World Journal of Pharmaceutical research Bouchra Meddah et ...

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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

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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parameters. In the forty five days study in mice the extract at 10 and 50 mg/kg/day showed no

toxicity, mortality, macroscopic or microscopic change of internal organs or tissues in either

sexe in comparison with the control group. The bodyweight of both sexes decreased at the

dose of 100, 500 mg/kg/day and clinical blood chemistry revealed slightly changes.The

extracts of Corrigiola telephiifolia showed a cytotoxic activity without mortality in

laboratory animals .They are good candidates for further investigations in the fields of new

anticancer drugs discovery.

Keywords: Corrigiola telephiifolia, acute toxicity, sub-acute toxicity, cytotoxic activity.

INTRODUCTION

The use of medicinal plants has always been part of human culture [1]. Today, medicinal

plants play a great role in human health services worldwide. Diverse medicinal species in

Morocco has constituted the main source of products used in folk medicine and have been

described in older Moroccan pharmacopoeia [2,3,4]. Herbal sources from medicinal plants are

presumed to be safe without any complications or toxicity however it is necessary to focus on

all aspects of medicinal plant research: from cultivation, ethno-pharmacology, utilization,

isolation andidentification of active constituents to efficacy evaluation, pharmacology, safety,

standardization, formulation and clinical evaluation, this scientific studies are presumed to

prove that natural products from remedies can be safe without any health effect, further

investigations are vitally needed and the same applies for Corrigiola telephiifolia Pourr. It is

a perennial species [5] woody distributed throughout the north of Africa. The root releases an

aromatic fume and used for medicinal and cosmetic purposes, it is the most effective part

traditionally described to treat flu, dermatological diseases, inflammation, ulcer, cough, and

jaundice; it is also used as an anasthenic and a diuretic [6], root decoction is reported be used

to treat the cancer in Morocco [7]. Some studies have reported that the extract of Corrigiola

telephiifolia have been investigated for a toxicological study [8], this study completed other

investigations by the identification of possible target organs involved in the plant toxicity.

The present study was carried out to evaluate the cytotoxic activity, acute and sub-acute

toxicity of the root parts of C. telephiifolia Pourr. in mice.

MATERIALS AND METHODS

Plant material: Corrigiola telephiifolia Pourr. (Family-Caryophyllaceae) was collected

based on ethnopharmacological information and traditional uses, in May 2009, 4km south of

Ben Slimane (Morocco) with the agreement from the authorities and respecting the United

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Nations Convention of Biodiversity. The plant was identified by a botanist from the Scientific

Institute. (Pr. M. Fennane). A voucher specimen (N° 77766) was deposited in the Herbarium

of Scientific Institute, University Mohammed V–Rabat–Morocco.

Extraction procedure: The dried roots (970g) of C. telephiifolia were extracted successively

with cyclohexane, dichloromethane and methanol by maceration at room temperature (22°C)

over period of 24h the extraction process was repeated again for a three time, the extracts

were then filtered through Whatman paper and the solvents were vacuum-distilled in a rotary

evaporator (Rotavap: Buchi). The remaining extracts were finally dried in the oven at 30°C

for 2h to ensure the removal of any residual solvent. Final extracts were a yellow powder in

percentage dry weight 0.11, 0.18 and 7.32% of cyclohexane, dichloromethane and methanol

extract respectively; the extracts was kept in deep freeze at -20°C until use. Phytochemical

screening of C. telephiifolia root parts was subjected to the tests tube to identify the major

groups of secondary metabolites [9].

Cytotoxic activity

Cell lines and culture medium: Cervical cancer cell lines (HeLa) and breast

adenocarcinoma cells (MCF-7) obtained from the American Type Culture Collection (ATCC)

were used in this study. Cells were grown at 37°C in humidified 5% CO2 and 100% relative

humidity atmosphere in Dulbecco’s Modified Eagle Media (DMEM) (1% glutamine, 100

U/ml Penicillin-Streptomycin mixtures and 10% fetal bovine serum).

