http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–10 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.957783
ORIGINAL ARTICLE
Hepatoprotective effects of Portulaca oleracea extract against CCl4-induced damage in rats Akram Eidi1, Pejman Mortazavi2, Jalal Zarringhalam Moghadam3, and Parisa Mousavi Mardani1
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
1
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran, 2Department of Pathology, Faculty of Specialized Veterinary Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran, and 3Department of Physiology, Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Abstract
Keywords
Context: Purslane (Portulaca oleracea L., Portulacaceae) has been traditionally used in folk medicine to afford protection against liver injury, although its actual efficacy remains uncertain. Objective: To evaluate purslane as a hepatoprotective agent, we investigated the protective effect of its ethanol extract against carbon tetrachloride (CCl4)-induced hepatic toxicity in rats. Materials and methods: A total of 108 male Wistar rats were randomly divided into 12 groups. The first group was maintained as normal control, whereas CCl4 (0.5 ml/kg bw, 50% CCl4 in olive oil, i.p.), purslane extract (0.005, 0.01, 0.05, 0.1, and 0.15 g/kg bw, intragastrically), and purslane extract (five doses as above) along with CCl4 were administered to the Groups II, III–VII, and VIII– XII, respectively. The rats were sacrificed on the 30th day, and blood was withdrawn by cardiac puncture. Liver damage was assessed by measuring hepatic marker enzymes (ALT, AST, ALP, GGT, and SOD) and histopathological observation. Results: Treatment with CCl4 resulted in increased serum activities of marker enzymes with a concomitant decrease in SOD. Histological alterations were also observed in the liver tissue upon CCl4 treatment. Administration of purslane extract (0.01, 0.05, 0.1, and 0.15 g/kg b.w.) significantly showed a marked tendency towards normalization of all measured biochemical parameters in CCl4-treated rats. Histopathological changes also paralleled the detected alteration in markers of liver function. Discussion and conclusion: These results demonstrate that purslane exerts protective effects against CCl4-induced damage in rat liver and supports a potential therapeutic use of purslane as an alternative for patients with liver diseases.
Carbon tetrachloride, detoxification, liver, purslane
Introduction Common purslane [Portulaca oleracea L., Portulacaceae] is a widespread weed, and has been ranked the eighth most common plants in the world (Liu et al., 2000). The genus name Portulaca originates from the Latin word ‘‘Portare,’’ which means ‘‘to carry,’’ and ‘‘lac,’’ which means ‘‘milk,’’ referring to the milky sap of this plant. The species name oleracea is also of Latin origin, and means ‘‘pertaining to kitchen gardens,’’ referring to its use as a vegetable (Boulos & El-Hadidi, 1984). It has long been used as human food, animal feed, and a folk medicine in many countries as a diuretic, febrifuge, antiseptic, antispasmodic, and vermifuge agent. It exhibits a wide range of pharmacological effects, including ameliorating cognitive deficits (Wang & Yang, 2010), antibacterial (Zhang et al., 2002), hypolipidemic, antiaging, analgesic, anti-inflammatory (Chan et al., 2000), antioxidative (Dkhil et al., 2011), skeletal muscle relaxant (Parry et al., 1987, 1993), wound healing (Rashed et al., Correspondence: Akram Eidi, Department of Biology, Science and Research Branch, Islamic Azad University, P.O. Box 16535-446, Tehran, Iran. Tel: +98 21 44865939. Fax: +9821 44865939. E-mail: eidi@ srbiau.ac.ir, [email protected]
History Received 11 November 2013 Accepted 5 August 2014 Published online 4 December 2014
2003), and in vitro antitumor (Yoon et al., 1999) activities. It has been reported to be rich in a-linolenic acid and b-carotene (Liu et al., 2000). Flavonoids, coumarins (Awad, 1994), a monoterpene glycoside (Sakai et al., 1996), and alkaloids have been reported to be important chemical constituents of this plant. The liver, a major site of metabolism and detoxification, plays a central role in the kinetics of absorption, distribution, and elimination of most drugs as well as several active or inactive metabolites. Hepatotoxic agents react with basic cellular components, and consequently, induce almost all types of liver lesions. Toxins and drugs are among the basic etiopathogenic agents of acute liver failure in several countries (Grattagliano et al., 2009); most adverse drug reactions, therefore, involve liver toxicity. It is not surprising that protection of liver against toxic injury remains a major challenge for clinical therapy. Chemical toxins such as acetaminophen, ethanol, carbon tetrachloride (CCl4), galactosamine, nitrosodiethylamine, and thioacetamide are often used as model substances to induce experimental hepatocyte injury under both in vivo and in vitro conditions (Domenicali et al., 2009; Kucera et al., 2006; Ledda-Columbano et al., 1991; Nwozo et al., 2014; Rousar et al., 2009; Sharma et al.,
2
A. Eidi et al.
2012; Song et al., 2013). Hepatic problems are responsible for a significant number of liver transplantations and deaths worldwide; however, pharmacotherapeutic options for liver diseases are very limited, resulting in a great demand for the development of new and effective drugs. Numerous studies have shown that plant extracts having antioxidant activities protect against CCl4-induced hepatotoxicity by inhibiting lipid peroxidation and enhancing antioxidant enzyme activity (Dhiman et al., 2012; Duh et al., 2011; Kuo et al., 2010; Quan et al., 2011; Shahjahan et al., 2004; Sheweita et al., 2001; Vuda et al., 2012; Yang et al., 2011). The present study investigates the hepatoprotective potential of purslane ethanolic extract against CCl4-induced liver toxicity in rats.
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
Materials and methods Chemicals CCl4 was obtained from Merck, Darmstadt, Germany. The kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were purchased from Parsazmoon Co., Tehran, Iran. The kits for g-glutamyltransferase (GGT) and superoxide dismutase (SOD) were obtained from Bionik, Lainate Milano, Italy, and Randox Co., England, UK, respectively. All other reagents used were of analytical grade. Preparation of plant extract The aerial parts of purslane were obtained from the local market (Voucher number: 052245), and identified in the Islamic Azad University herbarium. The aerial parts were dried at 25 C, and finely powdered. The powder (60 g) was extracted with 300 ml of aqueous ethanol (80% v/v) in a Soxhlet apparatus for 72 h. After extraction, the solvent was filtered and evaporated using a Rotavapor. The purslane ethanolic extract so obtained was stored at 20 C until further use. Phytochemical screening Phytochemical analysis using standard procedures was carried out to determine the active chemical constituents of purslane, such as tannins, terpenoids, alkaloids, saponins, and flavonoids. The extract was treated with dilute hydrochloric acid and filtered. The filtrate was used for the following tests. The extract (20 mg) was dissolved in 10 ml of ethanol and filtered. Concentrated HCl (0.5 ml) and magnesium ribbon were added to 2 ml of the filtrate. Development of pinktomato red color indicated the presence of flavonoids. For the detection of tannins, 20 mg of the purslane extract was dissolved in 2 ml of distilled water and filtered. FeCl3 (2 ml) was added to the filtrate; the development of a blue-black precipitate indicated the presence of tannins. For the detection of alkaloids, 20-mg extract was dissolved in 2 ml of distilled water and filtered. Steam was passed through the filtrate after addition of 2–4 drops of 1% HCl. To 1 ml of this solution, six drops of Wagner’s reagent were added; a brownish-red precipitate indicated the presence of alkaloids (Finar, 2003). Saponins were detected by adding 5 ml of distilled water to 0.5 ml of the filtrate obtained in the test for alkaloids. A persistent frothing indicated the presence of saponins (Parekh et al., 2006). Salkovski test was performed using a
Pharm Biol, Early Online: 1–10
small amount of extract solution. Five drops of conc. H2SO4 and 1 ml of chloroform were added to this solution. A change of color from yellow to red indicated the presence of terpenoids (Finar, 2003). Animals Male Wistar rats weighing 200–250 g were used. The animals were housed in groups of five per cage, with free access to standard laboratory chow (35% carbohydrates, 25% proteins, 7% lipids, and 3% vitamins) and tap water. The food was purchased from Pars-Dam food service, Tehran, Iran. The animals were housed in large, clean, polypropylene cages in a temperature-controlled room (22 ± 2 C) with 40–60% relative humidity under 12 h light and dark cycles for 1 week prior to and during the experiments. The study was conducted in accordance with ethical procedures and policies approved by the Animal Care and Use Committee of Islamic Azad University, Tehran, Iran. Experimental design A total of 108 rats were divided into 12 groups of nine each. Group I served as a normal control group, and received distilled water (intragastric route; daily) and olive oil (0.5 ml/kg bw; twice a week, i.p.) as vehicles for a period of 30 d. Group II served as the toxicity control group, and received distilled water (intragastrically; daily) and CCl4 (0.5 ml/kg bw, 50% CCl4 in olive oil; twice a week, i.p.) for a period of 30 d. Groups III–VII served as the purslane extract control group, and received purslane extract dissolved in distilled water (intragastrically; daily) at doses of 0.005, 0.01, 0.05, 0.1, and 0.15 g/kg bw, respectively, with olive oil (0.5 ml/kg bw; twice a week, i.p.) for a period of 30 d. Groups VIII–XII were the treatment groups, and received purslane extract dissolved in distilled water (intragastrically; daily) at doses of 0.005, 0.01, 0.05, 0.1, and 0.15 g/kg bw, with CCl4 (0.5 ml/kg bw, 50% CCl4 in olive oil; twice a week, i.p.) for 30 d. The dose of purslane ethanolic extract was selected based on previous published reports (Cheng-Jie et al., 2009; El-Sayed, 2011). At the end of the treatment period, the rats were anesthetized by ether inhalation 48 h after the administration of CCl4. Blood samples were collected from the heart and allowed to clot for 30 min. Serum was separated by centrifugation at 2500 rpm for 15 min and used for biochemical estimations. The absolute and relative (organ-to-body weight ratio) weights of the liver were also measured for all the rats when they were sacrificed. Measurement of serum biochemical parameters The activities of serum AST, ALT, ALP, and GGT were determined using the auto-analyzer (Shimadzu CL-7200, Shimadzu, Japan) (Reitman & Frankel, 1957). Measurement of hepatic SOD activity Following anesthesia, rat livers were quickly excised and perfused with chilled saline for the complete removal of
Hepatoprotective effects of Portulaca oleracea L. extract
DOI: 10.3109/13880209.2014.957783
blood cells. They were then cut into small pieces, placed in 50 mM phosphate buffer (pH 7.4), and homogenized using a glass-Teflon homogenizer to obtain 1:9 (w/v) whole tissue homogenate. The homogenate was centrifuged at 3000 rpm for 15 min at 4 C. The supernatant was collected and transferred to an Eppendorf tube, and was centrifuged at 12 000 rpm for 30 min to eliminate cell debris. The supernatant so obtained was used for the determination of SOD activity. Xanthine and xanthine oxidase were used for the generation of superoxide radicals, which subsequently reacted with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride to form a red formazan dye. SOD activity was measured by the degree of inhibition of this reaction (Sun et al., 1988).
