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Title: Hepatoprotective effect of leaf extracts from Citrus hystrix and C. maxima against paracetamol induced liver injury in rats
Author: Arumugam Abirami Gunasekaran Nagarani Perumal Siddhuraju
PII : DOI:
Reference:
S2213-4530(15)00014-2
http://dx.doi.org/doi:10.1016/j.fshw.2015.02.002
FSHW51
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Received date: Revised date: Accepted date:
12-11-2014
20-1-2015
23-2-2015
Please cite this article as: A. Abirami, G. Nagarani, P. Siddhuraju, Hepatoprotective effect of leaf extracts from Citrus hystrix and C. maxima against paracetamol induced liver injury in rats, Food Science and Human Wellness (2015), http://dx.doi.org/10.1016/j.fshw.2015.02.002
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1 Hepatoprotective effect of leaf extracts from Citrus hystrix and C. maxima
2 against paracetamol induced liver injury in rats
4 Arumugam Abirami, Gunasekaran Nagarani and Perumal Siddhuraju*
5 Bioresource Technology Lab, School of Life Sciences, Department of Environmental Sciences,
6 Bharathiar University, Coimbatore - 641 046, Tamil Nadu, India.
9 Corresponding Author: Ph: +91 422 2428394;
10 Fax No: +91 422 2422387;
11 E-mail: siddhurajubrt@yahoo.com
12 Abstract
13 The present investigation is aimed to evaluate the hepatoprotective effects of Citrus
14 hystrix and C. maxima (Red and White variety) methanolic leaf extracts on paracetamol induced
15 toxicity. Leaf extracts was given in the dose of 200 mg/kg b.w. for 7 days and toxicity was
16 induced by paracetamol (2 g/kg) on day 5. Silymarin (100 mg/kg b.w) was used as reference
17 standard. On the 7th day animals were sacrificed and liver function markers (ALT, AST, ALP),
18 total bilirubin and total protein in blood serums and hepatic antioxidants (SOD, CAT, GSH &
19 GPx) in liver homogenate were estimated. The leaf extracts restored the liver function markers
20 and hepatic antioxidants to the normal level than elevated levels noticed on paracetamol control
21 at ^<0.001. Reversal of hepatoarchitecture has also been registered. The present study shows that
22 C. hystrix and C. maxima leaf extracts possess hepatoprotective action against paracetamol
23 induced hepatotoxicity.
24 Keywords: Citrus hystrix, Citrus maxima, paracetamol, silymarin, hepatoprotection.
1. Introduction
Liver is the prime organ associated with various stages of metabolic and physiologic homeostasis of the organism. Free radicals, alcohol, xenobiotics, food additives and pollutants are the major risk factors which lead to hepatitis, cirrhosis and alcoholic liver diseases [1]. Treatment of diseases associated with the liver is necessary, and must be done with proper and extensive care. There are few conventional drugs that can stimulate liver function and offer hepatic protection or help in the regeneration of hepatic cells but they are proved to be hepatotoxic at particular dose [2]. Acetaminophen (N-acetyl-p-aminophenol, Paracetamol) induced toxicity in rats is one of the widely used experimental model to evaluate the hepatoprotective activity of plant extracts [3, 4]. At therapeutic doses, paracetamol is considered a safe drug. However, it can cause hepatic necrosis, nephrotoxicity, extra hepatic lesions, and even death in humans and experimental animals when taken in overdose [5]. So, there is a need to evaluate natural compounds as an effective alternative which are safer and cost effective.
C. hystrix (C. hystrix) DC (commonly known as Kaffir lime) and C. maxima (C. maxima) L are giant citrus (commonly known as Pummelo) originated from South East Asia, India and cultivated throughout the tropical and temperate regions for the fruits. C. hystrix is pear-shaped, bumpy, greenish yellow fruit with acidic flavor with very thorny bush, aromatic leaves and fruits. The leaves are strongly aromatic, one or two fresh leaves can be torn, chopped and used as a spice and for various flavouring purpose in Southeast Asian and Thai dishes. Also, small pieces of fresh leaves are added with butter milk to prevent the peroxidation of lipids due to the presence of associated bioactive compounds i.e. polyphenols and enhance the digestive system of stomach. It is used as traditional medicine for headache, flu, fever, sore throats, bad breath and indigestion [6]. The regular use of rubbing fresh leaves on the teeth and gum could aid in dental health. Many active compounds were isolated from leaves of this plant such as phenolic acids, flavonoids, limonoids, coumarins, glycerolipids and a-tocopherol that possess various pharmaceutical effects such as anti-tumor, antimicrobial, anti-inflammation and antioxidant activities [7-11].
