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Longo et al. World Journal of Pharmacy and Pharmaceutical Sciences

HEPATOPROTECTIVE EFFECTS OF CANNA INDICA L. RHIZOME

AGAINST ACETAMINOPHEN (PARACETAMOL)

F. Longo1*

, A. Teuwa1, S. Kouam Fogue

2, M. Spiteller

3 and L.S. Etoundi Ngoa

1

1Laboratory of Animal Physiology, Department of Biological Sciences, Higher Teachers’

Training College, University of Yaounde I. PO Box 47 Yaounde-Cameroon.

2Laboratory of Chemistry, Department of Chemistry, Higher Teachers’ Training College,

University of Yaounde I, PO Box 47 Yaounde-Cameroon.

3Institute of Environmental Research (INFU) of the Faculty of Chemistry TU Dortmund,

Otto-Hahn-Str. 6, D-44221 Dortmund, Germany.

ABSTRACT

Self-medication often leads to poisoning the body, especially the liver.

Canna indica is a Cannaceae empirically used to treat various types of

hepatitis in Cameroon. Liver toxicity was observed four hours after

oral administration of a single dose of Acetaminophen (APAP)

400mg.kg-1

. Effects of the aqueous extract of the rhizomes at doses 8,

16, 32, 64 and 128mg.kg-1

bw, and that of the antidote N-acetylcystein

[Mucomyst] were evaluated in male rats, pre-treated with APAP.

Results showed that Canna indica failed to increase rats’ body weight

(P>0.05), normalised rats’ behaviour (aggression and sensitivity to

touch and noise), and provoked a decrease (P<0.001) of the relative

liver weight. Canna indica also modelized, at all doses, the liver’

damages caused by inflammation, through a stimulation of hepatocyte

regeneration and a healing of inflamed areas. Its antioxidant effect was

revealed by a lower plasma activity of alanine aminotransferase transaminase. The dose

8mg.kg-1

and 16mg.kg-1

showed the most effective activity. The presence of flavonoids and

fatty acids, revealed by phytochemical analysis and two bioactive molecules: Carotol and 9-

Cedranone (Cedran-9-one) using high performance liquid chromatography, known to posses

anti-inflammatory activities, confirms the anti-inflammatory properties of Canna indica.

Canna indica at doses comprises between 8 and 16mg.kg-1

bw could be strongly

recommended.

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Article Received on

17 March 2015,

Revised on 08 April 2015,

Accepted on 29 April 2015

*Correspondence for

Author

F. Longo

Laboratory of Animal

Physiology, Department

of Biological Sciences,

Higher Teachers’ Training

College, University of

Yaounde I. POBox 47

Yaounde-Cameroon.


1610


KEYWORDS: Canna indica L., Liver toxicity, Acetaminophen, N-Acetylcystein, ALT, Rat.

INTRODUCTION

Self-medication is increasingly on the rise in Cameroon. When out of control, it causes an

overdose in the body, leading to a liver toxicity. Drugs overdose such as acetaminophen

(Paracetamol) and alcohol-based products (beer, whiskey, etc.), can provoke drug-induced

hepatitis.[1,2]

In fact, before tackling this disease, it’s worth mentioning that, drug intake could

lead to the development of various forms of liver diseases including, among others hepatitis,

cholestasis (liver tumor), steatosis (fat accumulation in the liver), phospholipidosis

(accumulation of phospholipids in the liver), sclerosing cholangitis and vascular disease. The

frequency of drug-induced liver disease is increasing.[3,4]

After absorption of a toxic dose,

severe acute hepatitis can occur with a procession of nonspecific clinical signs such as nausea

and vomiting in the early hours, followed by abdominal pain predominantly in the right

hypochondrium and jaundice.[5]

Cells have several defence mechanisms to protect against potentially toxic reactive

metabolites coming from the drug metabolism: antioxidant enzymes [superoxide dismutase

(SOD) and glutathione peroxidase], the absorption of vitamin E, ascorbic acid, and other free

radical scavengers and by a repairing mechanism of DNA damage.[6]

Because of their lipid

solubility, many drugs cannot be eliminated in native form by the body. They must firstly

being metabolized to water-soluble substances, before being excreted in urine or sweat. Drug

metabolism usually involves two phases: Phase I includes a variety of enzymatic systems

which mainly consists of the oxidation of the native molecule, under the control of a complex

enzyme system that includes cytochrome P450 and Phase II consists of a conjugation reaction

(mainly with glucuronic acid, under the control of uridine-glucuronosyltransferases (UGT)6.