Cytotoxicity assay: Cytotoxicity of sample on tumor cells was measured by microculture

tetrazolium (MTT) assay [10]. For the assays, 96-well microplates were seeded with 100 µl

medium containing 10, 000 cells in suspension. After 24 h incubation and attachment, the

cells were treated with 6 fourfold dilution of crude extracts. Exactly from the stock solution

(40 mg/ml), each extract sample was applied in a series of 6 dilutions (final concentrations

ranging from 31.25 to 1000 µg/ml) with a final DMSO concentration of 0.1% and was tested

in quadruplicate. After 48 h incubation, cell viability was determined by adding (Sigma)

tetrazolium salt as cytotoxicity indicator and by reading absorbance at 590 nm with a

scanning multiwell spectrophotometer (Spectra Count, Packard, Ont., Canada). Tetrazolium

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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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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collected into dried tubes the biochemical analyses of serum samples was performed using an

automatic chemistry analyzer (Architect 8000, Abbott). Biochemical parameters measured

were alanine aminotransferase (ALT), aspartate amino transferase (AST), blood urea g/l and

creatinine. A macroscopic examination of vital organs was carried out soon after sacrifice,

vital organs such as heart, kidneys, liver, lung and spleen were isolated, weighed and

examined for any lesions preserved in10% phosphate buffered formalin solution and

dissected out for histopathological examination, this organs have been fixed for 12 hours and

then embedded in paraffin using conventional methods [14] cut into 5 micrometer tick sections

and stained using hematoxylin eosin dye. The sections are then observed under microscope

for degeneration, necrotic changes and evidence of damage if any.

Statistical analysis: Results were expressed as the mean ± standard error of mean (SEM).

Statistical analysis of data was carried out using one-wayanalysis of variance (ANOVA)

followed by student’s t-test. Differencesin mean were considered to be significant when P <

0.05.

RESULTS AND DISCUSSION

Cytotoxic effect: The traditional medicine all over the world is nowadays revalued by an

extensive activity of research on different plant species and their therapeutic principles. Plant

kingdom is a potential source of chemical constituents with antitumor and cytotoxic activities

owing to their enormous propensity, which synthesize a variety of structurally diverse

bioactive compounds [15,16]. The rich and diverse plant sources of Morocco are likely to

provide effective anticancer agents. Using the ethnomedical data approach, C. telephiifolia is

used in the Moroccan traditional medicine for various diseases, including cancer, was

collected and evaluated for its cytotoxic effect. Uncontrolled proliferation is a universal

property of tumour cells. Investigation of the cellular growth control mechanisms has

contributed to the understanding of carcinogenesis and identification of compounds with

specific antitumoral activities. Thus, cytotoxicity screening models provide important

preliminary data to help select plant extracts with potential antitumoral properties for future

studies [17]. The cytotoxic activity was evaluated on two human cancer cell lines, HeLa and

MCF7. The cytotoxic effect of methanolic, dichloromethane and Cyclohexane extracts on

cancer cells was determined using the MTTassay. The MTT assays data are presented

respectively in figures 1. The cyclohexane extract of Corrigiola telephiifolia root displayed

moderate cytotoxicity (IC50 = 200 µg/ml) against HeLa cell line, whereas the

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dichloromethane and methanol extracts showed significantly lower IC50 values (more than

400 ug/ml). All the extracts tested are inactive against breast cancer cells, (IC50 350 to 400

ug/ul). To be a good drug candidate, the IC50 value of such agent should be sufficiently low

to avoid any possible unspecific effects. According to the US NCI plant screening program, a

crude extract is generally considered to have in vitro cytotoxic activity if the IC50 value is

less than 30 µg/ml [18,19]. Since the IC50 concentrations of C. telephiifolia extract in tree

cancerous cell lines were more than 200 µg/ml, we believe that they are not very potent as

anticancer therapeutic agent. Moreover, In terms of toxicity to the Chang cervical cell,

cyclohexane extract was more toxic than methanolic and dichloromethane extracts since the

