molecules-17-13937
TRANSCRIPT
Molecules 2012, 17, 13937-13947; doi:10.3390/molecules171213937
molecules ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Evaluation of the Hepatoprotective Effects of Lantadene A, a Pentacyclic Triterpenoid of Lantana Plants against Acetaminophen-induced Liver Damage
Chong Grace-Lynn 1, Yeng Chen 2, Lachimanan Yoga Latha 1, Jagat R. Kanwar 3,
Subramanion L. Jothy 1, Soundararajan Vijayarathna 1 and Sreenivasan Sasidharan 1,*
1 Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia,
Pulau Pinang 11800, Malaysia; E-Mails: [email protected] (C.G.-L.);
[email protected] (L.Y.L.); [email protected] (S.L.J.);
[email protected] (S.V.) 2 Dental Research & Training Unit and Oral Cancer Research and Coordinating Centre (OCRCC),
Faculty of Dentistry, University of Malaya, Kuala Lumpur 50603, Malaysia;
E-Mail: [email protected] 3 Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (LIMBR),
School of Medicine (SoM), Faculty of Health, Institute for Frontier Materials (IFM),
Deakin University, Waurn Ponds, Victoria 3217, Australia; E-Mail: [email protected]
* Authors to whom correspondence should be addressed; E-Mails: [email protected];
Tel.: +604-653-4820; Fax: +604-653-4803.
Received: 9 November 2012; in revised form: 15 November 2012 / Accepted: 19 November 2012 /
Published: 23 November 2012
Abstract: The aim of the present study was to evaluate the hepatoprotective activity of
lantadene A against acetaminophen-induced liver toxicity in mice was studied. Activity
was measured by monitoring the levels of aspartate aminotransferase (AST), alanine
aminotransferase (ALT), alkaline phosphatase (ALP) and bilirubin, along with histo-
pathological analysis. Silymarin was used as positive control. A bimodal pattern of
behavioural toxicity was exhibited by the lantadene A-treated group at the beginning of the
treatment. However, treatment with lantadene A and silymarin resulted in an increase in
the liver weight compared with the acetaminophen treated group. The results of the
acetaminophen-induced liver toxicity experiments showed that mice treated with lantadene
A (500 mg/kg) showed a significant decrease in the activity of ALT, AST and ALP and the
level of bilirubin, which were all elevated in the acetaminophen treated group (p < 0.05).
Histological studies supported the biochemical findings and a maximum improvement in
OPEN ACCESS
Molecules 2012, 17 13938
the histoarchitecture was seen. The lantadene A-treated group showed remarkable
protective effects against histopathological alterations, with comparable results to the
silymarin treated group. The current study confirmed the hepatoprotective effects of
lantadene A against the model hepatotoxicant acetaminophen, which is likely related to its
potent antioxidative activity.
Keywords: acetaminophen; hepatoprotective; antioxidant activity; lantadene A; triterpenes
1. Introduction
Pentacyclic triterpenes are extremely common compounds with the basic molecular formula C30H48
found in most plants. They are synthesized in many plants by the cyclization of squalene [1]. They are
obtained from plants and especially from the bark of trees such as plane, cork and birch but also in
liquorice roots, where they are particularly abundant. The most studied triterpenes are the pentacyclic
ones as their biological properties are considerable [2]. Pentacyclic triterpenes have contributed to the
development of modern therapeutic drugs. For example, triterpene acids (betulinic acid and ursolic
acid) have been shown to exhibit significant anticarcinogenic and anti-HIV activity [3,4], while lupeol is a
competitive inhibitor of both trypsin and chymotrypsin [5] and the antiphlogistic activity of betulin
was confirmed in various experimental models [6]. Lantadenes are the pentacyclic triterpenoids present in
the leaves of the plant Lantana camara (lantana) [7–9]. Previous studies showed that ingestion of
lantana foliage by grazing animals causes hepatotoxicity and photosensitization [9,10]. Our previous
results on L. camara leaf extracts which contain lantadene A (Figure 1) did not show any apparent
toxicity towards Vero cells. Conversely, the results of an oral acute toxicity study on L. camara leaf
showed a pro-toxic effect [11].