Acetaminophen (Paracetamol), is a combination of para-acetyl-amino-phenol and N-acetyl-

para-aminophenol (APAP); It is the active ingredient in many specialised drugs class of non

salicylates antipyretic analgesics.[7]

APAP is devoid of anti-inflammatory properties, and has

no effects on platelet aggregation, with the advantage to possess less restricted indications (as

age), and is devoid of serious side effects when used at recommended doses. However, in

case of overdoses, APAP becomes very noxious to the organism especially to the liver.[8,9]

It

primarily acts in the central nervous system,[10]

by inhibiting the production of prostaglandins

involved in the processes of pain and fever, through an inhibitory action on the prostaglandin

H2 synthase (PGHS). Other mechanisms of action have been invoked to explain the analgesic


1611


and antipyretic activity of APAP such as a central serotoninergic mechanism of action is

suspected by potentiating the effect of serotoninergic neurons in the spinal cord, thus exerting

an inhibitory control on pain pathways;[11]

it may also act by limiting the release of beta-

endorphins.[12]

Adverse reactions have been reported in most cases. The main adverse effects

are found such as rash rash or urticaria, probably allergic, thrombocytopenia and asthma,[13]

hypotension[14]

anaphylactic shock, vascular purpura[15]

rectal ulceration, agranulocytosis and

acute pancreatitis usually occurs when Paracetamol is combined with other drugs such as

codeine2. Chronic active hepatitis, granulomatous hepatitis and rhabdomyolysis are others

secondary effects of Paracetamol. In very young children, Paracetamol may increase the risk

of developing asthma.[16]

Canna indica L. belongs to the family of Cannaceae; with 10 species mostly are used for

decoration because of the beauty of their leaves and flowers (yellow, orange or red).[17]

C.

Indica is well known and widely used in traditional medicine for its diuretic[18]

and inhibitory

effects on enzymes involved in the mechanism of inflammation, among various diseases. In

the western region of Cameroon (Mbouda), C. indica is commonly used to treat hepatitis. The

decoction of its roots, in combination with fermented rice, is used in the treatment of

gonorrhoea and amenorrhea and the leaves burned off a smoke-owned insecticide.[19]

The

purpose of the following tests is therefore to assess the anti-inflammatory effects of the

aqueous extract of the rhizomes of Canna indica L., on rats’ liver and on the activity of ALT

(alanine aminotransferase), one of the main blood indicative of liver toxicity.

MATERIAL AND METHODS

The rhizomes of Canna indica L. were collected in a Yaounde suburb (Oyom-Abang).

Extraction was done as recommended by the traditional healer and the recommendations of

medicinal plants’ uses.[20,21]

The botanical identification of the plant specimen was made at

the national Herbarium of Cameroon in Yaounde by Mr Christian YOMBO, and found to be

identical to the voucher specimen N°42058HNC, deposited by Biholong in May 1979.

2-1. Plant materials

2-1.1. preparation of plant rhizomes’ extract

After cleaning, 03kg of fresh C. indica rhizomes were boiled in 4L of water during 30min;

after filtration and lyophilization, the 33.85g of a caramel colour powder (yield = 1.128%)

obtained was use for experiments after dilution in distilled water.


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2-1.2. Phytochemical Analysis

Phytochemical analysis of the aqueous extract of the rhizomes of Canna indica (ACI) was

conducted,[22]

to detect the presence of reducing substances: saponins and triterpene

glycosides, tannins (gallic and catechin); Chemical hydrolysis tests were also done to detect

phenols, coumarins glycosides, flavonoids, alkaloids, fatty acids, poly-uronides and

polysaccharides.

