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Moringa oleifera Lam prevents acetaminophen induced liver injury through restoration of glutathione level
Food and Chemical Toxicology 46 (2008) 2611–2615
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Moringa oleifera Lam prevents acetaminophen induced liver injury through restoration of glutathione level S. Fakurazi a,*, I. Hairuszah b, U. Nanthini a a b
Pharmacology Unit, Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
a r t i c l e
i n f o
Article history: Received 22 January 2008 Accepted 11 April 2008
Keywords: Acetaminophen Paracetamol Glutathione Hepatoprotective Hepatotoxicity
a b s t r a c t Initiation of acetaminophen (APAP) toxicities is believed to be promoted by oxidative stress during the event of overdosage. The aim of the present study was to evaluate the hepatoprotective action of Moringa oleifera Lam (MO), an Asian plant of high medicinal value, against a single high dose of APAP. Groups of five male Sprague–Dawley rats were pre-administered with MO (200 and 800 mg/kg) prior to a single dose of APAP (3 g/kg body weight; p.o). Silymarin was used as an established hepatoprotective drug against APAP induced liver injury. The hepatoprotective activity of MO extract was observed following significant histopathological analysis and reduction of the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in groups pretreated with MO compared to those treated with APAP alone. Meanwhile, the level of glutathione (GSH) was found to be restored in MO-treated animals compared to the groups treated with APAP alone. These observations were comparable to the group pretreated with silymarin prior to APAP administration. Group that was treated with APAP alone exhibited high level of transaminases and ALP activities besides reduction in the GSH level. The histological hepatocellular deterioration was also evidenced. The results from the present study suggested that the leaves of MO can prevent hepatic injuries from APAP induced through preventing the decline of glutathione level. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Acetaminophen (APAP) is an effective and widely used antipyretic-analgesic drug with excellent safety record when taken at therapeutic doses (Larson, 2007). Despite extensive efforts to conduct studies on the processes of APAP induced toxicity, the exact mechanisms are still incompletely understood. Most evidence suggested the depletion of glutathione and the formation of reactive metabolites is somehow triggers the cascade events of hepatotoxicity (Jaeschke and Bajt, 2006). Overdose of APAP results in the generation of free radicals following the depletion of glutathione (Jaeschke and Bajt, 2006). Recently, the use of herbal natural product has gained interest among the world population. Many of the herbs have been developed into herbal supplement which are claimed to assist in healthy lifestyle. Among those herbs, is Moringa oleifera Lam (MO) which is native to South Asia and with high potential medicinal value. The leaves have been reported to have antihypercholesterolemic action (Ghasi et al., 2000) and those with other risk factor, such as hypertension (Faizi et al., 1998) or diabetes melli-
* Corresponding author. Tel.: +60 3 89472352; fax: +60 3 89242341. E-mail addresses: [email protected], [email protected] (S. Fakurazi). 0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.04.018
tus (Kar et al., 2003). Reports have also described the plant to be highly potent anti-inflammatory agent (Ezeamuzle et al., 1996) and antitumour activity (Murakami et al., 1998). The plant has also been reported to be hepatoprotective against antitubercular drug such as isoniazid and rifampicin (Pari and Kumar, 2002). Therefore, the objective of this study is to evaluate the possible protection of aqueous extract of MO against hepatotoxicity induced by APAP. The hepatoprotective potential of the MO crude extract as was compared with silymarin, a known and commercially available hepatoprotective agent.
2. Materials and methods 2.1. Preparation of plant materials The fresh leaves of M. oleifera were collected from Bandar Sunggala Farm, Port Dickson and authenticated by a plant taxonomist at Institute Bioscience (IBS), Universiti Putra Malaysia. A voucher specimen has been kept at herbarium IBS for future reference. The leaves were shaded dried for 3 weeks. The dried leaves were grounded into powder form and stored at 4 °C until further use. The leaves powder were then extracted using 80% hydroalcoholic solvent (80% ethanol:20% distilled water) with constant shaking at room temperature for overnight. The extract was filtered and the residue was then resuspended in ethanol for 48 hours and refiltered. The filtrate was then concentrated using a rotary evaporator (Rotavor R-200, Buchi, Switzerland) under reduced pressure at 40 °C and then lyophilized using a freeze dryer (Freeze Dry System, FreeZone 4.5, Labconco, USA; 40 °C,
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120 bar). A dark green mass was obtained and stored at 20 °C until further use. The crude extract, was resuspended in distilled water before administration to the animals (Hossain et al., 1992). 2.2. Experimental animals and study design Male Sprague–Dawley rats (200–250 g) were purchased from Faculty of Veterinary Medicine, Universiti Putra Malaysia were acclimatized under control laboratory conditions for a week with free access to standard food and water ad libitum. The animals were handled in accordance to the rules and regulations by the Animal Care Unit Committee, Universiti Putra Malaysia. The rats were randomly divided into groups each consisting of 5 rats. Treatment consisted of pretreatment phase of MO in distilled water followed by the second phase in which the animals were given 3 g/kg APAP in 40% sucrose buffer on day 15. The animals were then euthanized 24 h after APAP administration. Group 1 received pretreatment with distilled water for 14 days prior to a single dose of APAP (3 g/kg body weight; p.o) served as hepatotoxin control group. Groups 2 and 3 were administered with aqueous extract of MO (200 and 800 mg/kg body weight; p.o) for 14 days before being intoxicated with APAP. Group 4 was treated with 200 mg/kg silymarin in distilled water prior to APAP administration. Twenty-four hours after APAP administration the rats were fasted and then anaesthetized with diethyl ether. Blood was collected via cardiac puncture and the animals were then sacrificed. Livers were then excised, washed in ice-cold saline to remove blood and duly weighed. A section from the median lobe was preserved in 10% formal saline. The remaining liver was quickly frozen in dry ice and stored at 80 °C for further analysis. 2.3. Histopathological examination The median lobe of the liver from each group was removed and fixed in 10% formalin and embedded in the paraffin. Microtome sections of 3–4 lm thickness were prepared according to the standard procedure and stained with haematoxylin and eosin. Sections were then examined for pathological findings of such as centrilobular necrosis, fatty and lymphocytes infiltration. 2.4. Liver function tests The liver function tests (LFTs) including ALT, AST and ALP were analysed by kinetic method kits from Roche Diagnostics using a double beam spectrophotometer. 2.5. Determination of reduced glutathione The liver glutathione (GSH) determination was performed according to the method of Ellman (1959) using 5,5,-dithiobis-2-nitrobenzoic (DTNB) as substrate. The liver homogenate was then prepared using Ultra Turax where liver tissue was homogenized in 0.01 M Phosphate Buffer (pH 7.4) at 4 °C. The absorbance was read at 420 nm and the concentration of glutathione is calculated and expressed in lmol/g protein. 2.6. Statistical analysis Data was presented as mean ± SEM. Statistical analysis was performed using ANOVA Statistical Package for the Social Sciences (SPSS) version 13.0. p-Value less than 0.05 (p < 0.05) was considered to be statistically significant.
