time induced by pentobarbital after all doses of
(mg/kg) Abdominal contrac- Maytenus given i.p.: however, only the responses
tions (n) seen with the three highest doses Were statistically
Saline -- 13.7 4- 5.4 different from the saline control. The same doses
Maytenus 680 7.5 4. 4.3 given orally did not have an effect on the sleeping
1360 11.7 4. 5.3 time.
LDso determination. Only two deaths were
observed in the 7 days following a single oral ad-
ministration of several different doses of Maytenus
TABLE 5 (340, 680, 1360, 2720, 5440 and 10880 mg/kg); one
EFFECTS OF MA YTENUS ON THE SLEEPING TIME IN
death occurred 7 days after the administration of
MICE I N D U C E D BY PENTOBARBITAL (30 mg/kg i.p.) 680 mg/kg of Maytenus; the other occurred 4 days
after the administration of 5440 mg/kg. No deaths
Treatment Dose Route Mean 4. S.D. were observed in the control group. These results
(mg/kg) durtion o f lost reflex
did not allow the calculation of the LDs0 ( > 1.09
(min)
g/kg) but they indirectly indicate the absence of
Saline Lp. 19.9 ± 17.2 severe toxic effects of Maytenus.
Maytenus a 6.8 I.p. 44.2 + 33.0
Anticonvulsant activity. Table 6 summarizes the
85.4 l.p. 43.0 + 29.5
effects of Maytenus against the tonic convulsions
170 Lp. 45.3 ± 27.3
340 I.p. 45.2 4. 27.9 and deaths induced by transcorneal electroshock.
68O l.p. 55.2 4- 24.6* There was no observable effect of 1360 mg/kg of
1360 I.p. 54.1 4. 21.6" Maytenus p.o. against the convulsions. There was
2720 l,p. 88.6 4. 21.6" a small increase in the number of deaths of animals
Saline -- p.o. 10.1 4- 14.3
treated with the plant, though it was not statistically
Maytenus 170 p.o. 15.1 ± 17.2 different from controls.
680 p.o. 12.9 + 20.4 As can be seen in Table 7, the p.o. dose of 1360
1360 p.o. 10.5 + 16.1 mg/kg of Maytenus did not protect the animals from
aANOVA /:'(7,72) = 5.505, followed by Dunnett's t-test *P < the convulsions and deaths induced by pentylene-
0.05. tetrazole.
34

TABLE 6 control group in regard to the rate of weight gain.

There were no differences between controls and
P E R C E N T A G E O F C O N V U L S I O N S A N D D E A T H S IN-
DUCED BY T R A N S C O R N E A L E L E C T R O S H O C K IN experimental rats in regard to glucose blood levels
MICE O R A L L Y P R E T R E A T E D WITH MA YTENUS (Table 9). In the same way, no differences were
observed on body temperature of rats as compared
Treatment Dose Mean -4- S.D. Tonic Deaths to controls, either at the beginning of treatment or
(mg/kg) E/F index convul- (%)
after 2, 6 and 8 weeks of Maytenus administration
sions
(%) (Table 10). However, in the 4th week, animals
treated with Maytenus showed a significantly lower
Saline 13.3 4- 3.1 100 30 body temperature when compared with controls.
Maytenus 1360 15.3 4- 3.3 100 50 However, this finding was not accompanied by any
other observable change in the physiology or
behavior of the animals, hence it is considered of
little biological significance.
TABLE 7 Table 11 and 12 summarize the results found on
EFFECTS OF MA YTENUS O R A L P R E T R E A T M E N T ON
serum analyses of urea (U), creatinine (CR), total
CONVULSIONS A N D DEATHS I N D U C E D BY PEN- lipids (TL), serum glutamic oxalacetic transaminase
T Y L E N E T E T R A Z O L IN MICE (SGOT), alkaline phosphatase (AP), sodium
(Na+), potassium (K+), total protein (TP), pan-
Treatment Dose Mean 4. S.D. Convul- Deaths creatic amylase (AMY) and the hematological
(mg/kg) latency sions (%)
evaluation of rats treated with Maytenus as com-
(s) (%)
pared to controls. Significant higher levels of urea
Saline -- 158 4. 30 100 100 and potassium were found in the animals treated
Maytenus 1360 147 4- 34 100 100 respectively with 272 and 136 mg/kg of Maytenus.
No other differences were observed between con-
trois and treated rats in regard to the parameters
measured, including the hematological evaluation.
There were no changes noted in differential white
Chronic effects blood cell count (data not shown). Lung congestion
Prolonged oral administration in rats. Table 8 with focal hemorrhage infra-alveoli as well as lym-
presents the changes in body weight of rats treated phocytic peribronchiolae infiltration were found in
daily with saline or Maytenus for two months. For all rats treated either with saline or Maytenus. pro-
this period, both test groups had significant in- bably due to the prolonged gavage administration.
creases in body weight. No significant differences No other alterations were found after examination
were found between the two groups and the saline of stomach, esophagus, duodenum, intestines, liver,