2-1.3. High performance liquid chromatography fingerprint

For HPLC analysis, the sample powder was weighed (1.0g) into a 100mL flask and 100%

methanol was added to the volumetric mark. The mixture was sonicated for 60min at room

temperature. After extraction, the mixture was filtered through a 0.2μm membrane filter. The

resulting solution was used for HPLC analysis. The qualitative assessment was performed by

HPLC coupled to a mass spectrophotometer, using a gradient method. Constituents were

detected at wavelength 205nm according to their through their retention time, relative

abundance and masses. The mass spectrum was obtained with Xcalibur 2.0.7., and structures

were drawn using CS Chemdraw ultra.[23]

2-2. Animals

Six to eight-week-old pathogen-free male Wistar rats were bred in our laboratory and

provided with water and food ad libitum. Experimental procedures were carried out

according to guidelines for the care of laboratory animals from the Cameroon National Ethics

Committee (Ref. N° FWA-IRB00001954) and with the NIH Guidelines for the Care and Use

of Laboratory animals.[24]

2.3. Experimental Design

After fasting during eighteen hours[25]

and divided into eight treatment groups (n = 5), except

in the healthy group, acute liver injury was induced (per os) with 400mg.kg- of APAP.

(Tablet of 500mg, Lot N°2F72134 were purchased in a local drugstore), and diluted in warm

phosphate-buffered saline (PBS, pH 7). Healthy and APAP groups received 1ml PBS; The

reference group (NAC) received N-acetylcystein, and test groups (ACI1 – ACI5) received

ACI (8, 16, 32, 64 and 128mg.kg-1

) per os during 72Hrs.[7]

2.4. Autopsy and diagnostic

On the behavioural changes: aggression, sensitivity to noise and touch, mortality and water

consumption were recorded throughout the experiment. Rats’ weights were recorded before


1613


and four hours after the final treatment and euthanized using ethyl ether. The artero-venous

blood of each rat was collected in a 5mL heparinized tube for transaminase analysis

(ALT).[26]

The liver was quickly isolated, weighed, filmed and the morphology was recorded

and a biopsy of the left liver was preserved in 10% neutralized formalin for histological

analysis. The relative weight of all livers was expressed in% [(liver weight /b.w.)*100].

2.5. Histological analysis

Liver biopsies were dehydrated, included in paraffin, cut (5µ), de-parrafined, rehydrated and

coloured with hematoxylin-eosin,[27]

and observed under a light microscope (Olympus) at

x400 magnification.

2.6. Evaluation of Alanine amino-transferase enzyme (ALT)

The ALT assessment was performed using a KIT (FORTRESS diagnostics, ALT (GPT)

IFCC KINETIC UV). Optical densities were measured using a spectrophotometer Genesis

brand, at the wavelength of 340nm.

2.7. Statistical analysis

Statistical analyses were performed using Sigma-Stat 3.2. Software; Means values ± S.E.M.

and the comparison between treated and controls groups were calculated by “t” student test.

Multiple groups’ comparison was also done by One Way ANOVA test, using Mann-Whitney

and Turkey methods, according to values. Results were considered significantly different at

P<0.05.

RESULTS

3.1. Phytochemical analysis and HPLC-SM

The phytochemical analysis of ACI, revealed the presence of phenolic compounds, catechin

gallic tannins, anthocyanins, alkaloids, fatty acids, coumarins and anthraquinons (tab.2).

Table 2: qualitative composition of the aqueous extract of Canna indica’s rhizomes

Canna

Indica

Poly

uro

nid

s

Poly

ose

s

Sap

onin

s

Phen

ols

Gal

lic

tannin

s

Cat

echin

tan

nin

s

Anth

ocy

anin

s

Tri

terp

ens

Fla

vonoïd

s

Alc

aloïd

s

Fat

ty a

cids

Coum

arin

s

Anth

raquin

ons

Gly

cosi

des

Antr

hac

éniq

ues

c

Canna Indica - - - + + - + - - + + + + -


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HPLC fingerprint coupled to the mass spectrometer after 28min, showed a chromatogram

spectrum with three main peaks corresponding respectively to: compound (A): retention time

0.61 and 0.7min, RT 0.61, [M+H] +

: m/z 360.1503; (calcd. for C27H20O 360.1504) and at the

retention time of 6.66min, two compounds appears: (B1) [M+H]+: m/z ; (calcd. For [M+H]

+

214.8362 C3H3O11 and (B2) [M+H]+: m/z; (calcd. for [M+H]

+ 212.8391C3HO11). Two known

bioactive sesquiterpenes former isolated from the essential oil of C. Indica28

, named (C)

Carotol: (3R,3aS,8aR)-3-Isopropyl-6,8 a-dimethyl-2,3,4,5,8,8a-hexahydro-3a(1H)-azulenol)

RT: 12.18, m/z 222.2054 (calcd. For [M+H]+, m/z 223.2055 C15H270) and (C’) 9-Cedranone

(Cedran-9-one) RT: 5.93, m/z 221.3583; (calcd. for (C15H250, [M+H]+ m/z 221.3552), were

identified (Fig.3).

