Journal of Ethnopharmacology 130 (2010) 470–476

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Toxicological evaluation of the hydroethanol extract of Tabernaemontana crassa

(Apocynaceae) stem bark
Victor Kuete a,∗ , Rostand N. Manfouo b , Véronique P. Beng c
a
Department of Biochemistry, University of Dschang, P.O. Box 67, Dschang, Cameroon
b
Department of Organic Chemistry, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon
c
Department of Biochemistry, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon

a r t i c l e i n f o a b s t r a c t

Article history: Aim: Tabernaemontana crassa Benth. is a medicinal plant widely used in Cameroon folk medicine to treat
Received 6 January 2010 a variety of affections. This study was aimed at evaluating its toxicological profile.
Received in revised form 1 May 2010 Materials and methods: The 70% (v/v) hydroethanol (HE) extract from the stem bark of this plant was
Accepted 19 May 2010
given to albino Wistar rats by oral gavage to study the acute and sub-acute toxicities.
Available online 27 May 2010
Results: The results of histopathological studies revealed that there was a dose-related effect in liver,
lungs and kidneys and that there was no difference in tissue profile of control group and those receiving
Keywords:
6 weeks daily treatment of 0.5 g/kg b.w. The result of the acute toxicity indicated the medium lethal
Tabernaemontana crassa
Apocynaceae
dose (LD50) of 6.75 g/kg body weight (b.w.) after 48 h of treatment and the significant variation (P < 0.05)
Acute toxicity of the relative body weight, serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), total
Sub-acute toxicity bilirubin (TBil), direct bilirubin (DBil) and creatinine (SCr) at the dose of 6 g/kg b.w. These results also
Biochemical parameters indicated significant variation of the liver alkaline phosphatase, aspartate aminotransferase (AST), ALT,
Rats total proteins (TP), glutathione (GSH) and malondialdehyde (MDA) and renal creatinine (RCr) and urea
(RU) at 6 g/kg b.w. The result of the sub-acute toxicity showed significant changes in the body weight but
no modification (P > 0.05) of blood and liver indices for the animal taking 6 weeks daily doses of the HE
compared to the control group.
Conclusions: The results showed that this extract was fairly non-toxic but that consumption of higher
doses up to 6 g/kg b.w. could cause organ injuries. Moderated consumption of small doses up to 0.5 g/kg
b.w. daily for 6 weeks appeared to be safe.
© 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction sinusitis, stomach disorders, hematuria, gonorrhea, wound infec-

Tabernaemontana crassa Benth. (Apocynaceae) is a native
species of the West African forest. It is frequently used in tradi-
tional medicine of several African countries. Extremely caustic, the
sap is an ingredient of an arrow-poison and may cause blindness;
it is commonly applied as a healing dressing to sores, abscesses,
furuncles and to anthrax pustules, and to dermal infections such as
filaria, ringworm and other fungal troubles (Burkill, 1985). The latex
is used to treat ringworm, wound infections, coryza, headaches,
abscesses, boils and carbuncles (Dalziel, 1937; Sandberg, 1965).
A paste of the fruit is applied topically in treating contusions
and sprained backs (Sandberg, 1965). The decoction of the leaves
is taken in West Africa as a tonic (Kerharo and Bouquet, 1950).
The stem bark is used to treat several ailments including coryza,
∗ Corresponding author. Tel.: +237 77355927; fax: +237 22319334.
E-mail address: kuetevictor@yahoo.fr (V. Kuete).
tion (Kerharo and Bouquet, 1950; Sandberg, 1965; Baumer, 1979).
A crude ethanolic extract of this plant was also reported to have
decreased motor activity and muscle relaxation effect (Sandberg
and Cronlund, 1982). Previously we demonstrated the antigonor-
rheal activity of this plant as well as its ability to inhibit in vitro
the growth of several Gram-negative and positive bacteria (Kuete
et al., 2005). It was also demonstrated that hot water extract of
the leaves of Tabernaemontana crassa exhibits good anaesthetic
effect on Guinea-pig skin (Agwu and Akah, 1990). A review of the
phytochemical constituents of this plant reported the presence of
several indole alkaloids such as pericyclivine, perivine, vobasine,
tabersonine, conopharyngine, oxocoronaridine, 3-oxocoronaridine
hydroxyindolenine, (−)-heyneanine, (−)-3-oxoheyneanine, (−)-
ibogamine, isovoacangine, voacristine, crassanine, conoduramine
(Van Beek et al., 1984). In the roots and bark of Tabernaemontana
crassa, indole alkaloids, ibogaine, isovoacangine, conopharyngine,
conodurine, conoduramine and coronaridine, and in the seeds,
voacamidine, coronaridine-hydroxyindolenine, tabersonine and