IC50 cyclohexane extract is around 200 µg/ml while the value of IC50 dichloromethane and

methanolic extracts were more than 200 µg/ml. The root cyclohexane of Corrigiola

telephiifolia could be a source of anticancer therapeutic agent against HeLa cell line. Well

that little remarkable, the cytotoxic effect of cyclohexane extract on the cervical line is

notable compared to that observed in cell lines of colon and breast. Thus, we are tempted in

perspective, to test the cyclohexane extract on other cell lines of the cervix, to examine the

specific effect on cancer of the cervix. It is useful and necessary to carry out other

investigations to better assess the cytotoxic effect of the cyclohexane extract of C.

telephiifolia.

Acute toxicity: The oral administration of a single dose (2000 mg/kg bodyweight) of C.

telephiifolia root extracts (cyclohexane, dichloromethane and methanol) to mice did not cause

death within the fourteen five days of the study. The evolution of the weight varied in the

mice weighed daily. Based on the symptoms observations animals under positive control

group treated orally with the C. telephiifolia, Mice were perturbed during the first 30 minutes

in 80% of mice. These effects are disparate in 1h after the treatment. Generally At a dose of

2000mg/kg, the extracts of the plants don’t lead to mortality by orally way. Under the system

of global harmonization of Chemicals (GHS), this product is classified Category 5, which the

higher LD50 is 2000mg/kg.Toxicological research and testing helps to live safely and to

derive benefit from natural and synthetic substances In this study no changes attributable to

treatment were found in body weight and any macroscopic changes that could point to the

cause of the death observed up to the maximum dose of 2000mg/kg body weight of the

extract administered orally which the single high dose is recommended by OECD guidelines

423 for testing acute toxicity, this result suggested that extracts of Corrigiola telephiifolia

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don’t cause any acute toxicity and the high safety margin through oral route justified it

therapeutic use by the traditional healers.

Sub-acute toxicity

Bodyweight, Mortality, and Clinical Signs: The results of in vivo toxicity show that the

treated groups at the administered doses of 10, 50 mg/kg/day appear normal, presented a

significant weight gain in both sexes of the mice. Weight loss was significant (p < 0.05) in

the 100 mg extract/kg group and in 500 extract/kg group in the 2nd and 3rd weeks of the

study, the reduction in body weight gain is a simple and sensitive index of toxicity after

exposure to toxic substances [20,21]. There was 3 mortality recorded in the group treated by the

extract at dose 500 and 100 mg/kg. The result of the effect of the extract on the bodyweight

and internal organs weight is presented in Table 2 and 3. There was no significant difference

in the water and food intake in the treatment group at dose 10 and 50 mg/kg/day when

compared to the control. Results showed that treatment with the extract at doses 100 and 500

mg/kg/day induced loss of appetite it then become normal and this may due to the stress of

receiving the oral administration of the extract and some significant changes are observed

skin effects, breathing, impairment in food intake and water consumption, abdominal

contraction, salivation and hair loss. Body weight is known to be one of the most sensitive

indicators of adverse effects may be due to the plant toxicity, especially to the presence of

some toxic principles in the extract such as saponins (table 1). It is a known fact that oral

administration of saponins containing compounds could cause bloat, thereby reducing

appetite in animals [22].