Figure 1. Lantadene A.
Individuals afflicted with serious idiosyncratic drug reactions are at risk of death without liver
transplantation. Some idiosyncratic responses can be attributed to unusual inheritance of or lack of
genes encoding key enzymes involved in drug metabolism. The mechanisms of some drug reactions
probably involve novel pathways that remain to be discovered. Drug-induced hepatotoxicity has now
become a significant cause of acute liver failure, accounting for 50% of cases. Toxic injury of the liver
is the most frequent impediment to development of candidate drugs by pharmaceutical companies and
Molecules 2012, 17 13939
is a major cause of withdrawal of approved drugs from the market [12]. There has been no consensus
on the identity of lantana toxins. Some research groups have reported lantadene A to be the
hepatotoxic principle, while others provided evidence that pure lantadene A did not elicit
hepatotoxicity. The action of lantadenes associated with hepatoprotection or hepatotoxicity is
contradictory due to the antioxidant activity of the compound. Our recent study showed that lantadene
A possessed good antioxidant activity with different mechanisms of action towards the different free
radicals tested [13]. However, there is no detailed study reported on lantadene A’s hepatoprotective or
hepatotoxic actions, therefore, there is a need to evaluate and verify these actions. Thus the current
study was conducted to investigate how lantadene A is associated with hepatoprotection or
hepatotoxicity as this compound is used in the development of drugs [1].
2. Results and Discussion
2.1. General Sign and Behavioural Analysis
The toxic effects exhibited by all treatment groups on the appearance and the general behavioural
pattern of mice are shown in Table 1. The behavioural patterns of animals were observed for the first 6 h
followed by 14 h and were continued for seven days after the various treatments. Animals treated with
saline water were normal and did not display any significant changes in behavior, skin effects,
breathing, impairment in food intake and water consumption. There was no postural abnormality or
hair loss. In the acetaminophen-, lantadene A- and silymarin-treated groups within the first 6 h rapid
heartbeat was observed after the administration, but it then become normal and this may due to the
stress of the oral administration of the drugs. In the acetaminophen-treated group bleeding of the eyes,
diarrhea and dark brown urine were noted.
Table 1. General appearance and behavioural observations for control and treated groups.
Observation Control Acetaminophen
Induced
Treated group
(Lantadene A)
Treated group
(Silymarin)
Time 6 h 14 h 6 h 14 h 6 h 14 h 6 h 14 h
Skin and fur Normal Normal Normal Normal Fur unkempt Fur unkempt Normal Normal
Eyes Normal Normal Normal Bleeding Normal Bleeding Normal Normal
Mucous
membrane Normal Normal Normal Normal Normal Normal Normal Normal
Behavioural
patterns Normal Normal Normal Normal
Loss of
appetite
Loss of
appetite Normal Normal
Salivation Normal Normal Normal Normal Normal Normal Normal Normal
Lethargy Normal Normal Present Present Present Present Present Normal
Sleep Normal Normal Normal Normal Present Present Normal Normal
Diarrhea Normal Normal Present Present Present Present Normal Normal
Coma Normal Normal Normal Normal Normal Normal Normal Normal
Tremors Normal Normal Present Present Present Present Normal Normal
Urine Normal Normal Dark
brown Dark brown Bloody Bloody
Dark
brown Normal
Molecules 2012, 17 13940
Conversely, the silymarin-treated group did not exhibit any significant changes in general signs and
behavioural analysis, except dark brown urine was noted. A bimodal pattern of behavorial toxicity was
exhibited by the lantadene A-treated group. This was characterized by drowsiness, unkempt
appearance, loss of appetite, diarrhea, tremors and blood in the urine. Tolerance gradually developed to
the initial depressant effects starting after 14 h. The possible mechanisms of tolerance to lantadene A
include uncoupling of receptor sites, alterations in gene expression, down-regulation of receptor sites,
and desensitisation of receptor sites to the effect of lantadene A, which deserve further detailed study.