0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2010.05.034
 V. Kuete et al. / Journal of Ethnopharmacology 130 (2010) 470–476
coronaridine have been reported (Dass et al., 1967; Van Beek et
al., 1985; Danieli and Palmisano, 1987).
Despite the available knowledge and the interesting therapeutic
uses, no toxicological study has been previously done until now on
Tabernaemontana crassa. This paper reports therefore for the first
time the acute and sub-acute toxicities of the HE extract of the stem
bark of this plant.
2. Experimental
2.1. Plant material
The stem bark of Tabernaemontana crassa Benth. was collected in
the Yaoundé outskirts, Centre Province of Cameroon. The botanical
identification of the plant with leaves was done by the National
Herbarium of Cameroon where the voucher specimen was con-
served under the reference number 43449/HNC.
2.2. Extract preparation
The plant powder (3 kg) was macerated in the water–ethanol
3:7 (v/v; 10 l) for 48 h at room temperature (27 ± 1 ◦ C). The filtrate
obtained using Whatman filter paper no. 1 was concentrated under
vacuum in a reduce pressure at 56 ◦ C to yield a paste that was then
dried in oven until complete disappearance of the solvent. This pro-
cess afforded a 70% green HE extract (9.91 ± 2.27% yield after three
extractions) that was then stored at 4 ◦ C for various toxicological
studies.
2.3. Experimental animals
Five weeks (90–100 g) and 10 weeks (140–160 g) old clinically
healthy Wistar rats bred in the Department of Biochemistry, Uni-
versity of Yaoundé I, Cameroon, were used. The animals were kept
under standard environmental condition (temperature: 27 ± 1 ◦ C).
They were fed with standard diet and water ad libitum. The
bioassay was conducted in accordance with European Community
guideline, EEC Directive of 1986; 86/609/EEC and WHO experimen-
tal guideline (WHO, 1992). The use of animals in the present
study received the approval of the University of Yaoundé I Ethics
Committee.
2.4. Acute toxicity
This assay was carried out to evaluate any possible toxic effect
or changes in normal behavior of Wistar rats. Four groups of 10
rats (5 males and 5 females) were used in the experiment. The
acute toxicity of the plant was studied by preparing four different
concentrations of the extract (2,4, 6 and 8 g/kg b.w.), and admin-
istered orally to four groups of animals. The fifth group was taken
as a control and given vehicle (0.5% Tween 80 in distilled water).
Animals were kept without food for 15 h prior to dosing and were
monitored continuously for 6 h after dosing for any sign of tox-
icity. The symptoms, motor activity, posture and mortality were
checked. Animals were kept under observation for 7 days and were
monitored daily for changes in body weight, signs of toxicity, and
mortality.
2.5. Sub-acute toxicity
Forty rats were divided into four groups of 10 animals each (5
males and 5 females). They were kept under the same conditions
as described above. The first group was given the vehicle of the
extract (0.5% Tween 80 in distilled water) and taken as control. The
remaining four groups were given orally 0.1, 0.5, and 1 g/kg b.w. of
HE extract of Tabernaemontana crassa daily for 6 weeks.
471
2.6. Body weight
The body weight of each rat was assessed during the acclima-
tization period, once before commencement of dosing, once every
7 days during the dosing period and once on the day of sacrifice.
The relative body weight (RBW) of each animal was then calculated
using the formula RBW = [absolute body weight of one time interval
(g)/body weight of rat on commencement of dosing day (g)] × 100.
2.7. Hematology
On day 42 (at the end of dosing period for the sub-acute study),
blood samples were collected from the ventral tail vein of each
rat into a heparin containing tube for hematological analysis. Red
blood cell, white blood cell and platelet counts, hemoglobin con-
centration (g/l) and hematocrit estimation were carried out using