3. Results The serum activities of liver alanine aminotransferase (ALT), aspartate transferase (AST) and alkaline phosphatase (ALP) were shown in Table 1. Rats treated with 3 g/kg APAP has significant elevation of ALT, AST and ALP activities when compared to those
groups pretreated with MO. Treatment of MO for 14 days prior to APAP administration has provided a significant protection to the liver, preventing the elevation of liver enzymes activities. The activities of ALT and AST in groups that were pretreated with lower dose of MO (200 mg/kg) were found to be 56.47 ± 2.15 IU/L and 171.40 ± 8.75 IU/L, respectively. The activity of both enzymes was significantly lower compared to the group that was treated with APAP alone, 136.97 ± 4.19 and 440 ± 20.87 IU/L, respectively. The activity of ALP was also significantly reduced to 179.00 ± 6.46 IU/L compared to group that was not pretreated with MO (228.67 ± 13.82 IU/L). When the rats were treated with higher doses of MO (800 mg/ kg), the reduction in the activities of the liver enzymes were more pronounced compared to the group that was given APAP alone. The level of ALT, AST and ALP was found to be significantly reduced in group treated with MO where the level was found to be 48.62 ± 1.25, 174.43 ± 27.04 and 178.50 ± 13.07 IU/L, respectively. The reduction in the activities of the enzymes was comparable to the groups pretreated with standard herbal supplement available commercially, silymarin. The level of glutathione was reduced with APAP treatment at 1.56 ± 1.19 lmol/g. When the rats were pretreated with MO, both treatment doses (200 and 800 mg/kg) have significantly elevated the level of glutathione in the liver compared to group receiving APAP only, at 2.59 ± 1.60 and 2.65 ± 0.21 lmol/g, respectively. This result is comparable to the animals that were pretreated with silymarin where the glutathione level was restored (2.79 ± 0.39 lmol/g). Pretreatment of 200 mg/kg and 800 mg/kg MO have significantly (p < 0.05) protected the liver from hepatocellular damage due to APAP (Fig. 1). The hepatoprotective of the extract was comparable to the effect seen with silymarin pretreatment. Acetaminophen (APAP) at 3 g/kg showed significant (p < 0.05) hepatocellular damage including the presence of prominent microvesiculation, moderate infiltration of monocytes and neutrophils with mild scattered focal necrosis. These changes were clearly observed around the perivenular (PV) hepatocytes (Fig. 1). The pathological hallmark after APAP treatment, such as bridging necrosis was not observed in the liver sections obtained from rats that were pretreated with MO (200 and 800 mg/kg). M. oleifera (MO) has provided a significant (p < 0.05) protection against hepatocytes injury. The protective effect seen when rats were pretreated with MO was similar to the protective effect offered by silymarin. 4. Discussion A single high dose of APAP administration has significantly elevated the serum transaminases and ALP activities besides reducing the activity of glutathione. The results has showed a promising likelihood that M. oleifera extract has exhibited a hepatoprotective effect following a significant decrease in serum transaminases and ALP activities and by preventing the changes seen in the treatment
Table 1 The effect of Moringa oleifera Lam (MO) extract on alkaline phosphatase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) activities and glutathione level in rats administered with a single dose of acetaminophen (APAP) Groups 40% sucrose Distilled water + APAP MO + APAP Silymarin + APAP * **
Treatment mg/kg
ALT (IU/L)
AST (IU/L)
ALP (IU/L)
Glutathione (lmol/g)
3000 200 800 200
47.75 ± 3.68 136.97 ± 4.19* 56.47 ± 2.15** 48.62 ± 1.25** 64.33 ± 7.27**
121.22 ± 10.04 440.00 ± 20.87* 171.40 ± 8.75** 174.43 ± 27.04** 183.30 ± 21.10**
178.67 ± 5.28 228.67 ± 13.82 179.00 ± 6.46** 178.50 ± 13.07** 181.67 ± 10.82**
2.40 ± 0.03 1.56 ± 0.19* 2.59 ± 1.60** 2.65 ± 0.21** 2.79 ± 0.39**
p < 0.05 significantly different from group treated with 40% sucrose. p < 0.05 significantly different from group treated with distilled water and APAP.