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
Histopathology Liver tissues, previously sectioned to 2 mm thickness, were fixed in 10% buffered formaldehyde for 24 h. The fixed tissues were embedded in paraffin, sectioned to 4–5 mm thickness, and rehydrated. The liver sections were stained with hematoxylin and eosin (H&E) stain, and histologically examined using conventional methods for assessing morphological changes in order to evaluate the index of CCl4-induced necrosis. Pathological assessment of the sections, such as fatty degeneration, necrosis, cell swelling, and lymphocyte infiltration, was also done for evaluating hepatotoxicity (Wills & Asha, 2006). Liver pathology was scored as described previously (French et al., 2000), as follows: Score 0 ¼ no visible cell damage. Score 1 ¼ focal hepatocyte damage in less than 25% of the tissue. Score 2 ¼ focal hepatocyte damage in 25–50% of the tissue. Score 3 ¼ extensive, but focal, hepatocyte lesions. Score 4 ¼ global hepatocyte necrosis. The morphology of observed lesions was classified and registered (Gray, 1958). Statistical analyses Results were expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by multiple comparisons using Tukey’s post-hoc test was used for the comparison of different parameters between the groups. A p value of50.05 was considered statistically significant.
Results Phytochemical analysis A percentage yield of 10.6% (w/w) was obtained for the extract from purslane aerial parts. Phytochemical analysis indicated the presence of flavonoids, tannins, saponins, terpenoids, and alkaloids in the plant extract (Table 1). Table 1. Phytochemical constituents of Portulaca oleracea. Extract Ethanolic
Phytochemical tests Flavonoids +
Tannins +
Saponins +
Terpenoids +
+: Presence of phytochemical; : absence of phytochemical.
Alkaloids +
3
Effects of purslane ethanolic extract administration on the body and relative liver weights of CCl4-treated rats The results of administration of purslane ethanolic extract on the body and relative liver weights of rats from each group are shown in Table 2. All the rats survived the experimental period of 30 d, till they were sacrificed. Daily observations over the experimental period showed no detectable alterations in the general states of the animals of all groups. Body weights of the rats significantly decreased in the CCl4-treated group, but increased in the normal control and purslane extract-treated groups. In contrast, relative liver weights significantly increased among rats of the CCl4-treated group. The administration of purslane extract reversed the increase in liver weight among the CCl4-treated rats. Effects of purslane ethanolic extract administration on biochemical parameters The hepatoprotective effects exerted by purslane ethanolic extract in CCl4-treated rats, as assessed by serum biochemical parameters, are shown in Figures 1 and 2. Rats treated with CCl4 (Group II) showed significantly higher serum AST, ALT, ALP, and GGT activities compared with normal control animals (Group I). Co-administration of purslane ethanolic extract (at doses of 0.01, 0.05, 0.1, and 0.15 g/kg bw) and CCl4 (Groups IX–XII) for 30 d resulted in significant hepatic protection, as measured by serum AST, ALT, ALP, and GGT activities, compared with the toxicity control group (Group II). SOD activity in CCl4-treated rats decreased drastically compared with the normal control group. Upon treatment with purslane ethanolic extract at doses of 0.01, 0.05, 0.1, and 0.15 g/kg bw (Groups IX–XII) for 30 d, this decrease in SOD activity (brought about by CCl4 treatment) showed recovery towards normalization in a purslane-extract dose-dependent manner. Therefore, treatment with purslane ethanolic extract showed a marked tendency towards normalization of all measured biochemical parameters in CCl4treated rats. Histopathological evaluation The liver lobules of the normal control and purslane extract control groups (Groups I and III–VII, respectively) showed a classical structure, with hepatocyte plates directed from the portal triads toward the central vein, where they freely anastomosed. Irregularly dilated liver sinusoids and space of Disse could also be observed. In contrast, liver sections from the rats treated with CCl4 alone (Group II) showed massive changes throughout the lobules, including fatty degeneration, cell swelling, necrosis, and infiltration of mononuclear inflammatory cells (lymphocyte infiltration) in the portal triads. However, upon co-treatment of the rats with CCl4 and purslane ethanolic extract at dose levels of 0.01, 0.05, 0.1, and 0.15 g/kg bw (Groups IX–XII), milder hepatocellular degeneration and better preservation of normal liver architecture could be observed, with moderate hepatocyte plate disorganizations and necrosis or inflammation. Moreover, animals in Groups XI and XII, which received the highest doses of purslane ethanolic extract, showed minimal lesions, which
4
A. Eidi et al.
Pharm Biol, Early Online: 1–10
Table 2. Effect of purslane ethanolic extract on body weight, liver weight, and liver index in normal and intoxicated rats.
Groups
Initial body weight (g)
Final body weight (g)
Weight gain (g)
Liver weight (g)
Liver weight/ Body weight 100
210 ± 7.5 215 ± 8.4
245 ± 8.5 218 ± 9.2***
35 ± 3.1 3 ± 0.6***
3.8 ± 0.4 5.2 ± 0.5***
1.55 ± 0.03 2.38 ± 0.12***
211 ± 9.3 216 ± 7.6 208 ± 6.9 214 ± 5.3 210 ± 6.8
248 ± 11.4 255 ± 10.7 247 ± 9.8 255 ± 11.7 250 ± 10.5
37 ± 3.6 39 ± 3.7 39 ± 3.5 41 ± 4.2 40 ± 4.1
3.7 ± 0.3 3.6 ± 0.4 3.5 ± 0.3 3.8 ± 0.5 3.6 ± 0.3
1.49 ± 0.04 1.41 ± 0.04 1.42 ± 0.04 1.49± 0.03 1.44 ± 0.04
216 ± 7.9 211 ± 4.8 209 ± 6.2 214 ± 5.1 217 ± 6.5
229 ± 8.6 226 ± 9.4 228 ± 9.1 237 ± 10.5+++ 242 ± 9.8+++
13 ± 1.2+ 15 ± 1.4++ 19 ± 2.1+++ 23 ± 2.4+++ 25 ± 2.8+++
4.6 ± 0.5 4.5 ± 0.6+ 4.3 ± 0.4+ 4.1 ± 0.3++ 4.1 ± 0.3++
2.01 ± 0.08 1.99 ± 0.07+ 1.89 ± 0.06+ 1.73 ± 0.05++ 1.69 ± 0.05+++
Values are mean ± S.E.M of nine rats. ***p50.001 significantly different from the normal control group. +p50.05 significantly different from the group treated with CCl4. ++p50.01 significantly different from the group treated with CCl4. +++p50.001 significantly different from the group treated with CCl4. (A) 250
***
Serum ALT (IU/L)
200
CCl4 +
150
+ 100
++ +++ +++
50
0 Normal CCI4
0.005
0.01
0.05
0.1
0.15
0.005
0.01
0.05
0.1
0.15
+++
+++
0.1
0.15
Purslane ethanolic extract (g/kg) (B) 300
*** CCl4 + Serum AST (IU/L)
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
Normal control CCl4 Purslane extract (g/kg) 0.005 0.01 0.05 0.1 0.15 Purslane extract (g/kg) + CCl4 0.005 0.01 0.05 0.1 0.15
200
++
++
0.01
0.05
100
0 Normal CCI4
0.005
0.01
0.05
0.1
0.15
0.005
Purslane ethanolic extract (g/kg)
Figure 1. Effect of oral administration of purslane extract at doses of 0.005, 0.01, 0.05, 0.1, and 0.15 g/kg bw on the activities of ALT (A), AST (B), ALP (C), and GGT (D) in the serum of CCl4-treated rats. Each column represents mean ± S.E.M. of data from nine rats. Normal control group was administered distilled water as a vehicle. The symbol ***represents p50.001 compared with the normal control group, whereas +, ++, and +++ represent p50.05, p50.01, and p50.001, respectively, compared with the CCl4-treated group.
Hepatoprotective effects of Portulaca oleracea L. extract
DOI: 10.3109/13880209.2014.957783
***
(C) 600
CCl4 +
Serum ALP (IU/L)
500
400
+ ++
300
+++
+++
0.1
0.15
200
100
Normal CCI4
0.005
0.01
0.05
0.1
0.15
0.005
0.01
0.05
Purslane ethanolic extract (g/kg)
(D) 12
***
10
Serum GGT (IU/L)
CCl4 +
8
++
6
+++ +++
4
2
0 Normal CCI4
0.005
0.01
0.05
0.1
0.15
0.005
0.01
0.05
0.1
0.15
Purslane ethanolic extract (g/kg)
Figure 1. Continued.
Figure 2. Effect of oral administration of purslane extract at doses of 0.005, 0.01, 0.05, 0.1, and 0.15 g/kg bw on SOD activity in the liver of CCl4-treated rats. Each column represents mean ± S.E.M. of data from nine rats. Normal control group was administered distilled water as a vehicle. The symbol ***represents p50.001 compared with the normal control group, whereas + and ++ represent p50.05 and p50.01 compared with the CCl4-treated group.