an automated globular counter HYCEL Diagnostics (Celly, type CA
4001, series no.: CA40D 1975).
2.8. Preparation of serum samples
On day 42 of the dosing period, all the animals were euthanized
by exsanguinations under ether anaesthesia and blood samples
were drawn from the jugular vein of each sacrificed animal. The
samples were collected in plastic test tubes and allowed to stand
for complete clotting. The clotted blood samples were centrifuged
at 3000 rpm for 15 min and serum samples were aspirated off and
frozen.
2.9. Relative organ weight and preparation of homogenate
samples
After taking the blood, the abdominal cavity of each animal was
opened and organs namely the heart, liver, lungs, spleen and kid-
neys were quickly removed, cleaned with ice-cold saline, weighed
and stored at −80 ◦ C. Liver and kidneys tissues were thawed and
homogenized 20 times (w/v) by homogenizer in ice-cold Tris–HCl
50 mM and KCl 50 mM buffer (pH 7.4). The homogenates were cen-
trifuged at 6000 rpm for 30 min and the supernatant was then used
for enzyme assays. The relative organ weight (ROW) of each ani-
mal was then calculated using the formula ROW = [absolute organ
weight (g)/body weight of rat on day of sacrifice (g)] × 100.
2.10. Microscopic examination
After sacrificing the animals, small pieces of liver, kidneys and
lungs were fixed in 10% formal saline for histopathological stud-
ies before storing the organs at −80 ◦ C. Tissues were processes
by conventional techniques using an automatic tissue processor
(Shandon, Sakura Fine Technical Co., Ltd., model 4634). The paraf-
fin embedded sections of 4–5 ␮m thickness were prepared with
the rotary microtome, stained with hematoxylin and eosin for
microscopic examination using optical Microscope (Leica DM LB
2, series no.: 1151200) with a Camera (Leica microsystem DI,
series no.: 007804503) linked to a computer for pictures-treatment
(Microsoft, no.:034013).
2.11. Serum and homogenate biochemistry
Serum samples were analyzed for the determination of total
proteins (TP), direct bilirubin (DBil), total bilirubin (TBil), and serum
urea (SU) and serum creatinine (SCr) respectively according to
the methods described by Gornall et al. (1949) and Cheesbrough
(1991). Serum concentration of glucose (SGlu) was determined
using Accu-Chek Active test strips (Randox Laboratories Ltd., Co.,
Antrim, UK). Enzymatic activities of aspartate aminotransferase
472 V. Kuete et al. / Journal of Ethnopharmacology 130 (2010) 470–476
(AST), alanine aminotransferase (ALT), and alkaline phosphatase
(ALP), were analyzed according to Reitman and Frankel (1957)
and Morgenstern et al. (1965). The homogenates of the liver tis-
sues were analyzed for the determination of malondialdehyde
(MDA) and hepatic glutathione (GSH) concentrations as described
by Wilbur et al. (1949) and Ellman (1959) respectively. The hepatic
TP, renal urea (RU) and renal creatinine (RCr) concentrations were
also determined using the method cited above.
2.12. Statistical analysis
Results were expressed as mean ± standard deviation (S.D.). Sta-
tistical analysis was determined by one-way analysis of variance
(ANOVA) and post hoc least significant difference (LDS) test. A
P < 0.05 was considered statistically significant.
3. Results
3.1. Acute toxicity
The animals treated with the dose of 0–4 g/kg b.w. did not
present any behavioral change after administration and during
the 7 days of observation. From 6 g/kg b.w., changes observed
20 min after the administration of the plant extract, included
slow response to external stimuli, prostation, stretching and slug-
gishness, rapid breathing all punctuated by a diarrhea. Two of
the ten (20%) animal’s death including one male and one female
were recorded at the first 2 h that followed the treatment and
no other deaths occurred later on. At the dose of 8 g/kg b.w.,
the signs of poisoning become more pronounced. The diarrhea
was more abundant and all animals (100%) died after 1 h. The