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Fig. 1. Hepatoprotective action of Moringa oleifera Lam (MO) against acetaminophen (APAP) induced hepatotoxicity in rats. (a) Pretreatment of distilled water + APAP. The liver section reveals submassive inflammation with cells undergoing necrosis (?) around perivenular area (PV); (b) pretreatment of MO (200 mg/kg) + APAP; (c) pretreatment of MO (800 mg/kg) + APAP. (d) Pretreatment of silymarin (200 mg/kg) + APAP; (b), (c) and (d) show overall normal liver architecture H & E Magnification 100.
with APAP. Interestingly, MO has been found to restore the activity of glutathione which was found diminished following APAP treatment. Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. It is selected as hepatotoxicant in inducing injury to the liver as it is known to cause hepatotoxicity in man and experimental animals when taken overdose which leads to elevation of liver enzymes (Kumar et al., 2004; Ahmed and Khater, 2001). Leakage of cellular enzymes into plasma indicates a hallmark sign of hepatic injury or damage (Ramaiah, 2007; Kumar et al., 2004). In addition, the extent and type of liver injury or damage can be assessed based on the presence or absence of specific enzymes in the bloodstream (Kumar et al., 2004). Generally, measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) are commonly used as marker enzymes in assessing APAP induced hepatotoxicity (Yanpallewar et al., 2002; Asha et al., 2004; Yen et al., 2007). Aminotransferases, are group of enzymes that catalyze reversible transfer of the amino acid group from an a-amino acid to an oxo acid. These enzymes are not normal components of plasma and its function outside organ of origin is unknown (James et al., 2003). The largest pool of ALT is found in cytosol of hepatic parenchymal cells (Okuda, 1997). Whereas, AST is found in cytosol and mitochondria of hepatocytes and also found in cardiac muscle, skeletal muscle, pancreas and kidney (Shyamal et al., 2006). Therefore, measurement of ALT is more liver specific to determine hepatocellular damage (Shyamal et al., 2006). Nevertheless, AST is still being commonly used as a laboratory test to assess liver function since it is considered to be a sensitive indicator of mitochondria damage particularly in the centrilobular regions of liver (Pantegh-
ini, 1990). Meanwhile, ALP comprises of a family of enzymes that hydrolyze phosphate esters at alkaline pH and is often used as a marker for cholestatic liver dysfunction. However, liver is not the sole source of ALP. Substantial amount are also present in the bone, placenta, kidney and intestine (Gaw et al., 1998). In this study, hepatoprotective effect of MO is evident by the restoration of ALT, AST and ALP. Concurrently, significant preservation of liver histology was observed in the groups that were pretreated with MO. Pretreatment with MO extract suppresses APAP induced AST and ALT elevations. Recovery towards normalization of the enzymes following MO pretreatment suggested that the plant extract have some roles in preserving structural integrity of hepatocellular membrane, thus prevented enzymes leakage into the blood circulation. Our results are consistent with generally accepted hypothesis that transaminase level return to normal with healing of hepatic parenchyma and the regeneration of hepatocytes (Ahmed and Khater, 2001). Previous study has reported that the root and flower of MO have prevented APAP induced ALT, AST and ALP elevation in rats and mice (Ruckmani et al., 1998). Meanwhile, another study has also showed that MO leaves extract has significantly restored the elevation of ALT and AST as well as ALP level that was induced by isoniazid to the normal level (Pari and Kumar, 2002). Recent study by Nadro et al. (2005) has demonstrated that MO leaves extract has prevented the release of these enzymes from hepatocytes into the bloodstream when induced with high level of ethanol administration in rats. Our results are consistent with the previous studies, which strongly suggest that MO may preserve the structural integrity of hepatocytes when challenge with hepatotoxicants and subsequently preventing enzyme leakage into plasma. The results
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have also showed that the plant extract is equally effective to silymarin in preventing the increase in liver enzymes when challenged with hepatotoxic dose of APAP. Silymarin is a well-established hepatoprotective drug reported to revert abnormal alterations of these enzymes in various drug induced hepatotoxicity (Upadhyay et al., 2007; Pradhan and Girish, 2006). The ability of silymarin in preventing drug induced hepatotoxicity is associated with its ability to act as a radical scavenger, thereby protecting membrane permeability (Song et al., 2006). Silymarin is used as a standard agent for comparing hepatoprotective effects of plant principle due to its proven hepatoprotective property (Dhiman and Chawla, 2005). The benefits of MO are then further confirmed by histopathalogical observations. It was well-established that overdoses of APAP lead to centrilobular necrosis, fatty infiltration, lymphocytic as well neutrophil infiltration. (Yen et al., 2007). In the present study, histological sections of the liver showed similar reported changes including centrilobular necrosis with inflammatory cell infiltration in rats treated with APAP alone. The cytochrome P450 system is abundantly distributed in Zone 3-centrilobular (Z3). The predominant distribution of CYP450 at Z3 inflicts localized production of toxic reactive metabolites of various drugs including APAP such as N-acetyl-p-benzoquinoneimine (NAPQI). This in turn induces hepatic necrosis around the centrilobular region (Chung et al., 2001). Our study is consistent with the reported observation, in which prominent centrilobular necrosis was observed around this area (Z3). Centrilobular necrosis, the hallmark feature of APAP induced liver damage was significantly reduced with MO pretreatment (Oliveira et al., 2005). The congestion and inflammatory cell infiltration evoked by APAP was considerably decreased following extract pretreatment. The possibility of MO extract 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. Furthermore, the ability of MO to reverse the hepatic lesions induced by the APAP is comparable to the treatment with sucrose and the silymarin, has strongly suggested the hepatoprotective effect of MO. The formation of toxic reactive metabolite of NAPQI of APAP is when sulfate and glucuronide conjugation pathway become saturated (Lee et al., 2001; Jodynis-Liebert et al., 2005). N-acetyl-p-benzoquinoneimine (NAPQI) conjugates rapidly with glutathione (GSH) forming mercapturic acid which subsequently is excreted in the urine. However, excess formation of NAPQI, leads to GSH depletion which subsequently results in covalent binding of NAPQI to liver macromolecules leading to cellular necrosis. Therefore, GSH is an important sulfhydryl group that maintains cellular macromolecules in functional states, serves as a key determinant of the extent of APAP induced hepatic injury (Yanpallewar et al., 2002). Our results showed that a high dose of APAP has caused remarkably reduced level of cellular GSH. GSH is a powerful scavenger of these radicals which also causes oxidation of GSH to GSSG that further depletes GSH stores. It has been shown that preservation of GSH from being depleted provides direct protection against APAP induced hepatotoxicity (Ahmed and Khater, 2001). In the current study, GSH depletion was prevented when MO was administered before APAP. This indicates protection against APAP mediated liver toxicity. Similar action of MO in preserving GSH level was reported by Kumar and Pari (2003) following administration of antitubercular drugs such as isoniazid, rifampicin and pyrazinamide. Apart from that, another probable mechanism related to the restoration of GSH level maybe due to the decrease in the biotransformation of APAP via cytochrome P450 (CYP450) pathway, which inhibits toxic metabolites generation which leads to GSH depletion following MO pretreatment. Previous reports showed that com-
pounds that inhibit CYP450 pathway or promotion of glucoronidation and sulphation also exhibit hepatoprotective property against APAP induced hepatotoxicity (Wang et al., 1996; Lee et al., 2001). The present study does not investigate activity of CYP450 dependent enzymes involved in bioactivation of APAP and elimination of APAP via sulphation and glucoronidation. However, it is feasible that protective effects afforded by MO against APAP induced hepatotoxicity is due to its ability to induce phase II detoxification pathway via promoting GSH conjugation with toxic metabolites generated from CYP450 pathway. Silymarin has already been reported to improve the GSH level (Fraschini et al., 2002; Pari and Murugan, 2004). Comparing both compounds, MO has the potential as an alternative hepatoprotective agent in preventing APAP mediated GSH depletion. Besides, significant protection offered by MO extract at a dose of 200 mg/ kg suggesting lower concentration of MO is sufficient in exerting protective against APAP induced hepatotoxicity. The results obtained from this study prompt further investigation of the mechanism of hepatoprotective activities of MO and the role of bioactive components of the plant extract responsible for this action. Each part of the plant has been reported useful with various medicinal properties (Anwar et al., 2007) and nutritional values (Seshadri and Nambiar, 2003). Therefore, scientifically proven hepatoprotective activities of MO leaves may therefore, certainly provides benefits following human consumption. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements A special acknowledgement is owed to Dr. Abdah Md. Akim who has generously provided advice and improvements of methodology of enzymatic study. We would like to express our gratitude for tremendous help and contribution of staff in the Department of Nutrition and Dietetic, Department of Human Anatomy, Department of Biomedical Sciences and Laboratory of Molecular Biomedicine, Institute Bioscience, Universiti Putra Malaysia for the technical assistance and advice as well as material provision. References Ahmed, M.B., Khater, M.R., 2001. Evaluation of the protective potential of Ambrosia maritime extract on acetaminophen-induced liver damage. J. Ethnopharmacol. 75, 169–174. Anwar, F., Latif, S., Ashraf, M., Gilani, A.H., 2007. Moringa oleifera: a food plant with multiple medicinal uses. Phytother. Res. 21 (1), 17–25. Asha, V.V., Akhila, S., Wills, P.J., Subramoniam, A., 2004. Further studies on the antihepatotoxic activity of Phyllanthus maderaspatensis Linn. J. Ethnopharmacol. 92, 67–70. Chung, Y.H., Kim, J.A., Song, B.C., Song, I.H., Koh, M.S., Lee, H.S., Yu, E., Lee, Y.S., Su, D.J., 2001. Centrilobular hepatic necrosis; isocitrate dehydrogenase as a marker of centrilobular model of rats. J. Gastroenterol. Hepatol. 16, 328–332. Dhiman, R.K., Chawla, Y.K., 2005. Herbal medicines for liver diseases. Dig. Dis. Sci 50, 1807–1812. Ellman, G.L., 1959. Tissue sulphydryl groups. Arch. Biochem. Biophys. 82, 70. Ezeamuzle, I.C., Ambedederomo, A.W., Shode, F.O., Ekwebelem, S.C., 1996. Antiinflammatory effects of Moringa oleifera root extract. Int. J. Pharmacogn. 34, 207–212. Faizi, S., Siddiqui, B.S., Saleem, R., Aftab, K., Shaheen, F., Gilani, A.H., 1998. Hypotensive constituents from the pods of Moringa oleifera. Planta Med. 64 (3), 225–228. Fraschini, F., Demartini, G., Esposti, D., 2002. Pharmacology of silymarin. Clin. Drug Invest. 22, 51–65. Gaw, A., Cowan, R.A., O’Reilly, D.S.T., Stewart, M.J., Shephed, J.S., 1998. Clinical Biochemistry. Churchill Livingstone, Edinburg. Ghasi, S., Nwobodo, E., Ofili, J.O., 2000. Hypocholestrolemic effects of crude extract of leaf of Moringa oleifera Lam in high fat diet fed Wister rats. J. Ethnopharmacol 69, 21–25. Hossain, M.Z., Shibib, B.A., Rahman, R., 1992. Hypoglycemic effects of Coccinia indica: inhibition of key gluconeogenic enzyme, glucose-6-phosphatase. Indian J. Exp. Biol. 30 (5), 418–420.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Moringa oleifera Lam prevents acetaminophen induced liver injury through restoration of glutathione level S. Fakurazi a,*, I. Hairuszah b, U. Nanthini a a b
Pharmacology Unit, Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
a r t i c l e
i n f o
Article history: Received 22 January 2008 Accepted 11 April 2008
Keywords: Acetaminophen Paracetamol Glutathione Hepatoprotective Hepatotoxicity
