www.wjpr.net 1 CYTOTOXIC ACTIVITY, ACUTE AND SUB-ACUTE TOXICITY OF METHANOLIC ROOT EXTRACT OF CORRIGIOLA TELEPHIIFOLIA POURR L. Doudach 1, 2 *, B. Meddah 1 , L. Rouas 3 , Mya Faouzi 1 , L. Benbacer 4 , M.Bouabdellah 5 , Z. Alhamany 3 , L.Chabraoui 5 , A. Elomri 2 , and Y. Cherrah 1 1 Laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy, Mohammed V Souissi University, Pharmacokinetic Research Team, Rabat, Morocco 2 Laboratory of Pharmacognosy, CNRS, UMR 6014, C.O.B.R.A, Faculty of Medicine and Pharmacy, University of Rouen, Rouen, France 3 Department of Anatomy and Cytology. Pediatric hospital of Rabat. CHU Ibn Sina. FMPR. UM5 Souissi. Rabat 4 Biology Unit and Medical Research CNESTEN, PB 1382 RP, 10001 Rabat, Morocco 5 Central Laboratory of Biochemistry, Ibn Sina Hospital Rabat, Morocco. ABSTRACT Corrigiola telephiifolia Pourr. (Caryophyllaceae) is an herbal plant commonly used in Moroccan traditional medicine for treatment of many disorders. In the present study, we investigated cytotoxic activity by an in vitro assay system of growth inhibition against a human cancer cell line, namely cervix adenocarcinoma (HeLa) and breast adenocarcinoma cells (MCF-7), the results demonstrated that cyclohexane extract show a moderate cytotoxic activity against HeLa cell lines . The acute and sub-acute toxicity of the crude methanolic extract of C. telephiifolia root parts was evaluated. For acute toxicity, a single oral administration was performed at a dose of 2000 mg/kg body weight (six females, six males mice). The study of sub-acute toxicity was evaluated by daily oral (five females, five males mice) with the extract at doses of 10, 50, 100 and 500 mg/kg/day for forty five days. No mortality or signs of toxicity were observed in the acute study.Mice were analyzed for final body and organ weights, necropsy, blood chemical and histopathological World Journal of Pharmaceutical research Volume 2, Issue 1, 1-15. Research Article ISSN 2277 – 7105 Article Received on 05 November 2012, Revised on 29 November2012, Accepted on 09 December 2012 *Correspondence for Author: * Pr. Bouchra Meddah, laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy, Mohammed V Souissi University, Rabat, Morocco
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Bouchra Meddah et al. World Journal of Pharmaceutical Research
CYTOTOXIC ACTIVITY, ACUTE AND SUB-ACUTE TOXICITY OF
METHANOLIC ROOT EXTRACT OF CORRIGIOLA TELEPHIIFOLIA
POURR
L. Doudach1, 2*, B. Meddah1, L. Rouas3, Mya Faouzi1, L. Benbacer4, M.Bouabdellah5,
Z. Alhamany3, L.Chabraoui5, A. Elomri2, and Y. Cherrah1
1Laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy,
Mohammed V Souissi University, Pharmacokinetic Research Team, Rabat, Morocco 2Laboratory of Pharmacognosy, CNRS, UMR 6014, C.O.B.R.A, Faculty of Medicine and
Pharmacy, University of Rouen, Rouen, France 3Department of Anatomy and Cytology. Pediatric hospital of Rabat. CHU Ibn Sina. FMPR.
UM5 Souissi. Rabat 4Biology Unit and Medical Research CNESTEN, PB 1382 RP, 10001 Rabat, Morocco
5Central Laboratory of Biochemistry, Ibn Sina Hospital Rabat, Morocco.
ABSTRACT
Corrigiola telephiifolia Pourr. (Caryophyllaceae) is an herbal plant
commonly used in Moroccan traditional medicine for treatment of
many disorders. In the present study, we investigated cytotoxic
activity by an in vitro assay system of growth inhibition against a
human cancer cell line, namely cervix adenocarcinoma (HeLa) and
breast adenocarcinoma cells (MCF-7), the results demonstrated that
cyclohexane extract show a moderate cytotoxic activity against HeLa
cell lines . The acute and sub-acute toxicity of the crude methanolic
extract of C. telephiifolia root parts was evaluated. For acute toxicity,
a single oral administration was performed at a dose of 2000 mg/kg
body weight (six females, six males mice). The study of sub-acute
toxicity was evaluated by daily oral (five females, five males mice)
with the extract at doses of 10, 50, 100 and 500 mg/kg/day for forty
five days. No mortality or signs of toxicity were observed in the acute study.Mice were
analyzed for final body and organ weights, necropsy, blood chemical and histopathological
salts are cleaved to formazan dye by cellular enzymes (only in the viable cells). The level of
absorbance directly correlates to the metabolically active cells. Vinblastine was used as a
positive control. Cells were observed before and after treatment by the plants extracts for 24
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Bouchra Meddah et al. World Journal of Pharmaceutical Research
and 48h using optic microscopy for any change in their morphology and to exanimate the
cellular process and the characteristics of the cells.