TABLE 8

BODY W E I G H T OF RATS T R E A T E D FOR TWO MON TH S WITH DALLY O R A L DOSES O F MA YTENUS

Treatment Dose Mean body weight + S.D. (g)

(mg/kg)
0 +2 +4 +6 +8 weeks

Saline -- 243 4. 26 268 4. 29 281 4. 31 303 4. 31 307 4. 3_.2

Maytenus 136 255 4. 18 272 4. 25 287 4. 23 301 4- 24 312 4. 24
272 267 4. 27 292 4- 31 305 4. 35 329 4. 35 330 4- 39
ANOVAF(2,29) 2.396 2.009 1.992 2.643 1.481
 35

TABLE 9

BLOOD G L U C O S E LEVELS OF RATS T R E A T E D DA ILY WITH MA YTENUS OR S A LIN E GIVEN O R A L L Y FOR 2

MONTHS

Treatment Dose Mean ± S.D. glucose (mg/dl)

(mg/kg)
0 +2 +4 +6 +8 weeks

Saline -- 90.7 ± 9.2 80.6 4- 9.0 90.5 ± 9.3 90.8 4- 11.5 80.5 ± 1.8
Maytenus 136 98.0 4- 10.6 89.6 4- II.0 92.5 4- 12.9 79.2 4- 5.3 82.3 ± 8.5
272 88.2 4- 8.4 84.5 4- 10.7 84.0 4- 5.7 81.5 4- 6.2 82.2 4- 5.7

spleen, kidneys, epididymides, seminal vesicles and
testicles of the rats.
Prolonged voluntary consumption by rats and mice.
As can be seen in Table 13, rats and mice receiving
Maytenus did not differ from controls on fluid in-
take during the three months of treatment. The
average daily dose for rats was 362 times higher than
the corresponding dose used by humans, while for
mice it was 100 times higher. During this period,
no differences were found in weight gain in both rats
and mice, in glucose blood levels and body
temperature of rats, in rota-rod performance of mice
on days 0, 15, 30, 45 and 60 of treatment, as well
as in biochemical, hematological and pathological
evaluations made on rats at the end of the 3 months
(data not shown).
Teratogenic screening
Effects on estrus cycle. As can be seen in Table
14, both control females and female rats treated with
Maytenus had an average of 6--7 estrus cycles dur-
ing the 30-day period, with an interval of 4 5 days
between cycles. No significant differences were
found between the two groups, but this could be due
to the fact that only 5 rats were used per treatment.
Treatment prior to mating. The oral administra-
tion of 544 mg/kg of Maytenus for 45 days to female
rats before mating did not alter the number and
duration of pregnancies or the number and weight
of pups (Table 15) when compared to controls. No
external congenital malformations were seen in
either control or Maytenus-treated progeny.
Treatment during pregnancy. Table 16 indicates
that treatment with Maytenus did not alter the
number of pregnancies completed, although the
mean weight of pups from rats treated with 272
mg/kg of Maytenus was significantly lower than con-
trois. This difference could be attributed to the
relatively higher number of pups per experimental
female. However, this weight difference was not seen