a b s t r a c t Initiation of acetaminophen (APAP) toxicities is believed to be promoted by oxidative stress during the event of overdosage. The aim of the present study was to evaluate the hepatoprotective action of Moringa oleifera Lam (MO), an Asian plant of high medicinal value, against a single high dose of APAP. Groups of five male Sprague–Dawley rats were pre-administered with MO (200 and 800 mg/kg) prior to a single dose of APAP (3 g/kg body weight; p.o). Silymarin was used as an established hepatoprotective drug against APAP induced liver injury. The hepatoprotective activity of MO extract was observed following significant histopathological analysis and reduction of the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in groups pretreated with MO compared to those treated with APAP alone. Meanwhile, the level of glutathione (GSH) was found to be restored in MO-treated animals compared to the groups treated with APAP alone. These observations were comparable to the group pretreated with silymarin prior to APAP administration. Group that was treated with APAP alone exhibited high level of transaminases and ALP activities besides reduction in the GSH level. The histological hepatocellular deterioration was also evidenced. The results from the present study suggested that the leaves of MO can prevent hepatic injuries from APAP induced through preventing the decline of glutathione level. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Acetaminophen (APAP) is an effective and widely used antipyretic-analgesic drug with excellent safety record when taken at therapeutic doses (Larson, 2007). Despite extensive efforts to conduct studies on the processes of APAP induced toxicity, the exact mechanisms are still incompletely understood. Most evidence suggested the depletion of glutathione and the formation of reactive metabolites is somehow triggers the cascade events of hepatotoxicity (Jaeschke and Bajt, 2006). Overdose of APAP results in the generation of free radicals following the depletion of glutathione (Jaeschke and Bajt, 2006). Recently, the use of herbal natural product has gained interest among the world population. Many of the herbs have been developed into herbal supplement which are claimed to assist in healthy lifestyle. Among those herbs, is Moringa oleifera Lam (MO) which is native to South Asia and with high potential medicinal value. The leaves have been reported to have antihypercholesterolemic action (Ghasi et al., 2000) and those with other risk factor, such as hypertension (Faizi et al., 1998) or diabetes melli-
* Corresponding author. Tel.: +60 3 89472352; fax: +60 3 89242341. E-mail addresses: [email protected], [email protected] (S. Fakurazi). 0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.04.018
tus (Kar et al., 2003). Reports have also described the plant to be highly potent anti-inflammatory agent (Ezeamuzle et al., 1996) and antitumour activity (Murakami et al., 1998). The plant has also been reported to be hepatoprotective against antitubercular drug such as isoniazid and rifampicin (Pari and Kumar, 2002). Therefore, the objective of this study is to evaluate the possible protection of aqueous extract of MO against hepatotoxicity induced by APAP. The hepatoprotective potential of the MO crude extract as was compared with silymarin, a known and commercially available hepatoprotective agent.
2. Materials and methods 2.1. Preparation of plant materials The fresh leaves of M. oleifera were collected from Bandar Sunggala Farm, Port Dickson and authenticated by a plant taxonomist at Institute Bioscience (IBS), Universiti Putra Malaysia. A voucher specimen has been kept at herbarium IBS for future reference. The leaves were shaded dried for 3 weeks. The dried leaves were grounded into powder form and stored at 4 °C until further use. The leaves powder were then extracted using 80% hydroalcoholic solvent (80% ethanol:20% distilled water) with constant shaking at room temperature for overnight. The extract was filtered and the residue was then resuspended in ethanol for 48 hours and refiltered. The filtrate was then concentrated using a rotary evaporator (Rotavor R-200, Buchi, Switzerland) under reduced pressure at 40 °C and then lyophilized using a freeze dryer (Freeze Dry System, FreeZone 4.5, Labconco, USA; 40 °C,
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120 bar). A dark green mass was obtained and stored at 20 °C until further use. The crude extract, was resuspended in distilled water before administration to the animals (Hossain et al., 1992). 2.2. Experimental animals and study design Male Sprague–Dawley rats (200–250 g) were purchased from Faculty of Veterinary Medicine, Universiti Putra Malaysia were acclimatized under control laboratory conditions for a week with free access to standard food and water ad libitum. The animals were handled in accordance to the rules and regulations by the Animal Care Unit Committee, Universiti Putra Malaysia. The rats were randomly divided into groups each consisting of 5 rats. Treatment consisted of pretreatment phase of MO in distilled water followed by the second phase in which the animals were given 3 g/kg APAP in 40% sucrose buffer on day 15. The animals were then euthanized 24 h after APAP administration. Group 1 received pretreatment with distilled water for 14 days prior to a single dose of APAP (3 g/kg body weight; p.o) served as hepatotoxin control group. Groups 2 and 3 were administered with aqueous extract of MO (200 and 800 mg/kg body weight; p.o) for 14 days before being intoxicated with APAP. Group 4 was treated with 200 mg/kg silymarin in distilled water prior to APAP administration. Twenty-four hours after APAP administration the rats were fasted and then anaesthetized with diethyl ether. Blood was collected via cardiac puncture and the animals were then sacrificed. Livers were then excised, washed in ice-cold saline to remove blood and duly weighed. A section from the median lobe was preserved in 10% formal saline. The remaining liver was quickly frozen in dry ice and stored at 80 °C for further analysis. 2.3. Histopathological examination The median lobe of the liver from each group was removed and fixed in 10% formalin and embedded in the paraffin. Microtome sections of 3–4 lm thickness were prepared according to the standard procedure and stained with haematoxylin and eosin. Sections were then examined for pathological findings of such as centrilobular necrosis, fatty and lymphocytes infiltration. 2.4. Liver function tests The liver function tests (LFTs) including ALT, AST and ALP were analysed by kinetic method kits from Roche Diagnostics using a double beam spectrophotometer. 2.5. Determination of reduced glutathione The liver glutathione (GSH) determination was performed according to the method of Ellman (1959) using 5,5,-dithiobis-2-nitrobenzoic (DTNB) as substrate. The liver homogenate was then prepared using Ultra Turax where liver tissue was homogenized in 0.01 M Phosphate Buffer (pH 7.4) at 4 °C. The absorbance was read at 420 nm and the concentration of glutathione is calculated and expressed in lmol/g protein. 2.6. Statistical analysis Data was presented as mean ± SEM. Statistical analysis was performed using ANOVA Statistical Package for the Social Sciences (SPSS) version 13.0. p-Value less than 0.05 (p < 0.05) was considered to be statistically significant.