Biochemical analysis: The data in Table 4 showed an elevation of readings for the various

biochemical parameters for most of the mice groups. The values for biochemistry analysis for

each data represent the value of n=10 for each group except for the group treated by the

extract at dose 500 (n=8) and 100 mg/kg (n=9). Significant reductions were recorded in the

serum creatinine concentration in directly suggested no kidney damage specifically by renal

filtration mechanism; Reduction in creatinine level is observed in cases of muscle wasting as

seen in malnutrition [23].The highly significant (P < 0.01) reduction in creatinine

concentration in all the extract-treated groups indicates that the extract does not exert

deleterious effect on the renal function and that the decrease in body weight of mice at 100

and 500 mg extract/kg body weight might be due to muscle wasting possibly due to toxic

effect of the extract. The extract produced a highly significant (p < 0.01) increase in the levels

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of alkaline phosphatase at all dose levels but there was no significant change in the levels of

the urea in all the treated groups. The liver and heart release ALP and an elevation in it

plasma concentrations are indicators of hepatic and cardiac damage and toxic activity in the

tissues [24,25]. The aminotransferases (formerly transaminases) are the most frequently utilized

and specific indicators of hepatocellular necrosis, the alanine amino transferase (ALT) is

primarily localized to the liver and the aspartate aminotransferase (AST) is present in a wide

variety of tissues like the heart, skeletal muscle, kidney, brain and liver [26,27]. The increase in

ALP level is observed in bone disorders involving osteoblastic activity. However, an

elevation in ALP activity is the first clue to intra hepatic and extra hepatic cholestasis (biliary

obstruction) [28,29]. A typical myocardial infarction gives an AST/ALT ratio greater than 1

while an AST/ALT ratio less than 1 is a result of release of ALT from the affected liver [30],

AST /ALT of more than 2 indicates alcoholic hepatitis or cirrhosis [30].These results indicated

that the methanolic extract of C. telephiifolia when taken for long periods of time might cause

a liver damage.

Histopathology: Figures 2-5 showed the histological studies of some vital organs of the

animals treated with root extract of C. telephiifolia. Histopathology results showed that

Corrigiola telephiifolia methanolic extract at 500mg/kg caused a significant damage in liver

as revealed by severe inflammatory granuloma subcapsular rounded shape with necrotic

debris in the center and clarified hepatocytes (hepatocytes signs of suffering). However, there

are no significant morphological changes detected in kidney and heart. Sub-acute toxicity of

medicinal plants help in the identification of possible target organs involved and the toxic

symptoms. The use of herbal preparations may lead to hypersensitivity reactions; sensitivity

to the toxic effects of plants differs with regard to the species [31,32,33]. Many widely used

medicinal plants have been implicated in the occurrence of liver lesions and tumors, lung and

kidney diseases [34]. Observations of liver and lung section tissues of control and animals in

group treated by extract at 500 mg/kg body weight shows possible toxics effects of the

methanolic extract of the plant. The severity was mostly noticed for the younger animals in

the liver. Lung showed no pathological changes; however Persistent inflammation may lead

to parenchymal necrosis and fibrosis. The liver is the main target for the toxicity of several

compounds among which many medicines, although drugs can cause hepatotoxicity through

different ways [35], mitochondrial dysfunction is one major mechanisms underlying

hepatotoxicity, many drug compounds can be metabolized and involve conjugation with

endogenous compounds via transferase enzymes and many chemicals damage mitochondria,

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an intracellular organelle that produce energy. Its dysfunction releases excessive amount of

oxidants which, in turn, injure hepatic cells [36]. In the development of necrosis the damage

first appears at cytoplasmatic level; next, it spreads to the rest of the cell involving

mitochondria and the nucleus, and finally results in swelling and loss of plasma membrane

integrity leading to lysis [37].

Table 1: Phytochemical screening of C. telephiifolia root parts

Chemical groups C. telephiifolia root

Flavonoids (-)

Tannins and polyphenols (-)

Saponins (+)

Anthocyanin (-)

Steroids / triterpenes (+

Alkaloids (-)

(+) : présence (-) : absence Table 2: Changes in the bodyweight of mice after daily oral treatment with Corrigiola

telephiifolia Methanolic extract.