TABLE 10

BODY T E M P E R A T U R E OF RATS T R E A T E D DAILY WITH MA YTENUS OR S A LIN E GIVEN O R A L L Y FOR 2 MONTHS

Treatment Dose Mean 4- S.D. body temperature (°C)

(mg/kg)
0 +2 +4 a +6 +8 weeks

Saline -- 38.3 4 - 0.3 38.7 + 0.4 38.7 ± 0.4 38.6 + 0.6 36.9 ± 0.3
Maytenus 136 38.2 4- 0.7 38.2 ± 0.5 38.1 ± 0.5 37.8 ± 0.6 37.3 4- 0.3
272 38.3 4- 0.3 38.3 4- 0.4 37.6 4- 0.5* 37.7 4- 0.5 37.0 ± 0.2

aANOVA FI2.1s~ = 9.038. followed by Dunnett's test: *P < 0.05.

T A B L E 11

SERUM BIOCHEMICAL EVALUATION OF RATS TREATED DAILY WITH MA YTENUS O R S A L I N E G I V E N O R A L L Y F O R 2 M O N T H S

Treatment Dose Ud CR TL SGOT AP Na + K +b TP AMY

(mg/kg) (mg%) (mg%) (mg%) (U/L) (U/L) (mmol/L) (mmol/L) (g%) (units/dl)

Saline -- 58.7 + 8.7 0.42 -4- 0.09 230 4. 47 131 + 37 69.0 4. 13.4 141 -4- 0.3 6.4 4. 0.3 6.33 4- 0.82 2 9 3 4 4- 301
Ma.rtemts 136 58.1 4- 2.8 0.49 4- 0.09 182 4- 58 179 4. 41 79.3 4. 23.5 140 + 2.2 7.3 4- 0.6* 6.32 4. 0.25 2661 4. 333
Muytt'nus 272 68.8 4. 1.6" 0.49 4- 0.03 182 -+ 18 117 4- 45 66.4 4- 19.3 138 + I1.1 6.5 + 0.85 6,38 -4- 0.15 2730 4- 202

Key: U = urea: C R = creatinine: T L = total lipids: S G O T = s e r u m g l u t a m i c oxalacetic t r a n s a m i n a s e : A P = alkaline p h o s p h a t a s e : T P = total protein: A M Y = p a n c r e a t i c amylase.
"~ANOVA F~2j4, = 4.620, f o l l o w e d by D u n n e t t ' s t-test: * P < 0.05.
hANOVA FI2~I4t= 4.084. f o l l o w e d by D u n n e t t ' s t-test: * P < 0.05.
 37

TABLE 12

H E M A T O L O G I C A L EVALUATION OF RATS T R E A T E D DALLY WITH M,4 YTENUS OR SALINE GIVEN ORALLY FOR
2 MONTHS

Treatment Dose RBC Hemoglobin Hematocrit WBC

(mg/kg) (x 106/mm3) (g%) (%) (x 103/mm 3)

Saline -- 7.24 ± 0.46 12.7 4- 0.4 40.4 ± 1.6 9.9 4- 2.7

Maytenus 136 7.83 ± 0.83 12.7 ± I.I 41.8 ± 2.6 13.0 ± 4.1
272 6.89 ± 0.94 12.7 ± 1.4 40.0 ± 1.4 9.6 4- 1.5

TABLE 13

VOLUNTARY CONSUMPTION (MEAN ± S.D.) OF WATER OR MA YTENUS BY RATS (17 mg/ml) A N D MICE (3.4 mg/ml)
FOR 3 MONTHS

Drinking Rats (6/group) Mice (I 5/group)

fluid
ml/rat ml/kg Maytenus ml/mouse ml/kg Mto'tenus
daily dose daily dose
(mg/kg) (mg/kg)