3. Results The serum activities of liver alanine aminotransferase (ALT), aspartate transferase (AST) and alkaline phosphatase (ALP) were shown in Table 1. Rats treated with 3 g/kg APAP has significant elevation of ALT, AST and ALP activities when compared to those
groups pretreated with MO. Treatment of MO for 14 days prior to APAP administration has provided a significant protection to the liver, preventing the elevation of liver enzymes activities. The activities of ALT and AST in groups that were pretreated with lower dose of MO (200 mg/kg) were found to be 56.47 ± 2.15 IU/L and 171.40 ± 8.75 IU/L, respectively. The activity of both enzymes was significantly lower compared to the group that was treated with APAP alone, 136.97 ± 4.19 and 440 ± 20.87 IU/L, respectively. The activity of ALP was also significantly reduced to 179.00 ± 6.46 IU/L compared to group that was not pretreated with MO (228.67 ± 13.82 IU/L). When the rats were treated with higher doses of MO (800 mg/ kg), the reduction in the activities of the liver enzymes were more pronounced compared to the group that was given APAP alone. The level of ALT, AST and ALP was found to be significantly reduced in group treated with MO where the level was found to be 48.62 ± 1.25, 174.43 ± 27.04 and 178.50 ± 13.07 IU/L, respectively. The reduction in the activities of the enzymes was comparable to the groups pretreated with standard herbal supplement available commercially, silymarin. The level of glutathione was reduced with APAP treatment at 1.56 ± 1.19 lmol/g. When the rats were pretreated with MO, both treatment doses (200 and 800 mg/kg) have significantly elevated the level of glutathione in the liver compared to group receiving APAP only, at 2.59 ± 1.60 and 2.65 ± 0.21 lmol/g, respectively. This result is comparable to the animals that were pretreated with silymarin where the glutathione level was restored (2.79 ± 0.39 lmol/g). Pretreatment of 200 mg/kg and 800 mg/kg MO have significantly (p < 0.05) protected the liver from hepatocellular damage due to APAP (Fig. 1). The hepatoprotective of the extract was comparable to the effect seen with silymarin pretreatment. Acetaminophen (APAP) at 3 g/kg showed significant (p < 0.05) hepatocellular damage including the presence of prominent microvesiculation, moderate infiltration of monocytes and neutrophils with mild scattered focal necrosis. These changes were clearly observed around the perivenular (PV) hepatocytes (Fig. 1). The pathological hallmark after APAP treatment, such as bridging necrosis was not observed in the liver sections obtained from rats that were pretreated with MO (200 and 800 mg/kg). M. oleifera (MO) has provided a significant (p < 0.05) protection against hepatocytes injury. The protective effect seen when rats were pretreated with MO was similar to the protective effect offered by silymarin. 4. Discussion A single high dose of APAP administration has significantly elevated the serum transaminases and ALP activities besides reducing the activity of glutathione. The results has showed a promising likelihood that M. oleifera extract has exhibited a hepatoprotective effect following a significant decrease in serum transaminases and ALP activities and by preventing the changes seen in the treatment
Table 1 The effect of Moringa oleifera Lam (MO) extract on alkaline phosphatase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) activities and glutathione level in rats administered with a single dose of acetaminophen (APAP) Groups 40% sucrose Distilled water + APAP MO + APAP Silymarin + APAP * **
Treatment mg/kg
ALT (IU/L)
AST (IU/L)
ALP (IU/L)
Glutathione (lmol/g)
3000 200 800 200
47.75 ± 3.68 136.97 ± 4.19* 56.47 ± 2.15** 48.62 ± 1.25** 64.33 ± 7.27**
121.22 ± 10.04 440.00 ± 20.87* 171.40 ± 8.75** 174.43 ± 27.04** 183.30 ± 21.10**
178.67 ± 5.28 228.67 ± 13.82 179.00 ± 6.46** 178.50 ± 13.07** 181.67 ± 10.82**
2.40 ± 0.03 1.56 ± 0.19* 2.59 ± 1.60** 2.65 ± 0.21** 2.79 ± 0.39**
p < 0.05 significantly different from group treated with 40% sucrose. p < 0.05 significantly different from group treated with distilled water and APAP.