2.2. Effects of Lantadene A on Serum Marker Enzymes
Blood biochemical parameters such as ALT, AST, ALP and total bilirubin level, of all the
experimental groups are shown in Table 2. Acetaminophen treatment significantly increased the serum
liver enzyme activity of ALT, AST, ALP and total bilirubin content. The activity of ALT
(36.33 ± 14.64 U/L), AST (151.33 ± 79.33 U/L), ALP (82.67 ± 11.50 U/L) and total bilirubin content
(7.47 ± 0.76 μmol/L) were significantly higher (p < 0.05) in the acetaminophen-treated group in
comparison to the normal control group (ALT 22.25 ± 5.38; AST 59.50 ± 10.88; ALP 73.75 ± 26.92 U/L;
and bilirubin 2.4 ± 0.58 μmol/L), indicating a marked hepatocellular injury (Table 2). Liver function
tests are used to determine the presence or absence of liver disease, to make specific diagnoses, to
determine severity, and to monitor the course of disease. Liver disease is classified broadly into two
categories of injury: cell necrosis and cholestasis [14]. ALT and AST are the most commonly used
indicators of cell necrosis. ALT is the more specific of the two because, for the most part, it is confined
to liver, whereas AST is present not only in liver, but also in skeletal and cardiac muscle and kidney
and red blood cells [15]. AST is present in both mitochondria (80%) and cytoplasm (20%). In contrast,
ALT is confined to the cytoplasm [15]. In general, increases in ALT and AST with lesser increases or
normal ALP favor cell necrosis, whereas the reverse points to cholestasis. This study exhibited ALT
and AST to be increasing while a lesser increment is seen in ALP, clearly indicating acetaminophen
caused cell necrosis at higher doses [14]. This is even observed in the histopathological analysis of
liver sections taken from the acetaminophen intoxicated group. Overdose of acetaminophen results in
the generation of free radicals following the depletion of endogenous antioxidants such as glutathione [16],
which is powerful scavenger of free radicals, and this leads to cellular damage. Bilirubin content has
been used to assess chemically-induced hepatic injury. Besides various normal functions, the liver
excretes the breakdown product of hemoglobin, namely bilirubin, into bile. It is well known that
necrotizing agents like acetaminophen produce sufficient injury to hepatic parenchyma to cause large
increases in bilirubin content [17].
Nevertheless, treatment with lantadene A (500 mg/kg bw) reduced the alterations in the activity of
ALT, AST, ALP and bilirubin level as against the acetaminophen induced group (Table 2). Similarly,
silymarin (50 mg/kg bw) also prevented the alterations in the activity of ALT, AST, ALP and bilirubin
level induced with acetaminophen. The findings of this study provide strong evidence that lantadene A
significantly inhibits the acute liver toxicity induced by high doses of acetaminophen in mice, as
shown by a reduction of serum liver enzyme activities and serum bilirubin content. The decrease in the
serum levels of these enzymes might possibly be due to the polyvalent antioxidant activity showed by
lantadene A with different mechanism of action towards different free radicals as reported in our recent
Molecules 2012, 17 13941
report [13]. Furthermore, the findings of this study also suggest that lantadene A has some functions in
preserving structural integrity of hepatocellular membrane, thus preventing enzyme leakage into the
blood circulation [18]. This is the first time, lantadene A is used in a study to assess the effect of
hepatoprotectivity induced by acetaminophen.
Table 2. Effect of lantadene A on liver marker enzymes and serum bilirubin content.