Water only 31.3 ± 5.4 120 + 26 -- 8.6 ± 2.3 221 ± 65 --

Maytenus 39.4 ± 9.1 145 q- 44 2461 ± 748 7.9 ± 2.3 193 ± 63 660 4- 211

during the development of pups, as the mean weight
per litter on days 7, 14 and 21 after birth did not
differ from controls (Table 17).
No differences were observed in all of the other
behavioral parameters evaluated in the pups:
righting reflex, ambulation and eye-opening (Table
17). Adult behavioral evaluation of rats from
mothers treated with Maytenus w a s measured
TABLE 14
through open-field activity and a learning task in
a T-maze. Again no differences were found between
controls and rats from mothers treated with
Maytenus (Tables 18 and 19, respectively).
Treatment of male rats before matingl As can be
seen in Table 20, no differences were found in the
number and duration of pregnancies, in the average
number of pups per litter and in the mean weight
per litter of female rats mated with male rats treated
with Maytenus for 2 months as compared to female
rats mated with rats treated with saline for the same
period.

MEAN ± S.D. EFFECTS OF ORAL DOSES OF Discussion

MA FTENUS OR SALINE GIVEN FOR 30 DAYS ON THE
ESTRUS CYCLE OF FEMALE RATS
This laboratory has previously shown clear anti-
Treatment Dose Estrus cycles Interval between ulcerogenic effects for oral and intraperitoneal
(mg/kg) (n) cycles (days) Maytenus in rats against indomethacin- and cold-
restraint-induced gastric ulcers (Souza-Formigoni
Saline -- 6.8 ± 0.4 3.9 ± 0.5
Maytenus 272 6.2 ± 0.5 4.1 ± 1.3
et al., 1991). The next logical step was to investigate
544 5.8 ± 1.0 4.6 ± 2.0 the potential toxic effects of Maytenus after acute
and chronic administration in laboratory animals.
38

TABLE 15

MEAN + S.D. EFFECTS OF O R A L ADMINISTRATION O F S A L I N E OR 544 mg/kg OF MA YFENUS FOR 45 DAYS

BE FORE M A T I N G ON THE N U M B E R OF P R E G N A N C I E S , MEA N N U M B E R O F PUPS A N D MEAN W E I G H T PER
LITTER OF F E M A L E RATS

Treatment Females Deliveries Pups/litter Weight/litter Median day of

mated (n) (n) (g) delivery

Saline 10 7 10.7 4- 2.4 6.5 4- 0.4 25 °

Maytenus 10 9 8.1 + 3.3 6.9 4- 1.4 25 ~

The data gathered in the present study indicate
that the different concentrations of Maytenus given
acutely were almost absent of toxic effects. Doses
equivalent to 400 times those used by humans (2720
mg/kg) did not induce any observable effect in mice.
This absence of toxicity was so striking that even
the LDs0 could not be estimated ( > 1.09 g/kg).
Maytenus did not have a sedative effect in mice
when given orally. In fact, the plant preparation did
not alter the performance of mice on the rota-rod
apparatus with an oral dose of 1360 mg/kg. This
dose also did not impair locomotor activity or
potentiate the loss of righting reflex induced by pen-
tobarbial in mice. However, when administered i.p.,
the results were somewhat different, revealing a
significant sedative effect, since doses of 680 mg/kg
and above clearly potentiated the sleeping time in-
duced by the barbiturate, These data can be inter-
preted in two ways. One is that several substances
have an non-specific sedating effect when given i.p.
as a result of peritoneal irritation or because of ab-
dominal discomfort following the injection (Riley
and Spinks, 1958). On the other hand, it is known
that H2 receptor blockers like cimetidine and
ranitidine have a secondary central depressant ac-
tivity (Douglas, 1980) and this may be a possible
mechanism of action of Maytenus against gastric
ulcers (Souza-formigoni et al., 1991).
More specific pharmacological testing indicated
that Maytenus has no analgesic effect, as indicated
by the hot-plate and acetic acid writhing tests, and
no anticonvulsant activity, as tested through the
transcorneal electroshock and chemoshock
methods. Overall, the results indicate that Maytenus