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Fig. 1. Hepatoprotective action of Moringa oleifera Lam (MO) against acetaminophen (APAP) induced hepatotoxicity in rats. (a) Pretreatment of distilled water + APAP. The liver section reveals submassive inflammation with cells undergoing necrosis (?) around perivenular area (PV); (b) pretreatment of MO (200 mg/kg) + APAP; (c) pretreatment of MO (800 mg/kg) + APAP. (d) Pretreatment of silymarin (200 mg/kg) + APAP; (b), (c) and (d) show overall normal liver architecture H & E Magnification 100.
with APAP. Interestingly, MO has been found to restore the activity of glutathione which was found diminished following APAP treatment. Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. It is selected as hepatotoxicant in inducing injury to the liver as it is known to cause hepatotoxicity in man and experimental animals when taken overdose which leads to elevation of liver enzymes (Kumar et al., 2004; Ahmed and Khater, 2001). Leakage of cellular enzymes into plasma indicates a hallmark sign of hepatic injury or damage (Ramaiah, 2007; Kumar et al., 2004). In addition, the extent and type of liver injury or damage can be assessed based on the presence or absence of specific enzymes in the bloodstream (Kumar et al., 2004). Generally, measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) are commonly used as marker enzymes in assessing APAP induced hepatotoxicity (Yanpallewar et al., 2002; Asha et al., 2004; Yen et al., 2007). Aminotransferases, are group of enzymes that catalyze reversible transfer of the amino acid group from an a-amino acid to an oxo acid. These enzymes are not normal components of plasma and its function outside organ of origin is unknown (James et al., 2003). The largest pool of ALT is found in cytosol of hepatic parenchymal cells (Okuda, 1997). Whereas, AST is found in cytosol and mitochondria of hepatocytes and also found in cardiac muscle, skeletal muscle, pancreas and kidney (Shyamal et al., 2006). Therefore, measurement of ALT is more liver specific to determine hepatocellular damage (Shyamal et al., 2006). Nevertheless, AST is still being commonly used as a laboratory test to assess liver function since it is considered to be a sensitive indicator of mitochondria damage particularly in the centrilobular regions of liver (Pantegh-
ini, 1990). Meanwhile, ALP comprises of a family of enzymes that hydrolyze phosphate esters at alkaline pH and is often used as a marker for cholestatic liver dysfunction. However, liver is not the sole source of ALP. Substantial amount are also present in the bone, placenta, kidney and intestine (Gaw et al., 1998). In this study, hepatoprotective effect of MO is evident by the restoration of ALT, AST and ALP. Concurrently, significant preservation of liver histology was observed in the groups that were pretreated with MO. Pretreatment with MO extract suppresses APAP induced AST and ALT elevations. Recovery towards normalization of the enzymes following MO pretreatment suggested that the plant extract have some roles in preserving structural integrity of hepatocellular membrane, thus prevented enzymes leakage into the blood circulation. Our results are consistent with generally accepted hypothesis that transaminase level return to normal with healing of hepatic parenchyma and the regeneration of hepatocytes (Ahmed and Khater, 2001). Previous study has reported that the root and flower of MO have prevented APAP induced ALT, AST and ALP elevation in rats and mice (Ruckmani et al., 1998). Meanwhile, another study has also showed that MO leaves extract has significantly restored the elevation of ALT and AST as well as ALP level that was induced by isoniazid to the normal level (Pari and Kumar, 2002). Recent study by Nadro et al. (2005) has demonstrated that MO leaves extract has prevented the release of these enzymes from hepatocytes into the bloodstream when induced with high level of ethanol administration in rats. Our results are consistent with the previous studies, which strongly suggest that MO may preserve the structural integrity of hepatocytes when challenge with hepatotoxicants and subsequently preventing enzyme leakage into plasma. The results
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have also showed that the plant extract is equally effective to silymarin in preventing the increase in liver enzymes when challenged with hepatotoxic dose of APAP. Silymarin is a well-established hepatoprotective drug reported to revert abnormal alterations of these enzymes in various drug induced hepatotoxicity (Upadhyay et al., 2007; Pradhan and Girish, 2006). The ability of silymarin in preventing drug induced hepatotoxicity is associated with its ability to act as a radical scavenger, thereby protecting membrane permeability (Song et al., 2006). Silymarin is used as a standard agent for comparing hepatoprotective effects of plant principle due to its proven hepatoprotective property (Dhiman and Chawla, 2005). The benefits of MO are then further confirmed by histopathalogical observations. It was well-established that overdoses of APAP lead to centrilobular necrosis, fatty infiltration, lymphocytic as well neutrophil infiltration. (Yen et al., 2007). In the present study, histological sections of the liver showed similar reported changes including centrilobular necrosis with inflammatory cell infiltration in rats treated with APAP alone. The cytochrome P450 system is abundantly distributed in Zone 3-centrilobular (Z3). The predominant distribution of CYP450 at Z3 inflicts localized production of toxic reactive metabolites of various drugs including APAP such as N-acetyl-p-benzoquinoneimine (NAPQI). This in turn induces hepatic necrosis around the centrilobular region (Chung et al., 2001). Our study is consistent with the reported observation, in which prominent centrilobular necrosis was observed around this area (Z3). Centrilobular necrosis, the hallmark feature of APAP induced liver damage was significantly reduced with MO pretreatment (Oliveira et al., 2005). The congestion and inflammatory cell infiltration