apparent causes of death were convulsion and respiratory distress.
The plot of this progressive increase in mortality with respect to
the dose of the extract showed that the oral LD50 values of the
extract were around 6.75 g/kg b.w. for both male and female rats
(Fig. 1).
3.2. Relative body weight
Fig. 2 shows the evolution of the animal’s body weight after sin-
gle administration of the HE extract of Tabernaemontana crassa. It
is observed that the difference in the body weight gain between
the animals of the control (0 g/kg b.w.) and the treated group did
not varied significantly (P > 0.05) up to a dose of 2 g/kg b.w. How-
ever, the decrease in body weight became significant (P < 0.05) at
4 g/kg b.w. and was more pronounced at 6 g/kg b.w. The plant
extract did not induced a significant modification (P > 0.05) in
Fig. 1. Overall mortality (%) observed in Wistar rats having received a single oral
dose (0, 2, 4, 6 and 8 g/kg b.w.) of the HE extract of Tabernaemontana crassa up to 7
days.
Fig. 2. Relative body weight trend for Wistar rats dosed once with the HE extract
of Tabernaemontana crassa at 0, 2, 4 and 6 g/kg b.w. Each data point represents the
mean ± S.D. (n = 10–8). Data points with different letters (at day 7) are significantly
different (P < 0.05).
the body weight gain between the control group rats and those
treated with the dose up to 0.5 g/kg b.w. following 6 weeks
daily administration (Fig. 3). Nevertheless, the difference between
treated and control rat weights was significant at 1 g/kg b.w.
(P < 0.05).
3.3. Relative organ weight
There were no significant changes in the relative weights of the
spleen and lung of all animals including those of the survival group
at 6 g/kg b.w. after single administration of the HE of Tabernaemon-
tana crassa (Fig. 4A). For other organs such as the liver, heart and the
kidneys, significant variations (P < 0.05) appeared between those of
controls and treated group at 6 g/kg b.w (Fig. 4A). However, no sig-
nificant changes were recorded with all those organs following the
6 weeks daily administration (Fig. 4B).
3.4. Hematology
The hematological parameters summarized in Fig. 5 did not
showed any significant changes in the red blood cell, white blood
cell and platelet counts of treated group compared with the control
group. Differences in hemoglobin concentration and hematocrit
percentage (P < 0.05) were observed with control group and that
receiving the dose of 1 g/kg b.w.
Fig. 3. Relative body weight pattern in Wistar rats fed with the HE extract of Taber-
naemontana crassa (at 0, 0.1, 0.5 and 1 g/kg b.w.) for 6 weeks. Each data point
represents the mean ± S.D. (n = 10). Data points (at week 6) with different letters
are significantly different (P < 0.05).
 V. Kuete et al. / Journal of Ethnopharmacology 130 (2010) 470–476
Fig. 4. Effect of the HE extract of Tabernaemontana crassa on the relative organ
weights dosed once (A) or after 6 weeks (B) oral dosing. Each data column rep-
resents the mean ± S.D. Data columns of the same organ with different superscript
letters are significantly different (P < 0.05).
3.5. Serum renal and hepatic biochemical parameters
The evolution of the blood, hepatic and kidney biochemical
indices in the acute toxicity study is presented respectively in
Tables 1–3. It is observed that the serum AST, TP, DBil, SGlu and the
SU of the treated animals did not changed significantly (P > 0.05)
compared to that of the control group. All others studied blood
indices varied significantly (P < 0.05). ALT was the most modified
biochemical value in the blood with about 44.60% (54.97 ± 4.20 IU/L
against 36.99 ± 1.76 IU/L) increase rate (at the dose of 6 g/kg b.w.)
Fig. 5. Mean hematological parameters: red blood cell count (100,000/␮l), white
blood cell count (1000/␮l), platelets count (10,000/␮l), hematocrit level (%) and
hemoglobin concentration (g/dl) in rats treated with the HE extract of Tabernae-