20
15
SOD (IU/mg protein)
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
0
CCl4 + 10
+
+
++
++
0.01
0.05
0.1
0.15
*** 5
0
Normal CCI4 0.005
0.01
0.05
0.1
0.15 0.005
Purslane ethanolic extract (g/kg)
5
6
A. Eidi et al.
Pharm Biol, Early Online: 1–10
Table 3. Histological injury score of liver under different doses of purslane ethanolic extract in rats treated with CCl4 (50% CCl4/olive oil). Injury of score Groups
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
Control CCl4 Purslane extract (g/kg) 0.005 0.01 0.05 0.1 0.15 Purslane extract (g/kg)+ CCl4 0.005 0.01 0.05 0.1 0.15
a
Fatty degeneration
Necrosis
Cell swelling
Inflammation
0.0 ± 0.00 3.4 ± 0.24***
0.0 ± 0.00 3.6 ± 0.24***
0.0 ± 0.00 3.6 ± 0.24***
0.0 ± 0.00 3.4 ± 0.24***
0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00
0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00
0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00
0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00
2.4 ± 0.24+ 2.2 ± 0.20+ 1.8 ± 0.20+++ 1.2 ± 0.20+++ 1.0 ± 0.32+++
2.4 ± 0.24++ 1.6 ± 0.24+++ 1.4 ± 0.24+++ 1.2 ± 0.20+++ 0.8 ± 0.20+++
3.2 ± 0.37 2.4 ± 0.24+ 2.2 ± 0.20++ 1.4 ± 0.24+++ 1.2 ± 0.20+++
2.2 ± 0.20+ 1.4 ± 0.24+++ 1.2 ± 0.20+++ 1.0 ± 0.31+++ 0.8 ± 0.20+++
a
Livers were scored for hepatic injury via light microscopy with score 0 ¼ no visible cell damage; score 1 ¼ focal hepatocyte damage on less than 25% of the tissue; score 2 ¼ focal hepatocyte damage on 25–50% of the tissue; score 3 ¼ extensive, but focal, hepatocyte lesions; score 4 ¼ global hepatocyte necrosis. ***p50.001 significantly different from the normal control group. +p50.05 significantly different from the group treated with CCl4. ++p50.01 significantly different from the group treated with CCl4. +++p50.001 significantly different from the group treated with CCl4.
included cell swelling towards the central vein, fatty degeneration, and necrosis (Table 3 and Figure 3).
Discussion CCl4-induced liver injury is the best-characterized model of xenobiotic-induced hepatotoxicity, and is commonly employed for screening antihepatotoxic/hepatoprotective activities of drugs (Brautbar & Williams, 2002; Brent & Rumack, 1993). The bio-activation of CCl4, primarily through the activity of CYP2E1, generates the free radicals CCl3 and CCl3OO, which results in hepatic damage. These free radicals initiate lipid peroxidation by abstracting a hydrogen atom from the polyunsaturated fatty acid of a phospholipid (Recknagel et al., 1989; Weber et al., 2003). The CCl4induced lipid peroxidation in turn increases the permeability of plasma membrane to Ca2+, leading to severe disruption of calcium homeostasis and necrotic cell death (Weber et al., 2003). The extent of hepatic damage is assessed by the increase in serum levels of the cytoplasmic enzymes AST, ALT, ALP, and GGT, and by histopathological examination. The increased serum levels of AST and ALT have been attributed to damages in the structural integrity of the liver, as these cytoplasmic enzymes are released into circulation after cellular damage (Recknagel et al., 1989, 1991). Elevation of AST has been reported to be an index of hepatocellular injury in rats, whereas ALT elevation is more commonly associated with the necrotic state (Navarro & Senior, 2006). Serum ALP and GGT, which are important enzymes for assessing obstructive liver injury (Bulle et al., 1990; Kaplan, 1986), were also found to be significantly elevated in CCl4-treated rats. ALP activity is related to the functioning of hepatocytes. Suppression of increased ALP activity is indicative of the stabilization of biliary dysfunction in rat liver during chronic hepatic injury induced by CCl4 (Mukherjee, 2002).
The present study demonstrates the hepatoprotective effects of purslane ethanolic extract against CCl4-induced liver injury in rats. The serum levels of certain important biochemical parameters were employed as diagnostic markers of hepatic injury. Increased levels of ALT, AST, ALP, and GGT in the serum of CCl4-treated animals were indicative of liver damage, due to the leakage of these enzymes from the liver into the blood stream during tissue damage, which is always associated with hepatonecrosis (Naik & Panda, 2008; Ree & Spector, 1961). Upon treatment with purslane extract, the levels of these markers were restored to near normal or were only slightly elevated, indicating protection against liver damage. Such a restoration of increased serum levels of hepatic enzymes to the normal range reflects protection against hepatic damage caused by hepatotoxins (Vogel, 2002). The female sex hormone 17b-estradiol has been reported to increase the sensitivity of hepatic Kupffer cells to endotoxins in rats (Ikejima et al., 1998). Moreover, the process of liver detoxification may also be sexually dimorphic (Rando & Wahli, 2011). Therefore, male Wistar rats were used in the present study, because of fluctuations in the levels of sex hormones as a result of the menstrual cycle in female rats. Treatment of animals with purslane extract resulted in the preservation of the histological integrity of liver cells, which correlated with a significant reversal of the CCl4-induced increase in serum levels of hepatic enzymes. Inflammation plays a central role in drug-induced hepatitis, and leukotrienes, which are products of 5-lipoxygenase pathway of arachidonic acid metabolism, have been shown to be extensively involved in inflammatory processes (Perez-Alvarez et al., 1993). Consequently, the inhibitors of leukotriene synthesis provide partial protection against acute liver damage induced by different hepatotoxins, including CCl4 (Neichi et al., 1983).
Hepatoprotective effects of Portulaca oleracea L. extract
7
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
DOI: 10.3109/13880209.2014.957783
Figure 3. Histopathology of various treatment groups. (a) In the normal control group (Group I), normal liver structure with central vein (cv) and hepatocyte column (arrow) is seen. (b) Liver sections from the toxicity control group (Group II) showed massive changes throughout the lobules, with fatty degeneration (arrow head), infiltration of mononuclear inflammatory cells (I), and necrosis (arrow). (c-g) Animals treated with purslane ethanolic extract at doses of 0.005, 0.01, 0.05, 0.1, and 0.15 g/kg bw (Groups III–VII) showed normal liver structure, with central vein (cv), sinusoid (s), and hepatocytes (arrow). (h-l) Animals treated with purslane ethanolic extract at doses of 0.005, 0.01, 0.05, 0.1, and 0.15 g/kg bw along with CCl4 (Groups VIII–XII) showed mild to moderate lesions indicative of hepatocyte necrosis (arrow and N), fatty degeneration (arrow head), and lymphocyte infiltration (*). Note that the lesions are very mild in k and l (Groups XI and XII) panels. The sections were stained with H&E prior to observation.
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
8
A. Eidi et al.
Pharm Biol, Early Online: 1–10
Figure 3. Continued.
Reactive oxygen species (ROS), including superoxide anion, hydroxyl radical, and hydrogen peroxide, have a causal relationship with oxidative stress. Antioxidant enzymes such as SOD form a part of the protective response against oxidative tissue damage (Halliwell & Gutteridge, 1990). SOD represents the first line of defense against free radicals, and was demonstrated to possess ROS-metabolizing activity; it efficiently and specifically catalyzes dismutation of O 2 to O2 and H2O2. H2O2 is further metabolized to water by catalase. Maintaining the balance between ROS and antioxidant enzymes is therefore crucial, and could serve as a major mechanism for damage prevention by oxidative stress (Taniguchi et al., 2004). In the present study, a decrease was observed in SOD activity in the liver upon CCl4 administration, which could be attributed to oxidative inactivation of the enzyme upon excessive ROS generation. These results are consistent with previous studies (Lee et al., 2007; Ohta et al., 2004; Taniguchi et al., 2004). Treatment with purslane extract resulted in improved antioxidant activity of SOD compared with the CCl4-treated control group. These results confirm previous data on the antioxidative properties of purslane extract in other tissues, including heart, where it was shown to increase SOD activity (Caballero-Salazar et al., 2002; Hongxing et al., 2007). Purslane has been classically characterized as an antioxidant (Dkhil et al., 2011), and is involved in the direct detoxification of rotenones, mediated by two flavonols and flavones present in purslane (Cai et al., 2004; Oliveira et al., 2009). Purslane also effectively detoxifies superoxide, either by superoxide dismutation or by directly reacting with it (Siriamornpun & Suttajit, 2010).
Histopathological changes also paralleled the detected alteration in markers of liver function. Fatty degeneration, lymphocyte infiltration, and extensive hepatocellular necrosis were observed upon CCl4 treatment, which were markedly reduced upon co-treatment with purslane extract. In conclusion, the results obtained in the current study confirm the protective effect of purslane extract against CCl4induced hepatic damage in rats, which could be deduced not only from biochemical parameters in serum and liver but also from the histological observation of liver tissues. Further studies to characterize the active components of purslane extract and to elucidate its mechanism of action are in progress.
Declaration of interest We would like to thank Deputy of Research of the Science and Research Branch, Islamic Azad University for financial support of the project.
References Awad NE. (1994). Lipid content and antimicrobial activity of phenolic constituents of cultivated Portulaca oleracea L. Bull Fac Pharm Cairo Univ 32:137–42. Boulos L, El-Hadidi MN. (1984). The Weed Flora of Egypt. Cairo, Egypt: The American University in Cairo Press. Brautbar N, Williams J. (2002). Industrial solvents and liver toxicity: Risk assessment, risk factors and mechanisms. Int J Hyg Environ Health 205:479–91. Brent JA, Rumack BH. (1993). Role of free radicals in toxic hepatic injury. Clin Toxicol 31:173–96.