Figure 3: chromatogram profile of Canna Indica rhizomes: (A): RT 0.61, [M+H]

+: m/z

360.1503; (calcd. for C27H20O 360.1504); (B1) RT 6.66, [M+H]+: m/z ; (calcd. For

[M+H]+ 214.8362 C3H3O11 , (B2) (A): RT 6.66, [M+H]

+: m/z 360.1503; (calcd. for

C27H20O 360.1504); (C) Carotol: (3R,3aS,8aR)-3-Isopropyl-6,8 a-dimethyl-2,3,4,5,8,8a-

hexahydro-3a(1H)-azulenol) RT: 12.18, m/z 222.2054 (calcd. For [M+H], m/z 223.2055

C15H270) and (C1) 9-Cedranone (Cedran-9-one) RT: 5.93, m/z 221.3583; (calcd. for

(C15H250, [M+H]+ m/z 221.3552).

[28]


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3.2. Canna indica effects on animal behaviour

After APAP per os administration, rats were gregarious with minimal movements; Four

hours later, they presented a cough coupled to breathing disorder. C. Indica caused few

behavioural disorders (ACI4 and ACI5) which disappeared 72 hours later, similarly to NAC

[Tab.3].

Table 3: Rats’ behavioural effects, 24 and 72 hours after administration of NAC and the

aqueous extract of rhizomes of Canna indica.

4 h after

Acetamino

phen

NAC

Canna indica

24h 72h

24

h

72

h

24

h

72

h

24

h

72

h

24

h

72

h

24

h

72

h

Doses (mg/Kg) 0 400 1330 8 (ACI1) 16

(ACI2)

32

(ACI3)

64

(ACI4)

128

(ACI5)

Locomotion N D N N N N N N N N D N D N

Sensitivity to

noise N D N N N N N N N N N N N N

Sensitivity to

touch N D N N N N N N N N N N N N

Aggression N D N N N N N N N N D N D N

Stool G G G G G G G G G G G G G G

Breathing N BD N N BD N RD N BD N BD N BD N

Cough NC C NC NC C NC C NC C NC C NC C NC

N=normal, C= Cough, NC= No Cough, D=decreased, G=granulous P=present, A=absent,

RD= Breathing disturbance

3.3. C. indica Effects on rat’s body weight

ACI caused failed to modify (P> 0.05) rat’s weight gain similarly to NAC.

3.4. C. indica on rat liver’s relative weight

ACI decreased rat’s liver relative weight, at all doses (vs APAP), especially at 8mg.kg-1

[4.54

± 0.49% (P<0.05)] equivalent to -29.61% (vs NAC -4.72 ± 0.45% (P <0.05)], lower than

NAC [fig.5].


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Figure 1: Canna indica rhizomes’ aqueous extract and NAC effects on rat’s liver

relative body weight (%). Each bar represents mean ±S.E.M. n=6 ; *P<0,05 Significant

difference vs APAP group.

3.5. Canna indica effects on ALT

APAP caused a plasmatic ALT increase up to 1870.16IU/L. ACI decreased (P<0.05) the

plasmatic level of ALT similarly to NAC. Meanwhile, ALT decrease induced by C. indica

occurred inversely proportional to the dose: the dose of 8mg/kg induced the highest decrease

value of 451.05 IU/L (P<0.001), while the dose of 128mg/kg induced a decrease of

1458.855IU/L. In groups ACI2 and ACI4, C. indica induced highest decreases equivalent to

71.98% and 75.88% respectively, compared to APAP group [1870.16IU/L]. Likewise,

compared to NAC group [520.308IU/L], ACI1 and ACI2 showed the highest efficiencies with

values inferior to NAC, while from ACI3 to ACI5, C. indica caused weak decreases.

Comparatively to Healthy rats [111.55IU/L], ALT values induced by ACI and NAC stayed

upper [fig.6].