C. maxima fruit is a largest of all citrus variety. It is globose, pear-shaped with 11-14 segments. The pulp appears as white or pinkish red with spindle-shaped juice sacks that separate easily from one another and sweetish-acidic flavor. The leaves are large 5-10 x 2-5 cm long size,
56 ovate to elliptical shape, frequently emarginated, obtusely acute apex and dotted glandular [12].
57 Traditionally leaves are used in the treatment of convulsive cough, cholera, epilepsy and
58 haemorrhagie diseases. Leaves possess the important classes of phytochemicals such as
59 alkaloids, saponins and carbohydrates [13]. The major essential oils such as DL-limonene, E-
60 citral, 1-hexene-4-methyl and Z-citral were analyzed through GC-MS in the leaves and they
61 could exhibit antifungal, antiaflatoxigenic and antioxidant activity [14]. The leaves exhibit a
62 variety of pharmaceutical effects such as antioxidant, hepatoprotection, anticancer, antimicrobial,
63 antihyperglycemic, antidepressant, anti-inflammation and analgesic activity [15-19]. Hence, the
64 aim of the present study was to investigate and compare the hepatoprotective effects of crude
65 methanolic extracts of C. hystrix and C. maxima (Red and White) leaves on paracetamol induced
66 acute liver toxicity in rats. The protective effects were compared with silymarin, a well known
67 hepatoprotective agent against paracetamol induced hepatotoxicity.
69 2. Materials and methods
71 2.1 Chemicals
73 Paracetamol (Cipla Ltd., Baddi, Himachal Pradesh, India), detection kits for alanine
74 transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) (Agappe
75 Diagnostics Ltd., Ernakulum, Kerala, India) were obtained for the studies. All the other
76 chemicals were obtained from HiMedia Laboratories (Mumbai, Maharashtra, India).
78 2.2. Preparation of test drugs
80 The leaves of Citrus hystrix & C. maxima (Red and White) fruits were collected from
81 Mayiladuthurai, Nagai district, Tamil Nadu during the month of April 2010. The leaves were
82 cleaned with tap water to wash out the sand particles, dried in oven at 40 °C and ground into fine
83 powder using laboratory blender (Remi Anupam Mixie Ltd., Mumbai, Maharashtra, India). The
84 powdered samples were extracted by stirring 100 mL 80:20 of aqueous methanol at 25 °C for 48
85 h and filtering through Whatmann No.4 filter paper. The residues were re extracted with
86 additional 75 mL of aqueous methanol, as described above, for 3 h. the solvent of the combined
87 extract were dried at 40 °C. They were dissolved in 0.5% carboxy methyl cellulose (CMC) in
88 (fixed dose in mg)/10 mL concentration and administrated to the desired volumes according to
89 the body weight of animals to the respective groups.
91 2.3. Animals housing and feeding conditions
93 Male Sprague-Dawley (SD) rats were procured from the Small Animal Breeding Centre,
94 College of Veterinary and Animal Sciences, Kerala Agricultural University, Mannuthy, Thrissur,
95 Kerala. Swiss albino mice were procured from animal house of Nandha College of Pharmacy
96 and Research Institute, Erode, TN, India. The animals were provided with adequate
97 environmental conditions (temperature 24 ± 2 °C; relative humidity 40 - 60%; and 12:12 light:
98 dark cycle) with the standard commercial pellets (M/s. Hindustan Lever Ltd., Mumbai,
99 Maharashtra, India) and purified water ad libitum. All the experiments were performed with the
100 permission from Institutional Animal Ethics Committee (688/2/C-CPCSEA) and were in
101 accordance with the guidelines of CPCSEA.