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
DOI: 10.3109/13880209.2014.957783
Bulle F, Mavier P, Zafrani ES, et al. (1990). Mechanism of gammaglutamyl transpeptidase release in serum during intrahepatic and extrahepatic cholestasis in the rat: A histochemical, biochemical and molecular approach. Hepatology 11:545–50. Caballero-Salazar S, Riveron-Negrete L, Ordaz-Tellez MG, et al. (2002). Evaluation of the antimutagenic activity of different vegetable extracts using an in vitro screening test. Proc West Pharmacol Soc 45:101–3. Cai Y, Luo Q, Sun M, Corke H. (2004). Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci 74:2157–84. Chan K, Islam MW, Kamil M, et al. (2000). The analgesic and antiinflammatory effects of Portulaca oleracea L. subsp. sativa (Haw.) Celak. J Ethnopharmacol 73:445–51. Cheng-Jie C, Wan-Yin W, Xiao-Li W, et al. (2009). Anti-hypoxic activity of the ethanol extract from Portulaca oleracea in mice. J Ethnopharmacol 124:246–50. Dhiman A, Nanda A, Akmad S. (2012). A recent update in research on the antihepatotoxic potential of medicinal plants. J Chin Integr Med 10:117–27. Dkhil MA, Abdel Moniem AE, Al-Quraishy S, Saleh RA. (2011). Antioxidant effect of purslane (Portulaca oleracea) and its mechanism of action. J Med Plants Res 5:1589–63. Domenicali M, Caraceni P, Giannone F, et al. (2009). A novel model of CCl4-induced cirrhosis with ascites in the mouse. J Hepatol 51: 991–9. Duh PD, Lin SL, Wu SC. (2011). Hepatoprotection of Graptopetalum paraguayense E. Walther on CCl4-induced liver damage and inflammation. J Ethnopharmacol 134:379–85. El-Sayed M-IK. (2011). Effects of Portulaca oleracea L. seeds in treatment of type-2 diabetes mellitus patients as adjunctive and alternative therapy. J Ethnopharmacol 137:643–51. Finar IL. (2003). Organic Chemistry. Stereochemistry and the Chemistry of Natural Products, vol. 2, 5th ed. Delhi: Pearson Education, 769–71. French SW, Miyamoto K, Ohta Y, Geoffrion Y. (2000). Pathogenesis of experimental alcoholic liver disease in the rat. Methods Achiev Exp Pathol 13:181–207. Grattagliano I, Bonfrate L, Catia VD, et al. (2009). Biochemical mechanisms in drug-induced liver injury. World J Gastroenterol 15: 4865–76. Gray P. (1958). Handbook of Basic Microtechnique. 3rd ed. New York: McGraw-Hill, 45–85. Halliwell B, Gutteridge JMC. (1990). Role of free radicals and catalytic metal irons in human disease: An overview. Methods Enzymol 186: 59–85. Hongxing Z, Nancai Y, Guofu H, et al. (2007). Neuroprotective effects of purslane herb aqueous extracts against D-galactose induced neurotoxicity. Chem-Biol Interact 170:145–52. Ikejima K, Enomoto N, Iimuro Y, et al. (1998). Estrogen increases sensitivity of hepatic Kupffer cells to endotoxin. Am J Physiol 274: G669–76. Kaplan MM. (1986). Serum alkaline phosphatase-another piece is added to the puzzle. Hepatology 6:526–8. Kucera O, Cervinkova Z, Lotkova H, et al. (2006). Protective effect of S-adenosylmethionine against galactosamine-induced injury of rat hepatocytes in primary culture. Physiol Res 55:551–60. Kuo DH, Kang WH, Shieh PC, et al. (2010). Protective effect of Pracparatum mungo extract on carbon tetrachloride-induced hepatotoxicity in rats. Food Chem 123:1007–12. Ledda-Columbano GM, Coni P, Curto M, et al. (1991). Induction of two different modes of cell death, apoptosis and necrosis, in rat liver after a single dose of thioacetamide. Am J Pathol 139:1099–109. Lee CP, Shih PH, Hsu CL, Yen GC. (2007). Hepatoprotection of tea seed oil (Camellia oleifera Abel.) against CCl4-induced oxidative damage in rats. Food Chem Toxicol 45:888–95. Liu LX, Howe P, Zhou YF, et al. (2000). Fatty acids and beta-carotene in Australian purslane (Portulaca oleracea) varieties. J Chromatogr A 893:207–13. Mukherjee PK. (2002). Quality Control of Herbal Drugs, 1st ed. New Delhi: Business Horizons Pharmaceutical Publication, 531. Naik SR, Panda VS. (2008). Hepatoprotective effect of Ginkgoselect phytosome in rifampicin induced liver injury in rats: Evidence of antioxidant activity. Fitoterapia 79:439–45. Navarro VJ, Senior JR. (2006). Drug related hepatotoxicity. N Engl J Med 354:731–9.
Hepatoprotective effects of Portulaca oleracea L. extract
9
Neichi T, Koshihara Y, Murota S. (1983). Inhibitory effect of esculetin on 5-lipoxygenase and leukotriene biosynthesis. Biochim Biophys Acta 753:130–2. Nwozo SO, Osunmadewa DA, Oyinloye BE. (2014). Anti-fatty liver effects of oils from Zingiber officinale and Curcuma longa on ethanolinduced fatty liver in rats. J Integr Med 12:59–65. Ohta Y, Kongo-Nishimura M, Matsura T, et al. (2004). Melatonin prevents disruption of hepatic reactive oxygen species metabolism in rats treated with carbon tetrachloride. J Pineal Res 36:10–17. Oliveira I, Valenta˜o P, Lopes R, et al. (2009). Phytochemical characterization and radical scavenging activity of Portulaca oleraceae L. leaves and stems. Microchem J 92:129–34. Parekh J, Karathia N, Chanda S. (2006). Antibacterial activity of Bauhinia variegata. J Biomed Res 9:53–6. Parry O, Marks JA, Okwuasaba FK. (1993). The skeletal muscle relaxant action of Portulaca oleracea: Role of potassium ions. J Ethnopharmacol 40:187–94. Parry O, Okwuasaba F, Ejike C. (1987). Skeletal muscle relaxant action of an aqueous extract of Portulaca oleracea in the rat. J Ethnopharmacol 19:247–53. Perez-Alvarez V, Bobadilla-Lugo RA, Murıel P, et al. (1993). Effects of leukotriene synthesis inhibition on acute liver damage induced by carbon tetrachloride. Pharmacology 47:330–6. Quan J, Yin X, Xu H. (2011). Boschniakia rossica prevents the carbon tetrachloride-induced hepatotoxicity in rat. Exp Toxicol Pathol 63: 53–9. Rando G, Wahli W. (2011). Sex differences in nuclear receptor-regulated liver metabolic pathways. Biochim Biophys Acta 1812:964–73. Rashed AN, Afifi FU, Disi AM. (2003). Simple evaluation of the wound healing activity of a crude extract of Portulaca oleracea L. (growing in Jordan) in Mus musculus JVI-1. J Ethnopharmacol 88: 131–6. Recknagel RO, Glende Jr EA, Britton RS. (1991). Free radical damage and lipid peroxidation. In: Meeks RG, ed. Hepatotoxicology. Florida: CRC Press, 401–36. Recknagel RO, Glende Jr EA, Dolak JA, Waller RL. (1989). Mechanisms of carbon tetrachloride toxicity. Pharmacol Ther 43: 139–54. Ree KR, Spector WG. (1961). Reversible nature of liver cell damage due to carbon tetrachloride as demonstrated by the use of phenergan. Nature 190:821–2. Reitman S, Frankel S. (1957). A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminase. Am J Pathol 28:57–60. Rousar T, Kucera O, Krivakova P, et al. (2009). Evaluation of oxidative status in acetaminophen treated rat hepatocytes in culture. Physiol Res 58:239–46. Sakai N, Inada K, Okamoto M, et al. (1996). Portuloside A, a monoterpene glucoside from Portulaca oleracea. Phytochemistry 42:1625–8. Shahjahan M, Sabitha KE, Jainu M, Devi CSS. (2004). Effect of Solanum trilobatum against carbon tetrachloride induced hepatic damage in albino rats. Indian J Med Res 120:194–8. Sharma V, Janmeda P, Paliwal R, Sharma S. (2012). Antihepatotoxic activity of Euphorbia neriifolia extract against N-nitrosodiethylamineinduced hepatocarcinogenesis in mice. J Chin Integr Med 10: 1303–9. Sheweita SA, El-Gabar MA, Bastawy M. (2001). Carbon tetrachlorideinduced changes in the activity of phase II drug-metabolizing enzyme in the liver of male rats: Role of antioxidants. Toxicology 165:217–24. Siriamornpun S, Suttajit M. (2010). Microchemical components and antioxidant activity of different morphological parts of Thai wild purslane (Portulaca oleracea). Weed Sci 58:182–8. Song HY, Zhang L, Pan JL, et al. (2013). Bioactivity of five components of Chinese herbal formula Jiangzhi granules against hepatocellular steatosis. J Integr Med 11:262–8. Sun Y, Oberley LW, Li Y. (1988). Simple method for clinical assay of superoxide dismutase. Clin Chem 34:497–500. Taniguchi M, Takeuchi T, Nakatsuka R, et al. (2004). Molecular process in acute liver injury and regeneration induced by carbon tetrachloride. Life Sci 75:1539–49. Vogel HG. (2002). Carbon tetrachloride induced liver fibrosis in rats. In: Vogel HG, Vogel WH, eds. Drug Discovery and Evaluation, Pharmacological Assays, 2nd ed. Berlin: Springer-Verlag, p. 942.
10
A. Eidi et al.
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
Vuda M, D’Souza R, Upadhya S, et al. (2012). Hepatoprotective and antioxidant activity of aqueous extract of Hybanthus enneaspermus against CCl4-induced liver injury in rats. Exp Toxicol Pathol 64: 855–9. Wang CQ, Yang GQ. (2010). Betacyanins from Portulaca oleracea L. ameliorate cognition deficits and attenuate oxidative damage induced by D-galactose in the brains of senescent mice. Phytomedicine 17: 527–32. Weber LW, Boll M, Stampfl A. (2003). Hepatotoxicity and mechanism of action of haloalkanes: Carbon tetrachloride as a toxicological model. Crit Rev Toxicol 33:105–36.
Pharm Biol, Early Online: 1–10
Wills PJ, Asha VV. (2006). Protective effect of Lygodium flexuosum (L.) Sw. extract against carbon tetrachloride-induced acute liver injury in rats. J Ethnopharmacol 108:320–6. Yang L, Wang CZ, Ye JZ, Li HT. (2011). Hepatoprotective effects of polyprenols from Ginkgo biloba L. leaves on CCl4-induced hepatotoxicity in rats. Fitoterapia 82:834–40. Yoon JW, Ham SS, Jun HS. (1999). Portulaca oleracea and tumor cell growth. US Patent. 5869060. Washington DC: Patent and Trademark Office. Zhang XJ, Ji YB, Qu ZY, et al. (2002). Experimental studies on antibiotic functions of Portulaca oleracea L. in vitro. Chin J Microbiol Immunol 14:277–80.