evoked by APAP was considerably decreased following extract pretreatment. The possibility of MO extract 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. Furthermore, the ability of MO to reverse the hepatic lesions induced by the APAP is comparable to the treatment with sucrose and the silymarin, has strongly suggested the hepatoprotective effect of MO. The formation of toxic reactive metabolite of NAPQI of APAP is when sulfate and glucuronide conjugation pathway become saturated (Lee et al., 2001; Jodynis-Liebert et al., 2005). N-acetyl-p-benzoquinoneimine (NAPQI) conjugates rapidly with glutathione (GSH) forming mercapturic acid which subsequently is excreted in the urine. However, excess formation of NAPQI, leads to GSH depletion which subsequently results in covalent binding of NAPQI to liver macromolecules leading to cellular necrosis. Therefore, GSH is an important sulfhydryl group that maintains cellular macromolecules in functional states, serves as a key determinant of the extent of APAP induced hepatic injury (Yanpallewar et al., 2002). Our results showed that a high dose of APAP has caused remarkably reduced level of cellular GSH. GSH is a powerful scavenger of these radicals which also causes oxidation of GSH to GSSG that further depletes GSH stores. It has been shown that preservation of GSH from being depleted provides direct protection against APAP induced hepatotoxicity (Ahmed and Khater, 2001). In the current study, GSH depletion was prevented when MO was administered before APAP. This indicates protection against APAP mediated liver toxicity. Similar action of MO in preserving GSH level was reported by Kumar and Pari (2003) following administration of antitubercular drugs such as isoniazid, rifampicin and pyrazinamide. Apart from that, another probable mechanism related to the restoration of GSH level maybe due to the decrease in the biotransformation of APAP via cytochrome P450 (CYP450) pathway, which inhibits toxic metabolites generation which leads to GSH depletion following MO pretreatment. Previous reports showed that com-
pounds that inhibit CYP450 pathway or promotion of glucoronidation and sulphation also exhibit hepatoprotective property against APAP induced hepatotoxicity (Wang et al., 1996; Lee et al., 2001). The present study does not investigate activity of CYP450 dependent enzymes involved in bioactivation of APAP and elimination of APAP via sulphation and glucoronidation. However, it is feasible that protective effects afforded by MO against APAP induced hepatotoxicity is due to its ability to induce phase II detoxification pathway via promoting GSH conjugation with toxic metabolites generated from CYP450 pathway. Silymarin has already been reported to improve the GSH level (Fraschini et al., 2002; Pari and Murugan, 2004). Comparing both compounds, MO has the potential as an alternative hepatoprotective agent in preventing APAP mediated GSH depletion. Besides, significant protection offered by MO extract at a dose of 200 mg/ kg suggesting lower concentration of MO is sufficient in exerting protective against APAP induced hepatotoxicity. The results obtained from this study prompt further investigation of the mechanism of hepatoprotective activities of MO and the role of bioactive components of the plant extract responsible for this action. Each part of the plant has been reported useful with various medicinal properties (Anwar et al., 2007) and nutritional values (Seshadri and Nambiar, 2003). Therefore, scientifically proven hepatoprotective activities of MO leaves may therefore, certainly provides benefits following human consumption. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements A special acknowledgement is owed to Dr. Abdah Md. Akim who has generously provided advice and improvements of methodology of enzymatic study. We would like to express our gratitude for tremendous help and contribution of staff in the Department of Nutrition and Dietetic, Department of Human Anatomy, Department of Biomedical Sciences and Laboratory of Molecular Biomedicine, Institute Bioscience, Universiti Putra Malaysia for the technical assistance and advice as well as material provision. References Ahmed, M.B., Khater, M.R., 2001. Evaluation of the protective potential of Ambrosia maritime extract on acetaminophen-induced liver damage. J. Ethnopharmacol. 75, 169–174. Anwar, F., Latif, S., Ashraf, M., Gilani, A.H., 2007. Moringa oleifera: a food plant with multiple medicinal uses. Phytother. Res. 21 (1), 17–25. Asha, V.V., Akhila, S., Wills, P.J., Subramoniam, A., 2004. Further studies on the antihepatotoxic activity of Phyllanthus maderaspatensis Linn. J. Ethnopharmacol. 92, 67–70. Chung, Y.H., Kim, J.A., Song, B.C., Song, I.H., Koh, M.S., Lee, H.S., Yu, E., Lee, Y.S., Su, D.J., 2001. Centrilobular hepatic necrosis; isocitrate dehydrogenase as a marker of centrilobular model of rats. J. Gastroenterol. Hepatol. 16, 328–332. Dhiman, R.K., Chawla, Y.K., 2005. Herbal medicines for liver diseases. Dig. Dis. Sci 50, 1807–1812. Ellman, G.L., 1959. Tissue sulphydryl groups. Arch. Biochem. Biophys. 82, 70. Ezeamuzle, I.C., Ambedederomo, A.W., Shode, F.O., Ekwebelem, S.C., 1996. Antiinflammatory effects of Moringa oleifera root extract. Int. J. Pharmacogn. 34, 207–212. Faizi, S., Siddiqui, B.S., Saleem, R., Aftab, K., Shaheen, F., Gilani, A.H., 1998. Hypotensive constituents from the pods of Moringa oleifera. Planta Med. 64 (3), 225–228. Fraschini, F., Demartini, G., Esposti, D., 2002. Pharmacology of silymarin. Clin. Drug Invest. 22, 51–65. Gaw, A., Cowan, R.A., O’Reilly, D.S.T., Stewart, M.J., Shephed, J.S., 1998. Clinical Biochemistry. Churchill Livingstone, Edinburg. Ghasi, S., Nwobodo, E., Ofili, J.O., 2000. Hypocholestrolemic effects of crude extract of leaf of Moringa oleifera Lam in high fat diet fed Wister rats. J. Ethnopharmacol 69, 21–25. Hossain, M.Z., Shibib, B.A., Rahman, R., 1992. Hypoglycemic effects of Coccinia indica: inhibition of key gluconeogenic enzyme, glucose-6-phosphatase. Indian J. Exp. Biol. 30 (5), 418–420.
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