103 2.4. Acute toxicity studies
105 The healthy Swiss albino mice (20-25 g) fasted for 3-4 h (provided only with water) and
106 were randomly transferred to five groups (n=3/group). They were fed orally with extracts in the
107 dose range of 5, 50, 300 and 2000 mg/kg body weight (b.w.) post oesophagus (p.o.) with the
108 control of 0.5% CMC. The study was carried out as per OECD guidelines-423 (acute toxic class
109 method) [20]. The animals were observed for any signs of toxicity, morbidity and mortality for
110 the first 24 h with the special attention during the first 4 h. they were also analyzed for the
111 changes in behavioral, neurological and autonomic profile. Further, they were observed for a
112 period of 72 h and till the completion of 14 days. Test dose was calculated as per Naskar et al.
113 [21].
115 2.5. Hepatoprotective activity
116 2.5.1. Experimental design and animal grouping
117 The SD rats weighing 100-150 g were used for the study. Animals were divided into six
118 groups (n=6/group) as follows:
119 GI - Normal control (NC) rats received distilled water 5 mL/kg b.w. p.o./7 days.
120 GII - Paracetamol control (PC) rats received distilled water 5 mL/kg b.w. p.o./7 days (except 5th
121 day).
122 GIII - Rats received the standard drug silymarin 100 mg/kg b.w. p.o./7 days.
123 GIV - Rats received test drug 1 (C. hystrix leaves) - 200 mg/kg b.w. p.o./7 days.
124 GV - Rats received test drug 1 (C. maxima (Red) leaves) - 200 mg/kg b.w. p.o./7 days.
125 GVI - Rats received test drug 1 (C. maxima (White) leaves) - 200 mg/kg b.w. p.o./7 days.
126 All the animals in the groups, GIII-GVI were pre-treated with their respective drugs for 5
127 consecutive days. On the fifth day of experimental period, after the drug administration of
128 respective treatments, all animals except those in GI were administered with paracetamol 2 g/kg
129 b.w. p.o. on the seventh day, after 2 h of respective drug treatments, animals were anaesthetized
130 using diethyl ether inhalation jar. Blood was collected through cardiac puncture and the serum
131 was separated.
133 2.5.2. Determination of key liver function biochemical markers
135 Liver function biochemical markers such as ALT, AST, ALP, total bilirubin and total
136 protein have been evaluated in the serum obtained from the experimental animals according to
137 the supplier's specifications from the standard kits.
139 2.5.3. Determination of key oxidative stress markers
141 One part of the liver tissue from the sacrificed experimental animals was washed and
142 homogenized (1:10, w/v) in ice-cold 50 mM Tris buffer (pH=7.4). the contents were centrifuged
143 at 10,000g for 20 min at 4 °C and the supernatant obtained was analyzed for superoxide
144 dismutase (SOD)[22], Catalase (CAT)[23], glutathione (GSH)[24] and glutathione peroxidase
145 (GPx)[25]. Lipid peroxidation byproduct malondialdehyde (MDA) was measured in the form of
146 thiobarbituric acid reactive substance (TBARS) by Ohkawa et al. [26].
148 2.5.4. Histopathological studies
150 Livers excised after sacrificing the animals were immediately washed with buffer and
151 fixed in10% buffered formalin. They were then dehydrated through graded alcohol series,
152 cleared in xylene and embedded in paraffin wax. Sections of 5-6 |im thickness were cut using
153 microtome and stained with hematoxylin-eosin. The histopathological changes were examined
154 under the microscope (Nikon, Japan) and the images were captured at the magnification of 10
155 and 40x
157 2.6. Statistical analysis
159 The values are expressed as mean ± standard deviation (SD) (n=6). The statistical
160 analysis was carried out by one way analysis of variance (ANOVA) followed by post hoc
161 Dunnett's multiple comparison test using the SPSS (Statistical Package for the social Sciences)
162 version 13.0 (SPSS Inc., Chicago, Illinois, USA). Significant difference were analyzed at three
163 levels; p < 0.05 (significant), 0.01 (most significant) and 0.001 (highly significant).