ORIGINAL ARTICLE
Hepatoprotective effects of Portulaca oleracea extract against CCl4-induced damage in rats Akram Eidi1, Pejman Mortazavi2, Jalal Zarringhalam Moghadam3, and Parisa Mousavi Mardani1
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
1
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran, 2Department of Pathology, Faculty of Specialized Veterinary Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran, and 3Department of Physiology, Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran Abstract
Keywords
Context: Purslane (Portulaca oleracea L., Portulacaceae) has been traditionally used in folk medicine to afford protection against liver injury, although its actual efficacy remains uncertain. Objective: To evaluate purslane as a hepatoprotective agent, we investigated the protective effect of its ethanol extract against carbon tetrachloride (CCl4)-induced hepatic toxicity in rats. Materials and methods: A total of 108 male Wistar rats were randomly divided into 12 groups. The first group was maintained as normal control, whereas CCl4 (0.5 ml/kg bw, 50% CCl4 in olive oil, i.p.), purslane extract (0.005, 0.01, 0.05, 0.1, and 0.15 g/kg bw, intragastrically), and purslane extract (five doses as above) along with CCl4 were administered to the Groups II, III–VII, and VIII– XII, respectively. The rats were sacrificed on the 30th day, and blood was withdrawn by cardiac puncture. Liver damage was assessed by measuring hepatic marker enzymes (ALT, AST, ALP, GGT, and SOD) and histopathological observation. Results: Treatment with CCl4 resulted in increased serum activities of marker enzymes with a concomitant decrease in SOD. Histological alterations were also observed in the liver tissue upon CCl4 treatment. Administration of purslane extract (0.01, 0.05, 0.1, and 0.15 g/kg b.w.) significantly showed a marked tendency towards normalization of all measured biochemical parameters in CCl4-treated rats. Histopathological changes also paralleled the detected alteration in markers of liver function. Discussion and conclusion: These results demonstrate that purslane exerts protective effects against CCl4-induced damage in rat liver and supports a potential therapeutic use of purslane as an alternative for patients with liver diseases.
Carbon tetrachloride, detoxification, liver, purslane
Introduction Common purslane [Portulaca oleracea L., Portulacaceae] is a widespread weed, and has been ranked the eighth most common plants in the world (Liu et al., 2000). The genus name Portulaca originates from the Latin word ‘‘Portare,’’ which means ‘‘to carry,’’ and ‘‘lac,’’ which means ‘‘milk,’’ referring to the milky sap of this plant. The species name oleracea is also of Latin origin, and means ‘‘pertaining to kitchen gardens,’’ referring to its use as a vegetable (Boulos & El-Hadidi, 1984). It has long been used as human food, animal feed, and a folk medicine in many countries as a diuretic, febrifuge, antiseptic, antispasmodic, and vermifuge agent. It exhibits a wide range of pharmacological effects, including ameliorating cognitive deficits (Wang & Yang, 2010), antibacterial (Zhang et al., 2002), hypolipidemic, antiaging, analgesic, anti-inflammatory (Chan et al., 2000), antioxidative (Dkhil et al., 2011), skeletal muscle relaxant (Parry et al., 1987, 1993), wound healing (Rashed et al., Correspondence: Akram Eidi, Department of Biology, Science and Research Branch, Islamic Azad University, P.O. Box 16535-446, Tehran, Iran. Tel: +98 21 44865939. Fax: +9821 44865939. E-mail: eidi@ srbiau.ac.ir, [email protected]
History Received 11 November 2013 Accepted 5 August 2014 Published online 4 December 2014
2003), and in vitro antitumor (Yoon et al., 1999) activities. It has been reported to be rich in a-linolenic acid and b-carotene (Liu et al., 2000). Flavonoids, coumarins (Awad, 1994), a monoterpene glycoside (Sakai et al., 1996), and alkaloids have been reported to be important chemical constituents of this plant. The liver, a major site of metabolism and detoxification, plays a central role in the kinetics of absorption, distribution, and elimination of most drugs as well as several active or inactive metabolites. Hepatotoxic agents react with basic cellular components, and consequently, induce almost all types of liver lesions. Toxins and drugs are among the basic etiopathogenic agents of acute liver failure in several countries (Grattagliano et al., 2009); most adverse drug reactions, therefore, involve liver toxicity. It is not surprising that protection of liver against toxic injury remains a major challenge for clinical therapy. Chemical toxins such as acetaminophen, ethanol, carbon tetrachloride (CCl4), galactosamine, nitrosodiethylamine, and thioacetamide are often used as model substances to induce experimental hepatocyte injury under both in vivo and in vitro conditions (Domenicali et al., 2009; Kucera et al., 2006; Ledda-Columbano et al., 1991; Nwozo et al., 2014; Rousar et al., 2009; Sharma et al.,
2
A. Eidi et al.
2012; Song et al., 2013). Hepatic problems are responsible for a significant number of liver transplantations and deaths worldwide; however, pharmacotherapeutic options for liver diseases are very limited, resulting in a great demand for the development of new and effective drugs. Numerous studies have shown that plant extracts having antioxidant activities protect against CCl4-induced hepatotoxicity by inhibiting lipid peroxidation and enhancing antioxidant enzyme activity (Dhiman et al., 2012; Duh et al., 2011; Kuo et al., 2010; Quan et al., 2011; Shahjahan et al., 2004; Sheweita et al., 2001; Vuda et al., 2012; Yang et al., 2011). The present study investigates the hepatoprotective potential of purslane ethanolic extract against CCl4-induced liver toxicity in rats.
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
Materials and methods Chemicals CCl4 was obtained from Merck, Darmstadt, Germany. The kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were purchased from Parsazmoon Co., Tehran, Iran. The kits for g-glutamyltransferase (GGT) and superoxide dismutase (SOD) were obtained from Bionik, Lainate Milano, Italy, and Randox Co., England, UK, respectively. All other reagents used were of analytical grade. Preparation of plant extract The aerial parts of purslane were obtained from the local market (Voucher number: 052245), and identified in the Islamic Azad University herbarium. The aerial parts were dried at 25 C, and finely powdered. The powder (60 g) was extracted with 300 ml of aqueous ethanol (80% v/v) in a Soxhlet apparatus for 72 h. After extraction, the solvent was filtered and evaporated using a Rotavapor. The purslane ethanolic extract so obtained was stored at 20 C until further use. Phytochemical screening Phytochemical analysis using standard procedures was carried out to determine the active chemical constituents of purslane, such as tannins, terpenoids, alkaloids, saponins, and flavonoids. The extract was treated with dilute hydrochloric acid and filtered. The filtrate was used for the following tests. The extract (20 mg) was dissolved in 10 ml of ethanol and filtered. Concentrated HCl (0.5 ml) and magnesium ribbon were added to 2 ml of the filtrate. Development of pinktomato red color indicated the presence of flavonoids. For the detection of tannins, 20 mg of the purslane extract was dissolved in 2 ml of distilled water and filtered. FeCl3 (2 ml) was added to the filtrate; the development of a blue-black precipitate indicated the presence of tannins. For the detection of alkaloids, 20-mg extract was dissolved in 2 ml of distilled water and filtered. Steam was passed through the filtrate after addition of 2–4 drops of 1% HCl. To 1 ml of this solution, six drops of Wagner’s reagent were added; a brownish-red precipitate indicated the presence of alkaloids (Finar, 2003). Saponins were detected by adding 5 ml of distilled water to 0.5 ml of the filtrate obtained in the test for alkaloids. A persistent frothing indicated the presence of saponins (Parekh et al., 2006). Salkovski test was performed using a
Pharm Biol, Early Online: 1–10
small amount of extract solution. Five drops of conc. H2SO4 and 1 ml of chloroform were added to this solution. A change of color from yellow to red indicated the presence of terpenoids (Finar, 2003). Animals Male Wistar rats weighing 200–250 g were used. The animals were housed in groups of five per cage, with free access to standard laboratory chow (35% carbohydrates, 25% proteins, 7% lipids, and 3% vitamins) and tap water. The food was purchased from Pars-Dam food service, Tehran, Iran. The animals were housed in large, clean, polypropylene cages in a temperature-controlled room (22 ± 2 C) with 40–60% relative humidity under 12 h light and dark cycles for 1 week prior to and during the experiments. The study was conducted in accordance with ethical procedures and policies approved by the Animal Care and Use Committee of Islamic Azad University, Tehran, Iran. Experimental design A total of 108 rats were divided into 12 groups of nine each. Group I served as a normal control group, and received distilled water (intragastric route; daily) and olive oil (0.5 ml/kg bw; twice a week, i.p.) as vehicles for a period of 30 d. Group II served as the toxicity control group, and received distilled water (intragastrically; daily) and CCl4 (0.5 ml/kg bw, 50% CCl4 in olive oil; twice a week, i.p.) for a period of 30 d. Groups III–VII served as the purslane extract control group, and received purslane extract dissolved in distilled water (intragastrically; daily) at doses of 0.005, 0.01, 0.05, 0.1, and 0.15 g/kg bw, respectively, with olive oil (0.5 ml/kg bw; twice a week, i.p.) for a period of 30 d. Groups VIII–XII were the treatment groups, and received purslane extract dissolved in distilled water (intragastrically; daily) at doses of 0.005, 0.01, 0.05, 0.1, and 0.15 g/kg bw, with CCl4 (0.5 ml/kg bw, 50% CCl4 in olive oil; twice a week, i.p.) for 30 d. The dose of purslane ethanolic extract was selected based on previous published reports (Cheng-Jie et al., 2009; El-Sayed, 2011). At the end of the treatment period, the rats were anesthetized by ether inhalation 48 h after the administration of CCl4. Blood samples were collected from the heart and allowed to clot for 30 min. Serum was separated by centrifugation at 2500 rpm for 15 min and used for biochemical estimations. The absolute and relative (organ-to-body weight ratio) weights of the liver were also measured for all the rats when they were sacrificed. Measurement of serum biochemical parameters The activities of serum AST, ALT, ALP, and GGT were determined using the auto-analyzer (Shimadzu CL-7200, Shimadzu, Japan) (Reitman & Frankel, 1957). Measurement of hepatic SOD activity Following anesthesia, rat livers were quickly excised and perfused with chilled saline for the complete removal of
Hepatoprotective effects of Portulaca oleracea L. extract
DOI: 10.3109/13880209.2014.957783
blood cells. They were then cut into small pieces, placed in 50 mM phosphate buffer (pH 7.4), and homogenized using a glass-Teflon homogenizer to obtain 1:9 (w/v) whole tissue homogenate. The homogenate was centrifuged at 3000 rpm for 15 min at 4 C. The supernatant was collected and transferred to an Eppendorf tube, and was centrifuged at 12 000 rpm for 30 min to eliminate cell debris. The supernatant so obtained was used for the determination of SOD activity. Xanthine and xanthine oxidase were used for the generation of superoxide radicals, which subsequently reacted with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride to form a red formazan dye. SOD activity was measured by the degree of inhibition of this reaction (Sun et al., 1988).