montana crassa (0, 0.1, 0.5 and 1 g/kg b.w.) after 6 weeks dosing. Each data column
represents the mean ± S.D. (n = 10). Data columns in the same parameter with dif-
ferent superscript letters are significantly different (P < 0.05).
473
compared to the values of control group. For all the investigated
liver biochemical indices (Table 2) significant changes (P < 0.05)
appeared between the treated and control groups. ALP, ALT and
MDA were the most modified parameters, recording 48.24%, 49.40%
reduction and 385.80% of increase rate respectively between values
of the survival (at the dose of 6 g/kg b.w.) and those of the control
group.
The evolution of blood and liver biochemical values for albino
Wistar rats having received 6 weeks daily administration in the sub-
acute toxicity study is shown in Tables 3 and 4. It can be observed
that changes recorded (P > 0.05) were not significant between the
treated and the control group rats for all the investigated biochem-
ical indices.
When exploring kidney function, we observed significant
changes (P < 0.05) in SU and SC from the dose of 0.5 g.kg
b.w. whereas 0.1 g/kg b.w. did not induced any modification
(Table 5).
3.6. Histopathological studies
The summary of histopathological studies revealed that there
were dose-related effects in liver, lungs and kidneys (Table 6). How-
ever, changes occur at 1 g/kg b.w in animal’s tissue profile compared
to that of control group.
4. Discussion
The HE extract was employed in this study because of its use in
the Cameroon folk medicine in the treatment of various ailments.
Evidence of the antimicrobial activity of this extract was lately
demonstrated (Kuete et al., 2005). In the present work, the oral
administration of the extract, up to a dose of 4 g/kg body weight did
not produce behavioral changes. As the DL50 of 6.75 g/kg recorded
is greater than 5 g/kg, this extract can be considered as fairly non-
toxic compound according to the OECD guideline (1981) for testing
chemicals. At 6 and 8 g/kg b.w., animals exhibited a characteristic
progression of signs including rapid breathing and death. Hence,
the death may be due to central nervous system/respiratory depres-
sion (Schmitt, 1976), as Tabernaemontana crassa was reported as
plant acting in the nervous system (Oliver-Bever, 1983). The change
in heart weight of treated animals as observed in this study is also
in consistence with such hypothesis. The toxicity of this plant at
higher dose might be related to the increase of the concentration of
toxic chemical constituents. However, the stem bark was reported
to contain several indole alkaloids (Van Beek et al., 1984; Van Beek
et al., 1985), numbers of compounds of such class being known
to have toxic effects (Porter et al., 1977; Levin and York, 1978;
Corcuera, 1984; Sarkera et al., 2001). The treatment with all doses
of HE of Tabernaemontana crassa did not affected the spleen relative
weight. This indicates that an immunotoxic effect is not predictable
since the spleen plays a major role in immunological mechanisms.
However, the hematocrit level was increased in treated animals
suggesting that Tabernaemontana crassa extract could stimulate
erythrocyte synthesis.
The biochemical blood and liver indices were determined in this
study in order to determine the mechanism by which toxic effect
of the extract appeared after administration and also to determine
the non-toxic effect dose. As no significant modification of these
parameters occurred in the blood or in the liver up to a dose of
2 g/kg b.w., this dose can be retained as the maximum non-toxic
effect dose.
The enzyme most often measured to indicate bile duct obstruc-
tion is ALP. Tissue sources of ALP are liver cells, osteoblast cell
of the bone, intestinal cells, and placental tissue. Measurement of
this enzyme is also done to investigate bone, liver and gall bladder
474 V. Kuete et al. / Journal of Ethnopharmacology 130 (2010) 470–476