165 3. Results
166 3.1. Acute toxicity and dose calculation
168 Animals showed good tolerance to the testing doses of methanolic extracts of the leaf
169 from the C. hystrix and C. maxima (Red & white) as high as 2 g/kg b.w.p.o. respectively. The
170 highest dose was found to be non-lethal and did not show any noticeable signs of toxicity and
171 mortality for 15 days. Generally 1/10th and 1/5th of lethal dose is chosen for the effective dose
172 calculation; hence, 200 mg/kg b.w. for all the samples has been scrutinized as test doses. In
173 addition, none of the toxic signs have been found with the selected test doses until the end of the
174 study period.
176 3.2. Effect of leaves from C. hystrix and C. maxima on serum biochemical parameters
178 The estimation of enzymes in the serum is a useful quantitative marker of the extent and
179 type of hepatocellular damage. The rats administered with overdose of paracetamol (2 g/kg)
180 caused significant liver damage and necrosis of cells as evidenced by the elevated serum hepatic
181 enzymes (ALT, AST and ALP) and reduced level of protein and increased level of total bilirubin
182 (Table 1). The level of enzyme markers ALT, AST and ALP in normal rats were found to be
183 61.9 ± 4.43, 79.43 ± 2.74, 159.77 ± 3.68 IU/L respectively; as expected, paracetamol intoxication
184 made their elevation to 4.3, 3.6 and 1.95-fold increment with the values of 266.93, 288.77 and
185 312.4 IU/L respectively. This indicates the hepatic injury and loss of structural integrity. Pre-
186 treatment with leaf extracts significantly (p < 0.001) reduced their elevations with the normal
187 values in the range of 79.97-90.20, 93.10-114.5, and 186.13-210.80 IU/L for ALT, AST and
188 ALP respectively. Treatments with methanolic extracts of leaf indicate the stabilization of
189 plasma membrane as well as repair of hepatic tissue damage caused by paracetamol.
190 Similarly, a distorted pattern for other markers by 2.9 fold increased bilirubin and 2.1
191 fold decreased protein content was observed in PC rats compared to the NC rats (2.18 vs. 0.74
192 mg/dL for bilirubin; 4.79 vs. 9.63 mg/dL for protein content) anticipate the impaired liver
193 function (Table 1). Significant (p < 0.001) restoration of these markers by the interventions C.
194 hystrix leaves extract (CHL), C. maxima (Red) leaves extract CMRL and C. maxima (White)
195 leaves extract CMWL to the comparable level of normal control and silymarin pre-treated rats
196 was registered by table values. Hepatocellular necrosis or membrane damage leads to very high
197 levels of serum AST and ALT released from liver to circulation and low level of protein and
198 high level of bilirubin respectively.
200 3.3. Effect of methanolic extracts of leaf from C. hystrix and C. maxima on liver biochemical
201 parameters
203 Liver biochemical parameters like SOD (52.6%) (13.75 U/mg protein), CAT (48.6%)
204 (57.06 |mol of H2O2 decomposed/mg protein), GPx (80%) (0.70 U/mg protein) and GSH (47%)
205 (3.26 |g/mg protein) were reduced and lipid peroxidation (3.3 fold) (5.19 nmol MDA/mg
206 protein) were increased in paracetamol induced rats (2 g/kg) compared to normal control (Table
207 2). In the present study, pretreatment with CHL, CMRL and CMWL extracts brought back these
208 oxidative stress markers in the range of 1.76-2.20 nmol MDA/mg protein, 21.86-24.14 U/mg
209 protein, 92.93-101.29 |imol of H2O2 decomposed/mg protein, 4.94-5.51 |ig/mg protein, 2.93210 3.44 U/mg protein for the parameters MDA, SOD, CAT, GSH and GPx respectively which is on
211 par with normal control rats and significantly different (p< 0.001) with the paracetamol
212 intoxicated rats. Glutathione removes free radical species such as hydrogen peroxide, superoxide
213 radicals and maintains membrane protein thiols.