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
Histopathology Liver tissues, previously sectioned to 2 mm thickness, were fixed in 10% buffered formaldehyde for 24 h. The fixed tissues were embedded in paraffin, sectioned to 4–5 mm thickness, and rehydrated. The liver sections were stained with hematoxylin and eosin (H&E) stain, and histologically examined using conventional methods for assessing morphological changes in order to evaluate the index of CCl4-induced necrosis. Pathological assessment of the sections, such as fatty degeneration, necrosis, cell swelling, and lymphocyte infiltration, was also done for evaluating hepatotoxicity (Wills & Asha, 2006). Liver pathology was scored as described previously (French et al., 2000), as follows: Score 0 ¼ no visible cell damage. Score 1 ¼ focal hepatocyte damage in less than 25% of the tissue. Score 2 ¼ focal hepatocyte damage in 25–50% of the tissue. Score 3 ¼ extensive, but focal, hepatocyte lesions. Score 4 ¼ global hepatocyte necrosis. The morphology of observed lesions was classified and registered (Gray, 1958). Statistical analyses Results were expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by multiple comparisons using Tukey’s post-hoc test was used for the comparison of different parameters between the groups. A p value of50.05 was considered statistically significant.
Results Phytochemical analysis A percentage yield of 10.6% (w/w) was obtained for the extract from purslane aerial parts. Phytochemical analysis indicated the presence of flavonoids, tannins, saponins, terpenoids, and alkaloids in the plant extract (Table 1). Table 1. Phytochemical constituents of Portulaca oleracea. Extract Ethanolic
Phytochemical tests Flavonoids +
Tannins +
Saponins +
Terpenoids +
+: Presence of phytochemical; : absence of phytochemical.
Alkaloids +
3
Effects of purslane ethanolic extract administration on the body and relative liver weights of CCl4-treated rats The results of administration of purslane ethanolic extract on the body and relative liver weights of rats from each group are shown in Table 2. All the rats survived the experimental period of 30 d, till they were sacrificed. Daily observations over the experimental period showed no detectable alterations in the general states of the animals of all groups. Body weights of the rats significantly decreased in the CCl4-treated group, but increased in the normal control and purslane extract-treated groups. In contrast, relative liver weights significantly increased among rats of the CCl4-treated group. The administration of purslane extract reversed the increase in liver weight among the CCl4-treated rats. Effects of purslane ethanolic extract administration on biochemical parameters The hepatoprotective effects exerted by purslane ethanolic extract in CCl4-treated rats, as assessed by serum biochemical parameters, are shown in Figures 1 and 2. Rats treated with CCl4 (Group II) showed significantly higher serum AST, ALT, ALP, and GGT activities compared with normal control animals (Group I). Co-administration of purslane ethanolic extract (at doses of 0.01, 0.05, 0.1, and 0.15 g/kg bw) and CCl4 (Groups IX–XII) for 30 d resulted in significant hepatic protection, as measured by serum AST, ALT, ALP, and GGT activities, compared with the toxicity control group (Group II). SOD activity in CCl4-treated rats decreased drastically compared with the normal control group. Upon treatment with purslane ethanolic extract at doses of 0.01, 0.05, 0.1, and 0.15 g/kg bw (Groups IX–XII) for 30 d, this decrease in SOD activity (brought about by CCl4 treatment) showed recovery towards normalization in a purslane-extract dose-dependent manner. Therefore, treatment with purslane ethanolic extract showed a marked tendency towards normalization of all measured biochemical parameters in CCl4treated rats. Histopathological evaluation The liver lobules of the normal control and purslane extract control groups (Groups I and III–VII, respectively) showed a classical structure, with hepatocyte plates directed from the portal triads toward the central vein, where they freely anastomosed. Irregularly dilated liver sinusoids and space of Disse could also be observed. In contrast, liver sections from the rats treated with CCl4 alone (Group II) showed massive changes throughout the lobules, including fatty degeneration, cell swelling, necrosis, and infiltration of mononuclear inflammatory cells (lymphocyte infiltration) in the portal triads. However, upon co-treatment of the rats with CCl4 and purslane ethanolic extract at dose levels of 0.01, 0.05, 0.1, and 0.15 g/kg bw (Groups IX–XII), milder hepatocellular degeneration and better preservation of normal liver architecture could be observed, with moderate hepatocyte plate disorganizations and necrosis or inflammation. Moreover, animals in Groups XI and XII, which received the highest doses of purslane ethanolic extract, showed minimal lesions, which
4
A. Eidi et al.
Pharm Biol, Early Online: 1–10
Table 2. Effect of purslane ethanolic extract on body weight, liver weight, and liver index in normal and intoxicated rats.
Groups
Initial body weight (g)
Final body weight (g)
Weight gain (g)
Liver weight (g)
Liver weight/ Body weight 100
210 ± 7.5 215 ± 8.4
245 ± 8.5 218 ± 9.2***
35 ± 3.1 3 ± 0.6***
3.8 ± 0.4 5.2 ± 0.5***
1.55 ± 0.03 2.38 ± 0.12***
211 ± 9.3 216 ± 7.6 208 ± 6.9 214 ± 5.3 210 ± 6.8
248 ± 11.4 255 ± 10.7 247 ± 9.8 255 ± 11.7 250 ± 10.5
37 ± 3.6 39 ± 3.7 39 ± 3.5 41 ± 4.2 40 ± 4.1
3.7 ± 0.3 3.6 ± 0.4 3.5 ± 0.3 3.8 ± 0.5 3.6 ± 0.3
1.49 ± 0.04 1.41 ± 0.04 1.42 ± 0.04 1.49± 0.03 1.44 ± 0.04
216 ± 7.9 211 ± 4.8 209 ± 6.2 214 ± 5.1 217 ± 6.5
229 ± 8.6 226 ± 9.4 228 ± 9.1 237 ± 10.5+++ 242 ± 9.8+++
13 ± 1.2+ 15 ± 1.4++ 19 ± 2.1+++ 23 ± 2.4+++ 25 ± 2.8+++
4.6 ± 0.5 4.5 ± 0.6+ 4.3 ± 0.4+ 4.1 ± 0.3++ 4.1 ± 0.3++
2.01 ± 0.08 1.99 ± 0.07+ 1.89 ± 0.06+ 1.73 ± 0.05++ 1.69 ± 0.05+++
Values are mean ± S.E.M of nine rats. ***p50.001 significantly different from the normal control group. +p50.05 significantly different from the group treated with CCl4. ++p50.01 significantly different from the group treated with CCl4. +++p50.001 significantly different from the group treated with CCl4. (A) 250
***
Serum ALT (IU/L)
200
CCl4 +
150
+ 100
++ +++ +++
50
0 Normal CCI4
0.005
0.01
0.05
0.1
0.15
0.005
0.01
0.05
0.1
0.15
+++
+++
0.1
0.15
Purslane ethanolic extract (g/kg) (B) 300
*** CCl4 + Serum AST (IU/L)
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
Normal control CCl4 Purslane extract (g/kg) 0.005 0.01 0.05 0.1 0.15 Purslane extract (g/kg) + CCl4 0.005 0.01 0.05 0.1 0.15
200
++
++
0.01
0.05
100
0 Normal CCI4
0.005
0.01
0.05
0.1
0.15
0.005
Purslane ethanolic extract (g/kg)
Figure 1. Effect of oral administration of purslane extract at doses of 0.005, 0.01, 0.05, 0.1, and 0.15 g/kg bw on the activities of ALT (A), AST (B), ALP (C), and GGT (D) in the serum of CCl4-treated rats. Each column represents mean ± S.E.M. of data from nine rats. Normal control group was administered distilled water as a vehicle. The symbol ***represents p50.001 compared with the normal control group, whereas +, ++, and +++ represent p50.05, p50.01, and p50.001, respectively, compared with the CCl4-treated group.
Hepatoprotective effects of Portulaca oleracea L. extract
DOI: 10.3109/13880209.2014.957783
***
(C) 600
CCl4 +
Serum ALP (IU/L)
500
400
+ ++
300
+++
+++
0.1
0.15
200
100
Normal CCI4
0.005
0.01
0.05
0.1
0.15
0.005
0.01
0.05
Purslane ethanolic extract (g/kg)
(D) 12
***
10
Serum GGT (IU/L)
CCl4 +
8
++
6
+++ +++
4
2
0 Normal CCI4
0.005
0.01
0.05
0.1
0.15
0.005
0.01
0.05
0.1
0.15
Purslane ethanolic extract (g/kg)
Figure 1. Continued.
Figure 2. Effect of oral administration of purslane extract at doses of 0.005, 0.01, 0.05, 0.1, and 0.15 g/kg bw on SOD activity in the liver of CCl4-treated rats. Each column represents mean ± S.E.M. of data from nine rats. Normal control group was administered distilled water as a vehicle. The symbol ***represents p50.001 compared with the normal control group, whereas + and ++ represent p50.05 and p50.01 compared with the CCl4-treated group.