TABLE 16

MEAN 4- S.D. EFFECTS OF O R A L A D M I N I S T R A T I O N OF MA YTENUS OR S A LIN E D U R I N G P R E G N A N C Y ON THE

N U M B E R OF DE LIVERIES, M E A N N U M B E R PUPS PER LITTER A N D MEAN W E I G H T PER LITTER O F F E M A L E
RATS

Treatment Dose Female Deliveries Pups/litter Weight/litter"

(mg/kg) mated (n) (nt Ig)
(n)

Saline 10 8 7.0 4- 3.8 5.6 + 0.4

Maytenus 136 10 8 10.3 4- 2.2 5.3 4- 0.3
272 10 8 10.9 4- 4.1 5 0 + 0.3*

aANOVA Fi2,~l~ = 4.677, followed by Dunnett's t-test: *P < 0,05.

TABLE 17

MEAN ± S.D. EFFECTS O F O R A L A D M I N I S T R A T I O N O F MA YTENUS OR SALINE TO F E M A L E RATS D U R I N G P R E G N A N C Y ON T H E W E I G H T

GAIN, R I G H T I N G R E F L E X OF PUPS A N D O T H E R P A R A M E T E R S

Mother's Dose Pup weight (g) Righting reflex (s) Median day Ambulation a count
treatment (mg/kg) of eye (n)
+7 +14 +21 days +1 +3 +7 days opening
+8 +13 days

Saline -- 12.1 .a- 1.2 22.8 ± 3.1 33.3 ± 4.1 10.3 ± 9.1 9.9 ± 8.3 4.6 ± 7.5 +15 7.1 ± 5.6 17.1 ± 7.2
Maytenus 136 II.0 ± I.I 21.2 ± 2.2 32.6 ± 2.9 10.8 ± 10.1 8.4 ± 8.8 2.5 ± 5.5 +14 8.9 ± 5.5 18.7 ± 9.8
272 11.5 ± 1.4 23.5 ± 2.2 34.9 ± 3.7 10.4 ± 9.9 10.3 ± 10.3 4.3 ± 6.3 +15 5.7 ± 5.1 18.4 ± 8.1

aMeasured by the number of squares crossed in the open-field apparatus.

40

TABLE 18

MEAN + S.D. BEHAVIOR OF ADULT OFFSPRING FROM MOTHERS TREATED WITH M A Y T E N U S OR SALINE
DURING PREGNANCY IN AN OPEN-FIELD APPARATUS

Mother's Dose 11 Ambulation" Rearing Median defecation

treatment (mg/kg) count (n) count (n) count (n)

Saline -- 10 65.0 :e 20.8 22.7 =t: 9.7 5.0

Ma.rlenus 136 10 65.8 :~ 10.6 30.5 =t: 18.9 3.5
272 10 61.4 ::t: 11.4 26.9 ± 6.2 6.0

"Measured by the number of squares crossed in the open-lleld apparatus.