215 3.4. Effect of methanolic extracts of leaffrom C. hystrix and C. maxima on histopathology
217 Histopathological liver sections of rats from all the six experimental groups were shown
218 in Fig 1A to 1F and they provide supportive evidence of biochemical analysis. The primary aim
219 is to understand how tissues are organized at all structural levels, including the molecular and
220 macromolecular, the entire cell and intercellular substances and tissues and organs. In this, liver
221 sections from normal control rats (Fig 1A (magnification under 10 X)) organized into lobules
222 which are roughly hexagonal in shape, with portal triads at the vertices and a central vein in the
223 middle. Within each lobule, hepatocytes are arranged into hepatic cords running radiantly from
224 the central vein and are separated by adjacent sinusoids. Portal tracts showed unremarkable
225 portal veins, bile ducts and no signs for inflammation, necrosis or fibrosis or toxic changes. Liver
226 sections from paracetamol treated rats (Fig 1B (magnification under 10 X)) showed
227 vacuolization of hepatocytes, mild sinusoidal dilation, mild portal tract inflammation, fatty
228 changes and necrosis. Pretreatment with silymarin, C. hystrix leaf and C. maxima (Red) leaf
229 extracts in paracetamol intoxicated rats showed recovery of the hepatocytes from necrosis
230 indicating that sample extracts preserved the structural integrity of the hepatocellular membrane
231 and liver cell architecture damaged by paracetamol which was confirmed by histopathological
232 examination. Liver sections of the rats treated with methanolic extract of C. maxima (White) leaf
233 and intoxicated paracetamol (Fig. 1F (magnification under 10 X)) showed moderate
234 hepatoprotective activity and the hepatocytes shows normal sinusoids, cytoplasmic clearing and
235 focal ballooning and binucleation.
237 4. Discussion
239 Plant medicines plays important role by their various formulations for the treatment of
240 various diseases. However some have been analyzed and scientifically validated for their
241 potentials. Here, we designed the experiments to examine the hepatoprotective activity of
242 methanolic extract of leaves from underutilized C. hystrix and C. maxima (Red and White) for
243 their development into safe natural drug candidates.
244 Paracetamol (acetaminophen) is widely consumed as antipyretic drug that is safe in
245 therapeutic doses but can cause fatal hepatic damage in human and animal at higher toxic doses.
246 Bioactivation of paracetamol by hepatic cytochrome P-450 leads to formation of a highly
247 reactive and toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). NAPQI is normally
248 detoxified by conjugation with reduced glutathione (GSH) to form mercapturic acid which is
249 excreted in urine. Toxic overdose of paracetamol depletes hepatic GSH content so that free
250 NAPQI binds covalently to cellular mitochondrial proteins which suppresses mitochondrial fatty
251 acid P-oxidation and results in massive necrosis and apoptosis of hepatocytes [27, 28]. An
252 obvious sign of hepatic injury is the leaking of cellular enzymes such as ALT, AST and ALP into
253 plasma due to the disturbance caused in the transport functions of hepatocytes. ALT is more
254 specific to the liver, and it is a better parameter for analyzing hepatic injury. High levels of AST
255 indicate the cellular leakage as well as loss of functional ability of cell membrane in liver. Serum
256 ALP is also related with liver cell damage. High concentration of ALP cause serious hepatic
257 damage in paracetamol treated rats [29]. The liver is the major source of most of the serum
258 proteins. Bilirubin is a product of heme within the reticuloendothelia system; its elevation in the
259 blood stream can be adduced to over production, increased hemolysis, decreased conjugation or
260 impaired bilirubin transport [30]. Bilirubin is an index that is used to assess the normal
261 functioning of the liver instead of the extent of hepatocellular injury.