Males Control 10 50 100 500 mg/kg

J1 18.81 ± 2.24 20.58 ± 1.36 20.78 ± 1.55 23.42 ± 1.03 22.47 ± 1.74

J5 19.27 ± 1.81 21.66 ±1.52 20.95 ±1.06 23.20 ±1.17 22.08 ±1.12

J10 19.76 ± 1.06 22.43 ±1.91 21.71 ±1.21 23.12 ±1.43 21.93 ±1.52

J15 20.44 ± 1.34 22.5 ±1.69 22.58 ±1.67 22.95 ±1.56 21.59 ±1.07

J20 21.09 ± 2.13 23.53 ±1.89 23.09 ±1.54 22.56 ±1.75 21.43 ±1.43

J25 22.61 ± 1.64 23.67 ± 2.12 23.53 ± 1.15 22.32 ± 1.12 * 21.08 ± 1.22 *

J30 23.12 ± 2.75 23.86 ± 1.13 23.70 ± 1.11 22.26 ± 1.54 * 20,89 ± 1.68 *

J35 23.53 ± 3.05 24.01 ± 1.45 24.12 ± 1.80 22.01 ± 1.09 * 20.72 ± 2,15 *

J40 23.88 ± 1.59 24.27 ± 2.29 24.29 ± 1.14 21.77 ± 2.08 * 20.57 ± 1.18 *

J45 24.14 ± 1.12 24.73 ±2.65 24.63 ±2.09 21.53 ±1.16 * 20.31 ±1.05 *

Values are expressed as mean ± S.D. of 10 mice in each group

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Femelles Control 10 50 100 500 mg/kg

J1 21.94 ± 1.23 25.02 ±1.06 21.94 ±1.45 24.53 ±1.08 25.82 ±1.12

J5 22.14 ±1.56 25.26 ±1.12 22.07 ±1.09 23.67 ±1.33 25.56 ±1.54

J10 22.38 ±1.50 25.41 ±1.21 22.13 ±1.22 23.52 ±1.07 25.44 ±1.87

J15 22.75 ±1.22 26.23 ±1.49 22.29 ±1.53 22.76 ±1.05 24.75 ±1.91

J20 23.32 ±1.09 26.43 ±1.57 23.34 ±1.67 22.50 ±1.43 * 24.56 ±1.23

J25 23.77 ± 1,13 26.79 ± 1,12 23.77 ± 2.09 22.37 ± 1.12 * 24.12 ± 1,13

J30 23.96 ± 1.37 26.81 ± 1.45 23.98 ± 2.13 21.75 ± 1.56 * 23.95 ± 1.53 *

J35 24.23 ± 1.08 27.18 ± 1.67 24.29 ± 1.42 21.23 ± 1.67 * 23.66 ± 1.45 *

J40 24.58 ± 1.17 27.33 ± 1.22 24.59 ± 1.26 21.10 ± 1.29 * 23.47 ± 1.10 *

J45 24.79±1.05 27.49 ±1.35 24.86 ±1.55 21.03 ±1.5 * 23.13 ±1.15 *

The different superscript letters indicate statistically significant differences (P <0.05) in the

Student’s t-test. As compared with the control group

Table 3: Effect of Oral administration of methanol extract of Corrigiola telephiifolia

Pourr.on organs weight (g) of mice

Dose mg/kg

bwt

Control 10mg/Kg bwt 50 mg/Kg bwt 100 mg/Kg bwt 500mg/Kg bwt

Liver

Heart

Lung

Kidney

2. 387 ± 0.05

0.144 ± 0.060

0.47± 0.057

0.217 ± 0.071

2.124 ± 0.086**

0.147 ± 0.05

0.488± 0.054*

0.217 ± 0.06

2.113 ± 0.086**

0.142 ± 0.065

0.49± 0.059*

0.216 ± 0.075

2.042± 0.030***

0.146 ± 0.043

0.73 ± 0.027**

0.218 ± 0.059

2.025 ± 0.025***

0.145 ± 0.051

0.753 ± 0.013**

0.217 ± 0.053

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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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.

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