Table 1
Blood biochemical values of Wistar rats having received a single oral dose of the HE extract of Tabernaemontana crassa up to 7 days.

Biochemical parameters Dose (g/kg b.w.) and parameter values

0 (n = 10) 2 (n = 10) 4 (n = 10) 6 (n = 8)

ALP (IU/L) 98.24 ± 8.63a 98.44 ± 9.47a 111.2 ± 9.99ab 134.66 ± 13.91b
AST (IU/L) 90.24 ± 2.72 90.12 ± 1.99 91.81 ± 2.11 94.37 ± 2.46
ALT (IU/L) 36.99 ± 1.76a 37.62 ± 1.77a 38.51 ± 1.37a 54.97 ± 4.20b
Total protein (mg/dl) 3.98 ± 0.17 4.05 ± 0.21 4.05 ± 0.10 3.84 ± 0.12
Total bilirubin (mg/dl) 0.32 ± 0.02a 0.33 ± 0.01a 0.34 ± 0.02a 0.39 ± 0.05b
Direct bilirubin (mg/l) 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00
Creatinine (mg/dl) 0.32 ± 0.00a 0.32 ± 0.00a 0.37 ± 0.01b 0.39 ± 0.01c
Glucose (mg/dl) 140.00 ± 1.08 140.13 ± 3.30 139.28 ± 0.9 137.59 ± 2.81
Urea (mg/dl) 10.96 ± 0.66 11.93 ± 0.86 12.11 ± 0.75 12.73 ± 0.92

Values are expressed as mean ± S.D. (n = 10–8).

Data lines in the same parameter with different superscript letters are significantly different (P < 0.05).

Table 2
Liver biochemical values of Wistar rats having received a single oral dose of the HE extract of Tabernaemontana crassa up to 7 days.

Dose (g/kg b.w.) and parameter values

0 (n = 10) 2 (n = 10) 4 (n = 10) 6 (n = 8)

ALP (IU/l) 26.18 ± 2.052a 26.11 ± 1.2a 20.17 ± 1.31b 13.55 ± 0.74c
AST (IU/l) 60.23 ± 2.27a 60.24 ± 1.32a 50.87 ± 2.41bc 44.39 ± 4.14c
ALT (IU/l) 28.33 ± 1.14a 28.39 ± 1.46a 20.37 ± 2.13b 14.34 ± 2.25c
TP (mg/g of the liver) 133.05 ± 2.12a 134.40 ± 2.95a 124.52 ± 2.41b 113.93 ± 6.67c
GSH (␮mol/g of the liver) 5.63 ± 0.23a 5.50 ± 0.27a 4.96 ± 0.20b 4.57 ± 0.42b
MDA (nmol/g of the liver) 6.87 ± 2.09a 8.13 ± 3.89ab 17.37 ± 5.29bc 26.38 ± 8.05c

Values are expressed as mean ± S.D. (n = 10–8).

Data lines in the same parameter with different superscript letters are significantly different (P < 0.05).

(Cheesbrough, 1991). In this study, ALP levels varied significantly in
the blood and liver from the dose of 4 g/kg b.w. This result showed
that the extract could be implicated in the bile duct obstruction
(Cheesbrough, 1991). When the bilirubin values were considered,
it revealed that the extract induce bile duct obstruction in the rats,
as the significant modification (P < 0.05) value in TBil was observed
at the dose of 6 g/kg b.w. Creatinine values of the treated rats
increased significantly at the dose of 6 g/kg b.w., but were not more
than onefold higher than values in the control group. The extract
may be implicated at low level in kidney damages as no signifi-
cant variation of SU occurred up to a dose of 6 g/kg b.w. following a
single administration. Nevertheless, prolong use of the HE extract
of Tabernaemontana crassa could induce some kidney damages, as
significant changes were recorded with the renal concentration of
both creatinine and urea (Cheesbrough, 1991). When inspecting the
liver, the two enzymes most often associated with hepatocellular
damage are AST and ALT. Large amount of AST is present in cardiac
muscle, liver, skeletal muscle and kidney. Small amount of enzyme

Table 3
Blood biochemical values following 6 weeks treatment of Wistar rats with the HE extract of Tabernaemontana crassa.