20
15
SOD (IU/mg protein)
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
0
CCl4 + 10
+
+
++
++
0.01
0.05
0.1
0.15
*** 5
0
Normal CCI4 0.005
0.01
0.05
0.1
0.15 0.005
Purslane ethanolic extract (g/kg)
5
6
A. Eidi et al.
Pharm Biol, Early Online: 1–10
Table 3. Histological injury score of liver under different doses of purslane ethanolic extract in rats treated with CCl4 (50% CCl4/olive oil). Injury of score Groups
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
Control CCl4 Purslane extract (g/kg) 0.005 0.01 0.05 0.1 0.15 Purslane extract (g/kg)+ CCl4 0.005 0.01 0.05 0.1 0.15
a
Fatty degeneration
Necrosis
Cell swelling
Inflammation
0.0 ± 0.00 3.4 ± 0.24***
0.0 ± 0.00 3.6 ± 0.24***
0.0 ± 0.00 3.6 ± 0.24***
0.0 ± 0.00 3.4 ± 0.24***
0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00
0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00
0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00
0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00
2.4 ± 0.24+ 2.2 ± 0.20+ 1.8 ± 0.20+++ 1.2 ± 0.20+++ 1.0 ± 0.32+++
2.4 ± 0.24++ 1.6 ± 0.24+++ 1.4 ± 0.24+++ 1.2 ± 0.20+++ 0.8 ± 0.20+++
3.2 ± 0.37 2.4 ± 0.24+ 2.2 ± 0.20++ 1.4 ± 0.24+++ 1.2 ± 0.20+++
2.2 ± 0.20+ 1.4 ± 0.24+++ 1.2 ± 0.20+++ 1.0 ± 0.31+++ 0.8 ± 0.20+++
a
Livers were scored for hepatic injury via light microscopy with score 0 ¼ no visible cell damage; score 1 ¼ focal hepatocyte damage on less than 25% of the tissue; score 2 ¼ focal hepatocyte damage on 25–50% of the tissue; score 3 ¼ extensive, but focal, hepatocyte lesions; score 4 ¼ global hepatocyte necrosis. ***p50.001 significantly different from the normal control group. +p50.05 significantly different from the group treated with CCl4. ++p50.01 significantly different from the group treated with CCl4. +++p50.001 significantly different from the group treated with CCl4.
included cell swelling towards the central vein, fatty degeneration, and necrosis (Table 3 and Figure 3).
Discussion CCl4-induced liver injury is the best-characterized model of xenobiotic-induced hepatotoxicity, and is commonly employed for screening antihepatotoxic/hepatoprotective activities of drugs (Brautbar & Williams, 2002; Brent & Rumack, 1993). The bio-activation of CCl4, primarily through the activity of CYP2E1, generates the free radicals CCl3 and CCl3OO, which results in hepatic damage. These free radicals initiate lipid peroxidation by abstracting a hydrogen atom from the polyunsaturated fatty acid of a phospholipid (Recknagel et al., 1989; Weber et al., 2003). The CCl4induced lipid peroxidation in turn increases the permeability of plasma membrane to Ca2+, leading to severe disruption of calcium homeostasis and necrotic cell death (Weber et al., 2003). The extent of hepatic damage is assessed by the increase in serum levels of the cytoplasmic enzymes AST, ALT, ALP, and GGT, and by histopathological examination. The increased serum levels of AST and ALT have been attributed to damages in the structural integrity of the liver, as these cytoplasmic enzymes are released into circulation after cellular damage (Recknagel et al., 1989, 1991). Elevation of AST has been reported to be an index of hepatocellular injury in rats, whereas ALT elevation is more commonly associated with the necrotic state (Navarro & Senior, 2006). Serum ALP and GGT, which are important enzymes for assessing obstructive liver injury (Bulle et al., 1990; Kaplan, 1986), were also found to be significantly elevated in CCl4-treated rats. ALP activity is related to the functioning of hepatocytes. Suppression of increased ALP activity is indicative of the stabilization of biliary dysfunction in rat liver during chronic hepatic injury induced by CCl4 (Mukherjee, 2002).
The present study demonstrates the hepatoprotective effects of purslane ethanolic extract against CCl4-induced liver injury in rats. The serum levels of certain important biochemical parameters were employed as diagnostic markers of hepatic injury. Increased levels of ALT, AST, ALP, and GGT in the serum of CCl4-treated animals were indicative of liver damage, due to the leakage of these enzymes from the liver into the blood stream during tissue damage, which is always associated with hepatonecrosis (Naik & Panda, 2008; Ree & Spector, 1961). Upon treatment with purslane extract, the levels of these markers were restored to near normal or were only slightly elevated, indicating protection against liver damage. Such a restoration of increased serum levels of hepatic enzymes to the normal range reflects protection against hepatic damage caused by hepatotoxins (Vogel, 2002). The female sex hormone 17b-estradiol has been reported to increase the sensitivity of hepatic Kupffer cells to endotoxins in rats (Ikejima et al., 1998). Moreover, the process of liver detoxification may also be sexually dimorphic (Rando & Wahli, 2011). Therefore, male Wistar rats were used in the present study, because of fluctuations in the levels of sex hormones as a result of the menstrual cycle in female rats. Treatment of animals with purslane extract resulted in the preservation of the histological integrity of liver cells, which correlated with a significant reversal of the CCl4-induced increase in serum levels of hepatic enzymes. Inflammation plays a central role in drug-induced hepatitis, and leukotrienes, which are products of 5-lipoxygenase pathway of arachidonic acid metabolism, have been shown to be extensively involved in inflammatory processes (Perez-Alvarez et al., 1993). Consequently, the inhibitors of leukotriene synthesis provide partial protection against acute liver damage induced by different hepatotoxins, including CCl4 (Neichi et al., 1983).
Hepatoprotective effects of Portulaca oleracea L. extract
7
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
DOI: 10.3109/13880209.2014.957783
Figure 3. Histopathology of various treatment groups. (a) In the normal control group (Group I), normal liver structure with central vein (cv) and hepatocyte column (arrow) is seen. (b) Liver sections from the toxicity control group (Group II) showed massive changes throughout the lobules, with fatty degeneration (arrow head), infiltration of mononuclear inflammatory cells (I), and necrosis (arrow). (c-g) Animals treated with purslane ethanolic extract at doses of 0.005, 0.01, 0.05, 0.1, and 0.15 g/kg bw (Groups III–VII) showed normal liver structure, with central vein (cv), sinusoid (s), and hepatocytes (arrow). (h-l) Animals treated with purslane ethanolic extract at doses of 0.005, 0.01, 0.05, 0.1, and 0.15 g/kg bw along with CCl4 (Groups VIII–XII) showed mild to moderate lesions indicative of hepatocyte necrosis (arrow and N), fatty degeneration (arrow head), and lymphocyte infiltration (*). Note that the lesions are very mild in k and l (Groups XI and XII) panels. The sections were stained with H&E prior to observation.
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
8
A. Eidi et al.
Pharm Biol, Early Online: 1–10
Figure 3. Continued.
Reactive oxygen species (ROS), including superoxide anion, hydroxyl radical, and hydrogen peroxide, have a causal relationship with oxidative stress. Antioxidant enzymes such as SOD form a part of the protective response against oxidative tissue damage (Halliwell & Gutteridge, 1990). SOD represents the first line of defense against free radicals, and was demonstrated to possess ROS-metabolizing activity; it efficiently and specifically catalyzes dismutation of O 2 to O2 and H2O2. H2O2 is further metabolized to water by catalase. Maintaining the balance between ROS and antioxidant enzymes is therefore crucial, and could serve as a major mechanism for damage prevention by oxidative stress (Taniguchi et al., 2004). In the present study, a decrease was observed in SOD activity in the liver upon CCl4 administration, which could be attributed to oxidative inactivation of the enzyme upon excessive ROS generation. These results are consistent with previous studies (Lee et al., 2007; Ohta et al., 2004; Taniguchi et al., 2004). Treatment with purslane extract resulted in improved antioxidant activity of SOD compared with the CCl4-treated control group. These results confirm previous data on the antioxidative properties of purslane extract in other tissues, including heart, where it was shown to increase SOD activity (Caballero-Salazar et al., 2002; Hongxing et al., 2007). Purslane has been classically characterized as an antioxidant (Dkhil et al., 2011), and is involved in the direct detoxification of rotenones, mediated by two flavonols and flavones present in purslane (Cai et al., 2004; Oliveira et al., 2009). Purslane also effectively detoxifies superoxide, either by superoxide dismutation or by directly reacting with it (Siriamornpun & Suttajit, 2010).
Histopathological changes also paralleled the detected alteration in markers of liver function. Fatty degeneration, lymphocyte infiltration, and extensive hepatocellular necrosis were observed upon CCl4 treatment, which were markedly reduced upon co-treatment with purslane extract. In conclusion, the results obtained in the current study confirm the protective effect of purslane extract against CCl4induced hepatic damage in rats, which could be deduced not only from biochemical parameters in serum and liver but also from the histological observation of liver tissues. Further studies to characterize the active components of purslane extract and to elucidate its mechanism of action are in progress.
Declaration of interest We would like to thank Deputy of Research of the Science and Research Branch, Islamic Azad University for financial support of the project.
References Awad NE. (1994). Lipid content and antimicrobial activity of phenolic constituents of cultivated Portulaca oleracea L. Bull Fac Pharm Cairo Univ 32:137–42. Boulos L, El-Hadidi MN. (1984). The Weed Flora of Egypt. Cairo, Egypt: The American University in Cairo Press. Brautbar N, Williams J. (2002). Industrial solvents and liver toxicity: Risk assessment, risk factors and mechanisms. Int J Hyg Environ Health 205:479–91. Brent JA, Rumack BH. (1993). Role of free radicals in toxic hepatic injury. Clin Toxicol 31:173–96.