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Figure 2: Canna indica rhizomes’ aqueous extract and NAC effects on ALT plasmatic

rate Each bar represents mean ±S.E.M. n=6 ; *P<0,05 ;**P<0,01 ;***P<0,001 :

Significant difference vs Paracetamol group (P); §P<0,05 ;

§§§P<0,001 : Significant

difference vs NAC group.

3.6. Canna indica effects on the liver morphology and histology

APAP provoked a liver tissue damage characterised by numerous nodules (oedemas) [vs. H

group] [fig.7H]. Histological analysis showed a cell degeneration at the portal vein with a

membrane’ lesion, resulting to a loss of cytoplasm [fig.7P'], while the healthy liver (H)

showed a normal liver tissue architecture, with normal hepatocytes, sinusoids with their

lining cells, normal portal vein, hepatic artery and the bile duct [fig.7H']. NAC administration

revealed a liver with a texture similar to a healthy liver. However, the presence of healing

points and small nodules [fig.7NAC] were recorded; at the tissue level, few inflamed

hepatocytes have also been recorded nearest the portal vein (fig.7NAC’).

C. indica significantly improved the inflammation caused by APAP 400mg/kg): On the

morphology of all rats (ACI1 to ACI5), livers were no longer shown nodules, but the presence

of healing points was observed, with a dark red colour [fig.7ACI1-5]. On the histology, rats’

liver from group ACI1 to ACI3 presented normal hepatocytes [fig.7ACI1’-3']; Group ACI4

(64mg/kg) presented a lack of hepatocyte vacuolation (no degeneration) accompanied by a

slight inflammation and abnormalities [fig.7ACI4’] and group ACI5 (128mg/kg) presented a

liver tissue with a structure similar to that of inflamed liver (APAP’) with the presence of bi-

nucleated hepatocyte with membrane lesions and numerous inflammation [fig.7ACI5’].


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Figure 7: effects of the aqueous extract of Canna indica rhizome extract [(8mg/kg (ACI1,

ACI1’); 16mg/kg (ACI2, ACI2’); 32mg/kg (ACI3, ACI3’); 64mg/kg (ACI4, ACI4’);

128mg/kg (ACI5, ACI5’))] and N-acetylcystein (NAC, NAC’) on the morphology and

histology of rat liver inflammation induced by APAP (400mg.kg-1

).; [HEx400]. 1 -

Nodule (oedema), 2-Door space (bile duct, Hepatic artery, and vein), 3-normal hepatocytes,

4- inflamed hepatocytes with flattened lining cells, 5- degenerated hepatocytes.

DISCUSSION

Evaluation of the anti-inflammatory effect of the aqueous extract of Canna indica rhizomes

was made on liver toxicity induced by APAP (Acetaminophen). Oral administration of an

acute dose of 400mg/kg APAP caused lesions resulting in the presence of granulomas,

oedema (small tumours of inflammatory origin) and numerous polymorphic cells, cells or

specific items scattered throughout the liver tissue; On the liver tissue, acetaminophen caused

a massive hepatocyte necrosis at the centrilobular region, with a dark red colour).[5,29]

C.

indica significantly improved the alteration induced by APAP. The improvement in

aggression, sensitivity to noise and locomotion demonstrated a tranquilizing effect exerted on

the central nervous system.[30]

Similarly, the liver’s relative weight decrease also revealed a

sign of behavioural improvement, especially at doses 8 and 16mg/kg. C. indica caused a

decrease in relative liver weight below that of the NAC group; Indeed, it is known that,

inflammation in general, and granulomas that appeared on liver tissue in particular, comes

from the fluid and cellular infiltration: inflammatory cells: leukocytes, macrophages, Kupffer

cells[2,6,30]

which certainly contributed to the relative liver weight increase on APAP livers. In

case of overdose, N-acetylcystein (NAC) is used to strengthen the body defence towards the


1619


toxic metabolites by reducing their level. Improvements in liver tissue damage by C. indica

were similar to NAC, especially at dose 64mg/kg, characterised by residual inflammatory

cells and some granulomas. In contrast, at doses 8 and 16mg/kg, the effects of C. indica were

significantly higher than that of NAC, with a complete elimination of inflammation,

demonstrated by the presence of scars, and the absence of nodules and granulomas.