Biochemical parameters Dose (g/kg b.w.) and parameter values

0 0.1 0.5 1

ALP (IU/L) 89.36 ± 8.75 88.88 ± 7.26 88.54 ± 4.37 88.42 ± 8.49
AST (IU/L) 89.85 ± 5.96 91.28 ± 6.53 92.16 ± 5.55 93.29 ± 6.94
ALT (IU/L) 38.36 ± 1.30 38.28 ± 2.25 38.98 ± 1.81 39.71 ± 2.67
Total protein (mg/dl) 7.55 ± 0. 40 7.80 ± 0.52 7.91 ± 0.44 7.83 ± 0.10
Total bilirubin (mg/dl) 0.32 ± 0.02 0.33 ± 0.01 0.35 ± 0.02 0.36 ± 0.03
Direct bilirubin (mg/dl) 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Creatinine (mg/dl) 0.59 ± 0.01 0.61 ± 0.01 0.61 ± 0.01 0.61 ± 0.01
Glucose (mg/dl) 146.00 ± 2.3 145.27 ± 3.87 146.61 ± 3.54 147.00 ± 4.86
Urea (mg/dl) 14.71 ± 1.2 1.41 ± 0.99 1.54 ± 1.12 1.50 ± 1.12

Values are expressed as mean ± S.D., n = 10.

No Significant difference from control P > 0.05.

Table 4
Liver biochemical values following 6 weeks treatment of Wistar rats with the HE extract of Tabernaemontana crassa.

Biochemical parameters Dose (g/kg b.w.) and parameter values

0 0.1 0.5 1

ALP (IU/L) 26.18 ± 3.01 26.18 ± 3. 7 26.20 ± 1.21 26.17 ± 3.59

AST (IU/L) 61.47 ± 8.15 60.48 ± 7.64 63.53 ± 9.21 64.55 ± 5.48
ALT (IU/L) 30.25 ± 2.4 32.48 ± 2.19 31.51 ± 1.24 32.38 ± 1.88
Total protein (mg/ml) 160.23 ± 8.46 160.02 ± 7.22 157.55 ± 7.07 154.93 ± 11.16
GSH (␮mol/g of the liver) 6.39 ± 0.20 6.24 ± 0.17 6.12 ± 0.16 6.14 ± 0.24
MDA (nmol/g of the liver) 17.48 ± 6.36 15.13 ± 5.11 18.63 ± 6.14 19.13 ± 6.03

Values are expressed as mean ± S.D., n = 10.

No significant difference from control P > 0.05.
 V. Kuete et al. / Journal of Ethnopharmacology 130 (2010) 470–476 475

Table 5
Renal biochemical values following 6 weeks treatment of Wistar rats with the HE extract of Tabernaemontana crassa.

Biochemical parameters Dose (g/kg b.w.) and parameter values (n = 10)

0 0.1 0.5 1

Urea (mg/g of kidney) 22.89 ± 3.10a 20.57 ± 2.84ab 16.92 ± 1.18b 12.83 ± 1.37c
Creatinine (mg/cg of kidney) 57.32 ± 2.76a 54.74 ± 3.91a 45.01 ± 2.39b 31.16 ± 0.98c

Values are expressed as mean ± S.D.

Values in the same column with different letters are significantly different (P < 0.05).

Table 6
Histological observation on Wistar rats given HE extract of Tabernaemontana crassa for 6 weeks.

