HEPATOPROTECTIVE EFFECTS OF CANNA INDICA L. …...Vo u e I su 05 16 1 2 . Vollumme 44,, Issuee 05,,...
Transcript of HEPATOPROTECTIVE EFFECTS OF CANNA INDICA L. …...Vo u e I su 05 16 1 2 . Vollumme 44,, Issuee 05,,...
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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.
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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
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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
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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
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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
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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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