Transaminases are usually measured in serum to detect pathogen. Generally, liver toxicity is

detected by assaying only the TGP (glutamic pyruvic Transaminase also called ALT), which

is preferentially localized in the liver (organ specificity). Serum ALT levels were elevated in

APAP rats, up to 6 times higher than that of NAC group. In addition, it has been shown that

NAPQI, a molecule produced during APAP poisoning31

, creates adducts (compounds

produced by addition to a DNA molecule), which can bind to liver protein. Degradation of

membrane lipids and disruption of calcium homeostasis can be resulted, causing necrosis and

cytolytic hepatitis up to the kidney by the same mechanism (1 to 2% of cases).[8,32]

Thus,

oedemas and necrosis of hepatocytes observed in the livers are evidence of an actual presence

of APAP poisoning. The fulminate hepatitis induced by a dose of 400mg/kg of APAP also

led to a considerable rise in plasma alanine aminotransferase concentration, up to 1870.16IU /

L. The ¾ decrease (P<0.05) clearly demonstrated an antioxidant effect (anti-inlammatory

effect) of C. indica. This result is similar to results which also showed a decrease in plasma

transaminase (ALT), and a reduction/absence of inflammation in the evaluation of

hepatoprotective effects of different extracts from medicinal plants in APAP hepatotoxicity

cases.[33]

Among the bioactive molecules from the rhizomes of C. indica, revealed by the

phytochemical analysis, phenolic compounds like coumarins and flavonoids detected are the

major antioxidants regularly founded in natural products.[34,35]

In addition, among all those

compounds, anthocyanins[36]

and alkaloids[37]

detected, are also known for their anti-oxidative

activity. The presence of fatty acids in rhizomes is also proven by the discovery of a red oil

called "Cannabiol" in red flowers of C. indica[38]

Those molecules have shown anti-ischemic,

anti-platelet, anti-inflammatory and anti-lipoperoxide, with biological antioxidants effects

mainly: radical-scavenging activity and enzyme inhibition through the action of enzymes

involved in oxidation systems such as: 5-lipoxygenases, cyclooxygenases, the

monooxygenases and an anti-lipoperoxidation), could be responsible of the so-observed

biological activities of C. indica, alone or synergistically. In addition, an antioxidant activity

of C. indica has already been demonstrated[39]

confirming once again, the anti-

inflammatory/antioxidative activity of C. indica rhizomes. The major components has been


1620


identified as steroids and the identification of Carotol.[28]

and 9-Cedranone, earlier discovered

in Marchantia Convoluta leaves with a proven cytotoxicity activity on human liver and lung

cancer cells.[40]

thus, those components of the essential oil of C. indica rhizomes, could take

part on the anti-inflammatory effect observed.

CONCLUSION

The behaviour normalization by C. indica showed that it affects the central nervous system.

Although, the lack of effect observed on rat’ body weight, linked with a decrease in liver

weight, contributed to demonstrate its curative effects without affecting animal weight. The

presence of healing marks and the decrease on ALT also confirmed its anti-inflammatory

effects. The extract revealed a greater efficiency at doses 8 and 16mg/kg. In addition,

previous work has determined to 22.56mg/L, the median lethal dose (LD50) of the same

extract41

. This, once again, confirmed our results which showed that the dosage 8 and

16mg/kg, actually belongs to the range of effective doses 100 (ED100) and lethal dose50

(LD50) of the aqueous extract of rhizomes of Canna indica L.

The most characteristic is that, C. indica rhizomes’ extract was more effective at low doses.

Indeed, the doses 8 and 16mg/kg induced greater reductions in the liver relative weight, with

a total regeneration of hepatocytes, and a complete reduction of inflammation. This result is

also confirmed by tests on plasma ALT levels, which showed that at both doses, the extract

induced the greatest oxydative activity.

ACKNOWLEDGMENTS

Special thanks to Dr D. Nzeufiet of the Laboratory of Animal Physiology (Faculty of

Sciences-UYI) for histological analysis, and to Dr G. Agbor and Mr R. Ajang Ngidi of the

Phytochemistry Laboratory of the Institute of Medical Research and Study of Medicinal

Plants of Cameroon (IMPM) for phytochemical analysis.

This work was supported, in part, by the German Academic Exchange Service (DAAD)

initiative ‘‘Welcome to Africa’’ and the Cameroonian High Education Ministry through the

special allocation account for the modernization of University research in Cameroon.

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