Parameters Control Acetaminophen Induced Group
Lantadene A Treated Group
Silymarin Treated Gruop
AST (U/L) 59.50 ± 10.88 151.33 ± 79.33 ** 64.00 ± 27.84 * 59.00 ± 18.53 * ALT (U/L) 22.25 ± 5.38 36.33 ± 14.64 ** 22.33 ± 5.51 * 24.00 ± 6.48 * ALP (U/L) 73.75 ± 26.92 82.67 ± 11.50 ** 77.00 ± 37.47 * 80.25 ± 30.82 *
Total Bilirubin (μmol/L) 2.4 ± 0.58 7.47 ± 0.76 ** 2.4 ± 0.44 * 3.57 ± 1.23 *
Results are expressed as mean ± S.E.M; * statistically significant compared to acetaminophen treated animals
(p < 0.05); ** statistically significant to control animals (p < 0.05).
2.3. Liver Weights of Mice in Acetaminophen Induced Hepatotoxicity
When a higher dose of acetaminophen (1g/kg p.o single dose) was used, the liver tissue, in
macroscopic appearance, showed extensive necrosis associated with haemorrhages. However, with the
treatment of lantadene A and silymarin, the gross appearance of the liver tissue showed complete
recovery with absence of punctate haemorrages, swelling and with smooth margins. With
acetaminophen treatment, the liver organ body weight index (OBWI) was decreased to 5.84 ± 0.66 g
from that of normal liver in the control group (5.99 ± 0.68), which was statistically (p <0.05)
significant (Table 3). Conversely the treatment with lantadene A and silymarin resulted in an increase
in the liver OBWI as compared to normal groups, which was statistically (p < 0.05) significant (6.64 ± 0.30
and 6.10 ± 0.27 respectively) (Table 3). The acetaminophen-treated group showed a reduction in the
liver OBWI compared to normal, silymarin- and lantadene A-treated groups mice liver OBWI. This
could possibly be the result of acute necrosis of hepatocytes in the absence of adequate an
hepatoprotective agent [19]. On that account, this finding proves that lantadene A and silymarin aided
liver parenchymal cell growth in acetaminophen-intoxicated mice consequentially helping to increased
liver OBWI.
Table 3. Effect of treatment with lantadene A on liver weights of mice in acetaminophen
induced hepatotoxicity.
P < 0.05, statistically significant. a–d were significant different (p < 0.05).
Groups Liver organ body weight index (OBWI) Control (saline only) 5.99 ± 0.68 ad
Acetaminophen Induced 5.84 ± 0.66 b Acetaminophen Induced + Lantadene A Treatment 6.64 ± 0.30 c
Acetaminophen aracetamol Induced + Silymarin Treatment 6.10 ± 0.27 cda
Molecules 2012, 17 13942
2.4. Liver Histopathology Analysis
The light microscopy examination of the transverse section of normal, acetaminophen-, lantadene
A- and silymarin-treated mice livers were shown in Figures 2–5. The findings of the liver
histopathology analysis corroborated the results of the biochemical analysis. Histopathological liver
sections of control group showed normal cellular architecture with distinct hepatic cells, sinusoidal
spaces, and central vein (Figure 2).
Figure 2. Light microphotographs of control liver. (H: hepatocytes; N: nucleus;
SS: sinusoid; CV: central vein).
Control 20× Control 40×
Figure 3 illustrates the liver of acetaminophen intoxicated mice showing wide necrosis across the
cells. The liver sections of these mice showed necrosis, ballooning and degeneration in hepatic plates
and loss of cellular boundaries. The accumulation of neutrophils was also seen. These neutrophils act
as an indicator of the occurrence of cell damage as they are absent in normal healthy tissues.
Figure 3. Light microphotographs of liver cell of mice exposed to acetaminophen.
(NP: Neutrophil; DS: dilated sinusoid; FN: focal necrosis; B: binucleated cells).