Toxicological studies
Animals: Animals were obtained from the animal experimental center of Mohammed V-
Souissi University, Medicine and Pharmacy Faculty – Rabat. They were housed three per
plastic cage for acute, five mice per cage for sub-acute toxicity, and under a controlled room
conditions: temperature (22 ± 1°C), humidity of about 60–80%, photo-periodicity of 12 h
day/12 h night and air changes. Mice were treated according to directives of the Official
Journal of the European Community about the care and of the use of the animals of
laboratory. All animals had free access to tap water and at ad-libitum feeding; the general
behavior of mice was observed continuously for 1h after treatment, intermittently for 6h and
over period of 24h [11] and all signs of toxicity and deaths and their latencies were recorded.
Acute toxicity: Acute toxicity study for the extracts is conducted according to the method of
Organization of Economic Co-operation and Development, as per 423 guidelines (OECD) [12,13] . Following the fasting period, body weight of the mice were determined and the dose
was calculated in reference to the body weight, C. telephiifolia extracts were dissolved in
distilled water and given by orally way in a single dose (2 000 mg / kg) of body weight. The
control group received only the water. Observations were made and recorded systemically
continuously observed for 6h and at 24h to detect any eventual symptoms of toxicity: changes
in physical appearance, skin, pain, stress, abdominal contraction or mortality and observed
for 14 days after administration of the substances. Care and treatment of the mice were in
compliance with the guidelines of the guide for the care and use of laboratory animals
(commission on life science, National Research Council 1996).
Sub-acute toxicity: We used 50 albino Swiss mice aged 10-12 weeks and weighing 22-30 g,
divided into five groups of mice (ten mice per dose, five males and five females) at single
doses of 0 (control, filtered water), 10, 50, 100 and 500 mg/kg bodyweight. The dose 200 mg
(methanolic extract) is equivalent to double the dose used by traditional healers and from the
acute toxicity study 2000 mg (the oral LD50 obtained higher than 2000mg/kg). The amount
of food and water consumed was measured daily from the quantity of food and water
supplied and the amount remaining after 24h for 45 days of the study period. The animals
were weighed and observed daily for clinical symptoms include hemorrhage, diarrhea,
convulsions, sedation, stimulation, colic and death. After 45 days of the treatment, blood was
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Bouchra Meddah et al. World Journal of Pharmaceutical Research
collected into dried tubes the biochemical analyses of serum samples was performed using an
Values are expressed as mean ± S.D of 4 mice in each group. Significance level: *P < 0.05,
**P<0.01, ***P<0.001
Table 4: Blood chemistry values of mice in sub-acute treatment with C. telephiifolia root
methanol extract
Parameter Control 10 mg/kg 50 mg/kg 100 mg/kg 500 mg/kg
ALT (UI/L)
AST (UI/L)
Urea (g/l)
Creatinine (mg/l)
6.4 ± 2.4
14.0 ± 2.0
0.42 ± 1.1
9.5 ± 0.1
45.1 ± 9.2 a
275.0 ± 59.9 a
0.3 ± 0.1
3.4 ± 0.2 a
53.5 ± 15.5 a
358.9 ± 91.2 a
0.3 ± 0.1
3.8 ± 0.7 a
45.7± 15.7 a
238.9 ± 91.5 a
0.3 ± 0.1
3.4 ± 0.6 a
38.4 ± 8.7 a
199.0 ± 48.6 a
0.3 ± 0.1
3.3 ± 0.2 a
Mean ± SEM, (n=10), p <0.01
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Bouchra Meddah et al. World Journal of Pharmaceutical Research
0,00
50,00
100,00
150,00
1000 500 250 125 62,5 31,25
Cel
l via
bilit
y %
Concentration (µg/ml)
Hella cell line (n=4)CyHex ext
Dich ext
MeOH ext
Fig. 1: Percentage cell viability curve of C. telephiifolia (CT) extracts again HeLa cell
lines.
Cell viability was plotted via the concentration.
All samples were run in quadruplicate (n=4).
Percentage viability = absorbance of test wells/absorbance of control wells) × 100) plotted
against the concentration of extract.
Fig. 2: Liver sections were stained with hematoxylin and eosin (H&E-stained 40×)
showing the effects of Corrigiola telephiifolia Pourr. extract on sub-acute toxicity study.
C0: Negative control, L1and L2(x10): C. telephiifolia 500 mg/kg body weight. Arrow shows
inflammatory granuloma sub capsular rounded shape with necrotic debris in the center and
clarified hepatocytes (hepatocytes signs of suffering).
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Fig.3: The cross section of renal tissue of the control group (CI, x4) and the group
treated with 500mg/kg body weight (K2x 40 and K3, x10) of the extract. No renal
abnormality was observed, an artery with inflammatory cells around an arteriole normal and
tubes not necrotic.
Fig. 4: Photomicrograph of the lung for control (C2) and of testis treated with 100mg/kg
(L1x4) and 500mg/kg (L2 x10) of the extract. Arrow shows inflammation and congestion in
the vascular parenchyma and pulmonary lymph node with normal appearance
Fig. 5: The histology of the cross section of the cardiac muscle of the control group C3 and H2 of the animals treated with 50mg/kg body weight. No abnormality was observed (x100).
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CONCLUSION
The toxicity study of C. telephiifolia in mice indicated that the methanol extract at the doses
used ethnomedicinally do not produce significant changes of biochemical parameters or
histopathology of internal organs. The highest dose of extract also induces sub acute severe
hepatotoxicity in mice liver. This was evidenced mainly by histopathological study on all
particular organs and has been supported by biochemistry findings this study provides
valuable data on the toxicity profile of Corrigiola telephiifolia that should be essential for
future study.
ACKNOWLEDGEMENTS
The authors wish to thank all the individuals and institutions who made this survey possible.