Dose (g/kg b.w.) Organs

Liver Lung Kidney

0 Normal histological feature Normal histological feature Normal histological feature

0.1 Normal histological feature Normal histological feature Normal histological feature
0.5 Normal histological feature Normal histological feature Normal histological feature
1 Normal histological feature Vascular congestion; alveolar dilatation; extensive Vascular congestion; important and diffuse
interstitial fibrosis; infiltration by inflammatory hemorrhage
cells

occurred in brain, pancreas and lung (Sacher and McPherson, 1991).
ALT is found principally in liver with only small amounts being
present in other organs. Therefore, ALT estimation is more specific
to evaluate liver damage than AST. In general with liver diseases,
serum AST and ALT rise and fall in parallel (Sacher and McPherson,
1991). In the acute toxicity study, the modifications of the AST and
ALT values were significant at the dose of 6 and 4 g/kg b.w. respec-
tively in the blood and in the liver. These results suggest that the
injury of the liver as well as that of the heart and other sources
organs of these enzymes might be induced by the dose close and
more. Their liberation in the blood might be mainly due to the per-
oxydation of the hepatic cells membrane lipids as shown by the
spectacular increase in the MDA rate of the treated rats which is an
essential marker of membrane degradation. Generally, blood level
of AST, ALT and ALP are higher than those found in the liver (Sacher
and McPherson, 1991) and this was respected in the present study.
The main serum proteins are albumin and globulin. Albumin
is synthetized entirely in the liver and is present in greater con-
centration than globulin in the plasma and serum. Globulin is
produced in the liver and also in the reticuloendothelial sys-
tem. An increase in serum total protein is not common. When it
occurs, it is usually due to haemoconcentration following shock,
severe vomiting, or diarrhea, increase in globulin production asso-
ciated with splenomegaly and chronic infections, and liver disease
(Cheesbrough, 1991). Decrease in total protein is associated with
high tissue demands as a result of an increase in body need for
protein. This is due to the serious damage, liver disease associ-
ated with a reduction in proteins synthesis, loss of protein from
the body in urine (as in the nephrotic syndrome, malabsorption,
chronic pancreatitis, coeliac diseases and sprue) and low protein
intake (Cheesbrough, 1991). In the present study, no significant
modification of the serum proteins was observed but that of the
liver was noted from the dose of 4 g/kg b.w., indicating that con-
sumption of high doses of the HE extract of Tabernaemontana
crassa could induce some liver damages or a disorder in protein
metabolism.
Glutathione is an endogenous compound also playing an impor-
tant role in drug metabolism. It is the main substrate for most of
the body purification enzymes (GSH-S-transferase and GSH per-
oxydase) (Kaplowitz et al., 1985). A significant reduction in the
liver concentrations between the treated and control group rats
was observed from the dose of 4 g/kg b.w. This might be due to a
faster use of this compound for the inactivation of reactive metabo-
lites formed during the biotransformation of the constituents of the
HE extract, compared to regeneration speed.
Three dose levels were used for the study of the sub-acute tox-
icity study. According to the OECD guideline (1981), if the acute
toxicity test at one dose level of at least 5 g/kg b.w. produced no
observable toxic effect then the full study using three dose levels is
considered not necessary. As the biochemical values changed sig-
nificantly from the dose of 4 g/kg b.w. in the acute toxicity study,
the doses of 0.1, 0.5 and 1 g/kg have been administrated daily for 6
weeks. After such treatment, no significant change of the inves-
tigated biochemical values in the sub-acute toxicity assay was
observed in liver. Changes in renal creatinine and urea were noted
from 0.5 g/kg b.w. These observations therefore indicate that pro-
long use of this extract at lower dose is safe for some key organs such
as liver, heart, spleen and kidney, but that the unique or chronic
administration of high doses could cause some injuries.
Finally, it can be concluded from the results of this work that
the HE of Tabernaemontana crassa is fairly non-toxic, but should be
taken with caution, bearing in mind that higher dose could induce
organs damage.
Disclosure statement
The authors declared that there is no conflict of interest for the
present work.
Acknowledgements
The authors acknowledge the technical support of the Univer-
sity of Yaoundé I Hospital Center. This paper is also dedicated to
the memory of Professor David LONTSI who died on December 21,
2008.
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