Acetaminophen 20× Acetaminophen 40×
CV
N H
SS
NP
FN
DS
B
B
Molecules 2012, 17 13943
The hepatocytes are disrupted and sinusoids are damaged as well. Additionally, hepatocytes also
demonstrated a focal necrosis with binucleated cells which reflected the signs of high regenerative
activity [20]. The macroscopic appearance of the acetaminophen intoxicated liver showed extensive
areas of necrosis which facilitate the leakage of these enzymes into the blood circulation, explaining
the rise in the activity of AST, ALT and bilirubin levels in this study,.
Figures 4,5 shows the histological architecture of lantadene A- and silymarin-treated livers. The
liver sections of the mice treated with 500 mg/kg body weight p.o of lantadene A (Figure 3) showed a
mild degree of degeneration and necrosis.
Figure 4. Light microphotographs of liver cells of mice treated with lantadene A
(H: hepatocytes; N: nucleus; SS: sinusoid; CV: central vein).
Lantadene A 20× Lantadene A 40×
Figure 5. Light microphotographs of liver cells of mice treated with silymarin
(H: hepatocytes; N: nucleus; SS: sinusoid; CV: central vein).
Silymarin 20× Silymarin 40×
The hepatocyte nucleases are at a recovery stage and there are very minimal numbers of neutrophils
surrounding the portal vein. Binucleate cells which reflected the signs of high regenerative activity
N
H
SS
N
H
SS
CV
CV
Molecules 2012, 17 13944
were dramatically reduced in lantadene A-treated group as compared to the acetaminophen intoxicated
group. The possibility of lantadene A accelerated recovery of hepatic cells was evidenced from the
histopathological observation, which suggests protection against membrane fragility thus decreased the
leakage of the marker enzymes into the circulation as observed in this study. The current
histopathological examination further verified the hepatoprotective effect of lantadene A against model
hepatotoxicant acetaminophen. Histopathological liver sections of lantadene A-treated group also
exhibited comparable results with the standard silymarin-treated group (Figure 5), hence confirming
the significant hepatoprotective effect of lantadene A at the dose of 500 mg/kg body weight.
3. Experimental
3.1. Preparation of the Lantadene A
Lantadene A was purchased from Chemtron, Penang, Malaysia. The single dose (500 mg/kg) was
determined in a separate toxicity study, with the LD50 value of 5,000 mg/kg BW (data not shown).
Therefore, 1/10 of the LD50 value (500 mg/kg) was chosen for the present study as the lantadene A
treatment dose. Lantadene A was dissolved in water to prepare the stock solution which was then
diluted to obtain the different concentrations used in further tests.
3.2. Hepatoprotective Activity of Lantadene A
3.2.1. Animals
Eighteen specific pathogen free and age-matched (7- to 10-week-old) male Wistar albino mice were
used to study the hepatoprotective activity of the Lantadene A. The Institution Animal Ethics
Committee of Universiti Sains Malaysia approved the animal study for this project (USM/Animal
Ethics Approval/2011/(74)(376). The animals were kept at 27 ± 2 °C, relative humidity 44–56% and
light and dark cycles of 10 and 14 h respectively, for a week before and during the experiments.
Animals were provided with standard diet (Lipton, Mumbai, India) and water ad libitum. The food was
withdrawn 18–24 h before starting the experiment. All experiments were performed in the morning
according to current guidelines for the care of the laboratory animals and the ethical guidelines for the
investigation of experimental pain in conscious animals [21].
3.2.2. Acetaminophen Dose Regimen
Acetaminophen tablets were obtained from a nearby clinic. Each tablet contains 500 mg of
acetaminophen. The dose administered to the mice was set as 1 g/kg. The acetaminophen was made
into fine powder using a mortar and pestle to increase the dissolution. The powdered acetaminophen
was suspended in saline and was administered orally according to the body weight of mice.