TABLE 19 they c o n t i n u e d to gain weight at the same rate as

controls. Selected biochemical and h e m a t o l o g i c a l
PERFORMANCE IN A T-MAZE LEARNING TEST OF parameters as well as p a t h o l o g i c a l e x a m i n a t i o n o f
ADULT OFFSPRING BORN FROM MOTHERS
different organs did not indicate any significant
TREATED WITH M A Y T E N U S OR SALINIE DURING
PREGNANCY alterations after 3 m o n t h s o f treatment. It must be
emphasized that toxicological testing o f plants often
Mother's Dose Quantal Trials to learn uses the oral route, since several plants have shown
treatment (mg/kg) learning (n) toxic effects after c h r o n i c a d m i n i s t r a t i o n ( G o r d o n
Saline 10/10 I1.1 ± 5.9 et al., 1982; Schoental, 1982; Swick et al., 1982:
Maytenus 136 9/9 11.4 + 4.0 G o e g e r et al., 1983).
272 7/9 10.0 ± 3.5 It is well known that several plant drugs c o m m o n -
ly used have adverse effects on the fertility o f rats
as well as teratogenic effects. Thus, for example,
mianserin prolongs the estrus cycle o f rats (Bueno
is not toxic when given acutely to l a b o r a t o r y et al., 1984); m o r p h i n e and caffeine given to male
animals. rats before mating decrease the n u m b e r o f pups and
The prolonged daily oral (gavage) administration frequently lead to death (Joffe, 1979). Synthetic
to rats o f doses 2 0 ~ 4 0 times g r e a te r than the ones drugs such as c h l o r d i a z e p o x i d e and a m p h e t a m i n e
used by h u m a n s (or 360 times g r e a te r when given are also known to decrease the body weight o f pups
as the only source o f fluids to rats and mice) in- at birth when given d u r i n g p r e g n a n c y ( M i d a u g h
dicated again the relative absence o f toxic effects. et al., 1974: Buttar, 1980) and a m p h e t a m i n e ,
The overall b e h a v i o r o f animals did not change and diazepam, c h l o r p r o m a z i n e , haloperidol, p h e n o b a r -

TABLE 20

EFFECTS OF TWO-MONTH ORAL ADMINISTRATION OF MAYTENUS OR SALINE TO MALE RATS BEFORE

MATING ON THEIR FERTILITY AND PROGENITY

Male Dose Quantal female Mean ± S.D. Mean ± S.D.

treatment (mg/kg) pregnancy weight of pups pups per litter
at birth (g) 01)