262 Antioxidant enzymes such as superoxide dismutase (SOD), catalase and glutathione
263 peroxidase (GPx) are very important in protecting organisms from reacting oxygen species. SOD
264 is a defense enzyme, which converts superoxide radicals to hydrogen peroxide. Catalase is a
265 hemeprotein found in peroxisomes of eukaryotic cells that catalyses the conversion of hydrogen
266 peroxide to water and oxygen. GPx plays a critical role in the maintaining balance in the redox
267 status of animals under acute oxidative stress and protect against chemically induced oxidative
268 destruction of lipid and proteins. Lipid peroxidation has been postulated to be the destructive
269 process in liver injury due to paracetamol administration. The increase in MDA level of liver
270 suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense
271 mechanisms to prevent formation of excessive free radicals. The decrease of glutathione, GPx,
272 SOD and catalase enzyme activity may indicate the toxic effects of reactive oxygen species
273 produced by toxicants. Reduced GSH level was depleted in paracetamol treated group may be
274 due to conjugation of glutathione with NAPQ1 to form mercapturic acid.
275 The mechanism of hepatoprotection by methanolic extracts of C. hystrix and C. maxima
276 (Red & White) leaves is due to their antioxidant potential. This suggests that leaf extracts can
277 reduce ROS that may lessen the oxidative damage to the hepatocytes and improve the activities
278 of the liver antioxidant enzymes, thus protecting the liver from paracetamol induced damage.
279 Also, the possible mechanism could be by the stimulation of hepatic regeneration through an
280 improved synthesis of protein or accelerated detoxification and excretion. Akachi et al. [31]
281 demonstrated the hepatoprotective effect of polymethoxy flavonoids such as citromitin,
282 tangeretin and nobiletin from juice of C. depressa could act against D-Galactosamine induced
283 liver injury in rats. Park et al. [32] evaluated the oral administration of narirutin fraction from the
284 peel of C. unshiu against alcohol induced hepatic damage in rats could block the development of
285 alcoholic fatty liver and hepatic tissue damage. Mahmoud et al. [33] reported that the
286 hepatoprotective effect of limonin from the seed of C. aurantium var bigaradia on D-
287 Galactosamine induced liver injury in rats. They have also been noted as rich phytoconstituents
288 such as dietary phenolics and flavonoids which are mainly responsible for the antioxidant power.
290 5. Conclusions
292 Based on these all profiles of hepatoprotective analysis, it indicates the C. hystrix and C.
293 maxima (Red and White) leaf can serve as hepatoprotectants as they restored all the liver
294 function and oxidative stress markers to the desirable levels. Further verification by their
295 histological micrographs reveals the attenuation of liver damage. This is aided by the superior
296 antioxidant potential of the leaf extracts against the sequential events of free radical toxicity by
297 paracetamol. It can be concluded from the observations in our study that C. hystrix and C.
298 maxima (Red and White) leaf extract may have a protective effect against paracetamol induced
299 hepatotoxicity in rats. However, further studies using some more models of experimental hepatic
300 damage are required to elucidate exact molecular and biochemical mechanisms involved and to
301 establish its therapeutic role as a hepatoprotective agent.
303 Acknowledgement
304 The authors would like to thank Dr. T. Sivakumar, Principal, Nandha College of Pharmacy
305 and Research Institute, Erode, TN, India for necessary permission to carry out the study and Dr.
306 S. Sengottuvelu, Head, Department of Pharmacology, and Mrs. V. Lalitha, Department of
307 Pharmacology, Nandha College of Pharmacy and Research Institute for their support and
308 suggestions.
310 Conflict of Interest
311 The authors declare that there are no conflicts of interest.
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Figure caption
Fig 1 Photomicrographs of hematoxylin and eosin stained histological sections of normal, paracetamol intoxicated, standard and test drug treated rats liver. 1) Normal control (x10) 2) Paracetamol treated rat liver (x10) 3) Silimarin + paracetamol treated rat liver (x10) 4) C. hystrix leaf extract + paracetamol treated rat liver (x40) 5) C. maxima (Red) leaf extract + paracetamol treated rat liver (x40) 6) C. maxima (White) leaf extract + paracetamol treated rat liver (x40). (N-nucleus; H- hepatocytes; CV- central vein; NC- necrosis; SD- sinusoidal dilation, FC- fatty change; V- vacuole; PRI- portal tract inflammation; B- ballooning and binucleation.