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
DOI: 10.3109/13880209.2014.957783
Bulle F, Mavier P, Zafrani ES, et al. (1990). Mechanism of gammaglutamyl transpeptidase release in serum during intrahepatic and extrahepatic cholestasis in the rat: A histochemical, biochemical and molecular approach. Hepatology 11:545–50. Caballero-Salazar S, Riveron-Negrete L, Ordaz-Tellez MG, et al. (2002). Evaluation of the antimutagenic activity of different vegetable extracts using an in vitro screening test. Proc West Pharmacol Soc 45:101–3. Cai Y, Luo Q, Sun M, Corke H. (2004). Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci 74:2157–84. Chan K, Islam MW, Kamil M, et al. (2000). The analgesic and antiinflammatory effects of Portulaca oleracea L. subsp. sativa (Haw.) Celak. J Ethnopharmacol 73:445–51. Cheng-Jie C, Wan-Yin W, Xiao-Li W, et al. (2009). Anti-hypoxic activity of the ethanol extract from Portulaca oleracea in mice. J Ethnopharmacol 124:246–50. Dhiman A, Nanda A, Akmad S. (2012). A recent update in research on the antihepatotoxic potential of medicinal plants. J Chin Integr Med 10:117–27. Dkhil MA, Abdel Moniem AE, Al-Quraishy S, Saleh RA. (2011). Antioxidant effect of purslane (Portulaca oleracea) and its mechanism of action. J Med Plants Res 5:1589–63. Domenicali M, Caraceni P, Giannone F, et al. (2009). A novel model of CCl4-induced cirrhosis with ascites in the mouse. J Hepatol 51: 991–9. Duh PD, Lin SL, Wu SC. (2011). Hepatoprotection of Graptopetalum paraguayense E. Walther on CCl4-induced liver damage and inflammation. J Ethnopharmacol 134:379–85. El-Sayed M-IK. (2011). Effects of Portulaca oleracea L. seeds in treatment of type-2 diabetes mellitus patients as adjunctive and alternative therapy. J Ethnopharmacol 137:643–51. Finar IL. (2003). Organic Chemistry. Stereochemistry and the Chemistry of Natural Products, vol. 2, 5th ed. Delhi: Pearson Education, 769–71. French SW, Miyamoto K, Ohta Y, Geoffrion Y. (2000). Pathogenesis of experimental alcoholic liver disease in the rat. Methods Achiev Exp Pathol 13:181–207. Grattagliano I, Bonfrate L, Catia VD, et al. (2009). Biochemical mechanisms in drug-induced liver injury. World J Gastroenterol 15: 4865–76. Gray P. (1958). Handbook of Basic Microtechnique. 3rd ed. New York: McGraw-Hill, 45–85. Halliwell B, Gutteridge JMC. (1990). Role of free radicals and catalytic metal irons in human disease: An overview. Methods Enzymol 186: 59–85. Hongxing Z, Nancai Y, Guofu H, et al. (2007). Neuroprotective effects of purslane herb aqueous extracts against D-galactose induced neurotoxicity. Chem-Biol Interact 170:145–52. Ikejima K, Enomoto N, Iimuro Y, et al. (1998). Estrogen increases sensitivity of hepatic Kupffer cells to endotoxin. Am J Physiol 274: G669–76. Kaplan MM. (1986). Serum alkaline phosphatase-another piece is added to the puzzle. Hepatology 6:526–8. Kucera O, Cervinkova Z, Lotkova H, et al. (2006). Protective effect of S-adenosylmethionine against galactosamine-induced injury of rat hepatocytes in primary culture. Physiol Res 55:551–60. Kuo DH, Kang WH, Shieh PC, et al. (2010). Protective effect of Pracparatum mungo extract on carbon tetrachloride-induced hepatotoxicity in rats. Food Chem 123:1007–12. Ledda-Columbano GM, Coni P, Curto M, et al. (1991). Induction of two different modes of cell death, apoptosis and necrosis, in rat liver after a single dose of thioacetamide. Am J Pathol 139:1099–109. Lee CP, Shih PH, Hsu CL, Yen GC. (2007). Hepatoprotection of tea seed oil (Camellia oleifera Abel.) against CCl4-induced oxidative damage in rats. Food Chem Toxicol 45:888–95. Liu LX, Howe P, Zhou YF, et al. (2000). Fatty acids and beta-carotene in Australian purslane (Portulaca oleracea) varieties. J Chromatogr A 893:207–13. Mukherjee PK. (2002). Quality Control of Herbal Drugs, 1st ed. New Delhi: Business Horizons Pharmaceutical Publication, 531. Naik SR, Panda VS. (2008). Hepatoprotective effect of Ginkgoselect phytosome in rifampicin induced liver injury in rats: Evidence of antioxidant activity. Fitoterapia 79:439–45. Navarro VJ, Senior JR. (2006). Drug related hepatotoxicity. N Engl J Med 354:731–9.
Hepatoprotective effects of Portulaca oleracea L. extract
9
Neichi T, Koshihara Y, Murota S. (1983). Inhibitory effect of esculetin on 5-lipoxygenase and leukotriene biosynthesis. Biochim Biophys Acta 753:130–2. Nwozo SO, Osunmadewa DA, Oyinloye BE. (2014). Anti-fatty liver effects of oils from Zingiber officinale and Curcuma longa on ethanolinduced fatty liver in rats. J Integr Med 12:59–65. Ohta Y, Kongo-Nishimura M, Matsura T, et al. (2004). Melatonin prevents disruption of hepatic reactive oxygen species metabolism in rats treated with carbon tetrachloride. J Pineal Res 36:10–17. Oliveira I, Valenta˜o P, Lopes R, et al. (2009). Phytochemical characterization and radical scavenging activity of Portulaca oleraceae L. leaves and stems. Microchem J 92:129–34. Parekh J, Karathia N, Chanda S. (2006). Antibacterial activity of Bauhinia variegata. J Biomed Res 9:53–6. Parry O, Marks JA, Okwuasaba FK. (1993). The skeletal muscle relaxant action of Portulaca oleracea: Role of potassium ions. J Ethnopharmacol 40:187–94. Parry O, Okwuasaba F, Ejike C. (1987). Skeletal muscle relaxant action of an aqueous extract of Portulaca oleracea in the rat. J Ethnopharmacol 19:247–53. Perez-Alvarez V, Bobadilla-Lugo RA, Murıel P, et al. (1993). Effects of leukotriene synthesis inhibition on acute liver damage induced by carbon tetrachloride. Pharmacology 47:330–6. Quan J, Yin X, Xu H. (2011). Boschniakia rossica prevents the carbon tetrachloride-induced hepatotoxicity in rat. Exp Toxicol Pathol 63: 53–9. Rando G, Wahli W. (2011). Sex differences in nuclear receptor-regulated liver metabolic pathways. Biochim Biophys Acta 1812:964–73. Rashed AN, Afifi FU, Disi AM. (2003). Simple evaluation of the wound healing activity of a crude extract of Portulaca oleracea L. (growing in Jordan) in Mus musculus JVI-1. J Ethnopharmacol 88: 131–6. Recknagel RO, Glende Jr EA, Britton RS. (1991). Free radical damage and lipid peroxidation. In: Meeks RG, ed. Hepatotoxicology. Florida: CRC Press, 401–36. Recknagel RO, Glende Jr EA, Dolak JA, Waller RL. (1989). Mechanisms of carbon tetrachloride toxicity. Pharmacol Ther 43: 139–54. Ree KR, Spector WG. (1961). Reversible nature of liver cell damage due to carbon tetrachloride as demonstrated by the use of phenergan. Nature 190:821–2. Reitman S, Frankel S. (1957). A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminase. Am J Pathol 28:57–60. Rousar T, Kucera O, Krivakova P, et al. (2009). Evaluation of oxidative status in acetaminophen treated rat hepatocytes in culture. Physiol Res 58:239–46. Sakai N, Inada K, Okamoto M, et al. (1996). Portuloside A, a monoterpene glucoside from Portulaca oleracea. Phytochemistry 42:1625–8. Shahjahan M, Sabitha KE, Jainu M, Devi CSS. (2004). Effect of Solanum trilobatum against carbon tetrachloride induced hepatic damage in albino rats. Indian J Med Res 120:194–8. Sharma V, Janmeda P, Paliwal R, Sharma S. (2012). Antihepatotoxic activity of Euphorbia neriifolia extract against N-nitrosodiethylamineinduced hepatocarcinogenesis in mice. J Chin Integr Med 10: 1303–9. Sheweita SA, El-Gabar MA, Bastawy M. (2001). Carbon tetrachlorideinduced changes in the activity of phase II drug-metabolizing enzyme in the liver of male rats: Role of antioxidants. Toxicology 165:217–24. Siriamornpun S, Suttajit M. (2010). Microchemical components and antioxidant activity of different morphological parts of Thai wild purslane (Portulaca oleracea). Weed Sci 58:182–8. Song HY, Zhang L, Pan JL, et al. (2013). Bioactivity of five components of Chinese herbal formula Jiangzhi granules against hepatocellular steatosis. J Integr Med 11:262–8. Sun Y, Oberley LW, Li Y. (1988). Simple method for clinical assay of superoxide dismutase. Clin Chem 34:497–500. Taniguchi M, Takeuchi T, Nakatsuka R, et al. (2004). Molecular process in acute liver injury and regeneration induced by carbon tetrachloride. Life Sci 75:1539–49. Vogel HG. (2002). Carbon tetrachloride induced liver fibrosis in rats. In: Vogel HG, Vogel WH, eds. Drug Discovery and Evaluation, Pharmacological Assays, 2nd ed. Berlin: Springer-Verlag, p. 942.
10
A. Eidi et al.
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Otago on 01/09/15 For personal use only.
Vuda M, D’Souza R, Upadhya S, et al. (2012). Hepatoprotective and antioxidant activity of aqueous extract of Hybanthus enneaspermus against CCl4-induced liver injury in rats. Exp Toxicol Pathol 64: 855–9. Wang CQ, Yang GQ. (2010). Betacyanins from Portulaca oleracea L. ameliorate cognition deficits and attenuate oxidative damage induced by D-galactose in the brains of senescent mice. Phytomedicine 17: 527–32. Weber LW, Boll M, Stampfl A. (2003). Hepatotoxicity and mechanism of action of haloalkanes: Carbon tetrachloride as a toxicological model. Crit Rev Toxicol 33:105–36.
Pharm Biol, Early Online: 1–10
Wills PJ, Asha VV. (2006). Protective effect of Lygodium flexuosum (L.) Sw. extract against carbon tetrachloride-induced acute liver injury in rats. J Ethnopharmacol 108:320–6. Yang L, Wang CZ, Ye JZ, Li HT. (2011). Hepatoprotective effects of polyprenols from Ginkgo biloba L. leaves on CCl4-induced hepatotoxicity in rats. Fitoterapia 82:834–40. Yoon JW, Ham SS, Jun HS. (1999). Portulaca oleracea and tumor cell growth. US Patent. 5869060. Washington DC: Patent and Trademark Office. Zhang XJ, Ji YB, Qu ZY, et al. (2002). Experimental studies on antibiotic functions of Portulaca oleracea L. in vitro. Chin J Microbiol Immunol 14:277–80.