3.2.3. Grouping of Mice and Treatments
Eighteen mice (25–30 g) were randomly divided into three groups and each group consisted of six
mice. The first group received a single daily dose of 1 mL/kg of saline orally (control group). Group II
was given a single daily dose of acetaminophen (1.0 g/kg) orally (induced group), group III received
Molecules 2012, 17 13945
orally a single daily dose of both 1.0 g/kg acetaminophen [22] and 500 mg/kg of lantadene A
(treated group) and group IV received orally a single daily dose of both 1.0 g/kg acetaminophen and
50 mg/kg of silymarin (positive control treated group). Lantadene A was administered three hours
after the administration of acetaminophen. Acetaminophen 1g/kg was given to mice to induce
hepatotoxicity. The treatments were continued for seven days and on the eighth day of the experiment;
all animals were anesthetized and dissected [23].
3.2.4. General Sign and Behavioural Analysis
The mice were observed in detail for any indications of toxicity within the first six hours after the
treatment period, and further for a period of 24 h. Surviving animals were weighed and visual
observations for mortality, behavioral pattern, changes in physical appearance, injury, pain and signs
of illness were conducted daily during the period.
3.2.5. Sacrifice and Organ Harvesting
The liver was removed carefully after euthanizing and killing the animals by cervical dislocation.
The livers were weighed and fixed using 10% buffered formalin. After fixation, the livers were
dehydrated in a graded series of alcohol, cleared in xylene and embedded in paraffin wax. Multiple 5 μm
sections from each block were mounted on slides and stained with hematoxylin and eosin. The
remaining liver was quickly frozen in dry ice and stored at −80 °C for further analysis.
3.2.6. Liver Function Tests
The mice of each group were anaesthetized with ether, and blood was collected directly from the
heart. It was centrifuged at 2,000 g for 10 min at 4 °C to separate the serum and kept at 4 °C to assay
the activities of serum enzymes. Aspartate aminotransferase (AST) and alanine aminotransferase
(ALT) were determined by the method described by Reitman and Frankel [24]. Alkaline phosphatase
(ALP) was determined by the method described by Kind et al. [25]. Serum bilirubin level was
estimated according to Malloy and Evelyn [26].
3.3. Statistical Analysis
All values are mean ± S.E.M. obtained from six animals. For statistical analysis, one-way ANOVA
with Duncan’s variance (SPSS 15) was used to compare the groups. In all the cases a difference was
considered significant when p <0.05.
4. Conclusions
Taken together, the data of this research offer direct evidence that lantadene A is a potential
hepatoprotective agent candidate, which possesses good in vivo hepatoprotective activity. An overdose
of acetaminophen results in liver cell damage, however, mice treated with lantadene A, displayed
lower biochemical activities compared to the group that received acetaminophen. Some studies have
reported lantadene A to be the hepatotoxic principle of lantana plants, while others have provided
evidence that pure lantadene A did not show hepatotoxicity. Interestingly, the results of this study
Molecules 2012, 17 13946
confirmed that the lantadene A is a potential hepatoprotective agent. The hepatoprotective activity of
lantadene A may be due to its free radical-scavenging and antioxidant activity, as reported by us
earlier. Nonetheless, toxic drugs may produce acute changes for which long term follow-up has not
been done. Since lantadene A showed an effective hepatoprotective activity at the dosage of 500
mg/kg bw in this study, thus justifies its usage as hepatoprotective agent. However, there was no
toxicity data for lantadene A at very high doses. Hence there is a need for a separate chronic toxicity
study to be conducted to understand its toxicity at high dose with long term follow-up. Further studies
are in progress to better understand the mechanism of action of lantadene A responsible for the
observed hepatoprotective and the toxicity study at high dose.
Acknowledgments
This project was funded by USM Short Term Grant (304 /CIPPM /6312034) from Universiti Sains
Malaysia. Chong Grace-Lynn is supported by the MyMaster fellowship from Ministry of Higher
Education of Malaysian Government.
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Sample Availability: Samples of the compound lantadene A is available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
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