Saline 8/10 5.8 + 0.3 11.4 + 1.7

Maytenus 136 9/10 5.3 ± (/.4 11.0 ± 2.0
272 10/10 5.3 ± 0.6 10.9 ± 2,6

bital and diphenylhydantoin are examples of drugs
which induce behavioral teratogenesis in laboratory
animals (Gauron and Rowley, 1970; Fonseca et al.,
1976; Midaugh et al., 1981). Our search for the
potential effects of Maytenus on the fertility of
female and male rats in the course of pregnancy as
well as for potential teratogenic effects revealed
nothing of an adverse nature. No alterations were
found in the number of estrus cycles, the interval
between cycles, the duration of pregnancy or the
number of pups per litter. Pups from treated and
control mothers had similar weight gains, and
neurological and behavioral development until
adulthood. No congenital malformations were
detected in the progeny of treated female rats as
well. When Maytenus was given to male rats no
adverse signs were obtained. All of these data, taken
together, reinforce the possibility that Maytenus
may be a safe plant for human use and deserving
of further investigations.
Acknowledgements
This work has been supported by Central de
Medicamentos (CEME) and Associag~.o Fundo de
Incentivo ~ Psicofarmacologia (AFIP).
References
Balbach, A.V. (1980} A flora nacional na medicina dom6stica.
In: A edi[~ca¢9o do lar, Ilth Edn. S~o Paulo, p. 885.
Buena, O.F.A., Carlini, E.A., Leite, J.R., Silveira Filho, N.G.,
Souza, M.L.O. and Tufik, S. (1984) Estudos pre-clinicos
cam mianserina. Efeitos agudos e cr6nicos em ratos e
camundongos. Jornal Brasileiro tie Psiquiatria 33,266~278.
Buttar, H.S. (1980} Effects of clordiazepoxide on the pre- and
post-natal development of rats. Toxicology 17, 311--321.
Carlini (1972) Screening farmacologico de plantas brasileiras.
Revista Brasileira de Biologia 32, 265--274.
Cruz, G.L. (19821 Diciondrio das plantas iiteis do Brasil. 2nd
Edn. Civilizaq~o Brasileira, Rio de Janeiro, pp. 335--336.
Douglas, W.W. (19801 Histamine and 5-hydroxytryptamine
(serotonin) and their antagonists. In: A.G. Gilman, L.S.
Goodman and L. Gilman (Eds.), The Pharmacological Basis
~/" Therapeutics. Sixth edition, Macmillan Publishing Co.
Inc., New York, pp. 609--646.
Dunham, N.W. and Miya. T.S. (19571 A note on a simple ap-
paratus for detecting neurological deficit in rats and mice.
41
Journal of the American Pharmaceutical Association (Scien-
tific Edition) 46, 208--209.
Fonesca, N.M., Sell, A.B. and Carlini, E.A. [1976) Differential
behavioral responses of male and female rats treated with
five psychotropic drugs in the neonatal stage. Psychophar-
macologia (Berlin) 46, 263--268,
Gauron, E.F. and Rowley, V.N. (1970) Chronic administration
of trifluoperazine in multiple dosages and multiple dura-
tions. European Journal of Pharmacology 13, 35--39.
Gunby, P. (19791 Plant known for centuries still causes pro-
blems today. Journal of the American Medical Association
241, 224~-2247.
Goeger, D.E., Cheeke, P.R., Ramsdell, H.S., Nicholson, S.S.
and Buhler, D.R. (1983) Comparison of the toxicities of
Senecio jacobada. Senecio vulgaris and Senecio glabellus in
rats. Toxicology Letters 15, 19--23.
Gordon, W.P., Forte, A.J., MacMurtry, R.J., Gal, J. and
Nelson, S.D. (19821 Hepatoxicity and pulmonary toxicity of
pennyroyal oil and constituent terpenes in the mouse. Tox-
icology and Applied Pharmacology 65, 4 1 3 4 2 4 .
Gyires, K. and Torma, Z. (1984) The use of the writhing test
in mice for screening different types of analgesics. Archives
internationales de Pharmaeodynamie et de Th~rapie 267,
131--140.
Huxtable, R.J. (10801 Herbal teas and toxins: Novel aspects of
pyrrolizidine poisoning in the United States. Perspectives in
Biology 24, 1-- 13.
Joffe, J.M. (19791 Influence of drug exposure of the father on
perinatal outcome. Clinics in Perinatology 67. 21--36.
Masur, J. (1972} Sex differences in emotionality and behavior
of rats in the open-field. Behavior Biology 7, 749--754.
Midaugh, L.D., Blackwell, L.A., Santos, C.A. and Zemp, J.W.
(19741 Effects of d-amphetamine sulfate given to pregnant
mice on activity and on catecholamines in the brain of off-
spring. Developmental Psychobiology 7, 429~438.
Midaugh, L.D., Simpson, L.W., Thomas, T.N. and Zemp, J.W~
(1981) Prenatal maternal phenobarbital increases reactivity
and retards habituation of mature offspring to environment
stimuli. Psvchopharmaeology 74, 349--363,
Riley, H. and Spinks, A. (19581 Biological assessment of tran-
quilizers. Part I. Journal of Pharmacy and Pharmacology 10,
657--671.
Schoental, R. (1982) Health hazards of pyrrolizidine alkaloids:
A short review. Toxicology Letters 10, 323--326.
Silva Araujo, J.E. and Lucas, V. (19301 Cat61ogo de Extratos
Fluiidos. Nova Ediq~o, Silva Arafjo e Cia, Ltda., Rio de
Janeiro, pp. 126--129.
Souza-Formigoni, M.L.O., Oliveira, M.G., Monteiro, M.G..
Silveira-Filho, N.G., Braz, S. and Carlini, E,A. (19911 An-
tiulcerogenic effects of two Maytenus species in laboratory
animals. Journal of Ethnopharmacology in press.
Swick, R.A., Cheeke, P.R., Goeger, D.E. and Buhler, D.R.
(19821 Effect of dietary Senecio jacobada and injected
Senecio alkaloids and monocrotaline on guinea pigs. Jour-
nal of Animal Science 55, 1411--1416.