Gini C.
Kuriakose†
*a and
Muraleedhara G.
Kurup
*b
aDepartment of Applied Biochemistry, Mahatma Gandhi University, Kottayam, Kerala, India. E-mail: ginibesant@gmail.com
bDepartment of Biochemistry, Kerala University, Palayam Campus, Thiruvananthapuram, 695035, Kerala, India. E-mail: muraleedharakurup@gmail.com; Tel: +91 471 2443456
First published on 1st February 2011
The vast biodiversity of nature provides bioactive compounds that may be useful in the fight against chronic diseases. This study was designed to investigate the protective effects of the ethanol extract of Spirulina laxissima West (Pseudanabaenaceae) (EESL) against carbon tetrachloride (CCl4) induced hepatotoxicities in rats. Male albino rats of Sprague-Dawley strain were treated orally with the ethanol extract of S. laxissima (50, 100 mg kg−1 body wt.) 1 h before each CCl4 administration. The ethanol extract of S. laxissima showed the maximum antioxidant property in vitro. There were statistically significant losses in the activities of antioxidant enzymes and an increase in TBARS and liver function marker enzymes in the serum of the CCl4-treated group compared with the control group. However, all the tested groups were able to counteract these effects. The antioxidant activity of the extracts might be attributable to its proton-donating ability, as evidenced by DPPH. In the present study, the decline in the level of antioxidant observed in CCl4-treated rats is a clear manifestation of excessive formation of radicals and activation of the lipid peroxidation system resulting in tissue damage. The significant increases in the concentration of antioxidant enzymes in tissues of animals treated with CCl4 + EESL indicate the antioxidant effect of EESL. This study suggests that EESL can protect the liver against CCl4-induced oxidative damage in rats, and the hepatoprotective effect might be correlated with its antioxidant and radical-scavenging effects.
Focusing our attention on natural and bioavailable sources of antioxidants, we undertook to investigate the antioxidant properties of the cyanophyte Spirulina laxissima West (Pseudanabaenaceae), a unicellular blue-green alga and that is consumed as a nutrient-dense food source and for its health-enhancing properties. Spirulina is an important source of the blue photosynthetic pigment phycocyanin (PC), which has been described as a strong antioxidant2 and anti-inflammatory3 natural compound, as evidenced by in vitro and in vivo studies on PC from the cyanophyte Spirulina platensis Geitler (Pseudanabaenaceae). PC is a water-soluble phycobiliprotein composed of a and h subunit polypeptides which associate into ah monomers4 in turn, have a high affinity to assemble together to form (ah)3 trimers and finally (ah)6 hexamers. The a and h subunits are constituted of a protein backbone to which linear tetrapyrrole chromophores are covalently bound.5 The chromophore, named phycocyanobilin, is similar in chemical structure to bilirubin, and like the latter acts as a powerful scavenger of reactive oxygen species.6
Carbon tetrachloride (CCl4), a potent hepatotoxic agent, is biotransformed to a trichloromethyl radical by the cytochrome P450 system in liver microsomes, and consequently causes lipid peroxidation of membranes that leads to liver injury.7 The present study was designed to evaluate the putative antioxidant action of ethanol extract of S. laxissima in an experimental model of CCl4-induced hepatotoxicity in albino rats.
Inhibition of DPPH activity = [(A − B)/A] × 100% |
Group I – Vehicle
Group II – CCl4 in paraffin oil (1:5 v/v, 1.5 ml kg−1 body weight, i.p.)
Group III – Ethanol extract (oral) of S. laxissima (50 mg kg−1 body weight) + CCl4 in paraffin oil
Group IV – Ethanol extract (oral) of S. laxissima (100 mg kg−1 body weight) + CCl4 in paraffin oil
Group V – Silymarin (oral) (100 mg kg−1 body weight) + CCl4 in paraffin oil
Animals in Groups II, III and IV and V were treated with CCl4 three times a week for five weeks (total 15 doses). Groups III, IV and V were treated orally with ethanol extract of S. laxissima (50, 100 mg) and silymarin respectively one hour before each CCl4 administration. The group treated with the vehicle was kept as normal. Group II, treated with CCl4 alone, was kept as an untreated control. Twenty-four hours after the last dose of CCl4, the animals were sacrificed by decapitation, and the blood was collected by cutting the jugular vein. A portion of the liver was used for histopathological analysis. The liver in each case was dissected out, the blood blotted off, and washed in cold saline. The serum was separated from the blood, and the serum and liver samples were stored at −80 °C until analysis.
Fig. 1 Percent DPPH radical-scavenging capacity of different extracts of S. laxissima (ethanol extract (EESL), methanol extract (MESF) and water extract (WESF)). |
A significant increase in the activity of the serum enzymes AST, ALT, GGT and ALP was observed in rats receiving CCl4 in the vehicle (Group II) when compared to normal (Group I) rats administered with the vehicle alone (Table 1). However, the activities of these serum enzymes were significantly (P < 0.01) lower in rats treated with the EESL (Group III and IV) than in Group II rats. The protection offered by silymarin was found to be higher.
Group | AST (U/L serum) | ALT (U/L serum) | ALP (U/L serum) | GGT (U/L serum) |
---|---|---|---|---|
a Values are mean ± SD of 8 animals in each group. Statistical analysis: ANOVA followed by Dunnett’s t-test. P < 0.01 as compared with Group I (*), Group II (†). | ||||
I – Normal | 38.36 ± 7.09 | 29.60 ± 8.35 | 89.61 ± 12.98 | 3.94 ± 0.96 |
II – CCl4 + LP | 137.61 ± 11.54* | 178.38 ± 9.09* | 196.92 ± 13.92* | 9.64 ± 0.34* |
III – CCl4 + EESL (50 mg) | 90.32 ± 10.58† | 86.19 ± 12.45† | 114.69 ± 7.87† | 7.34 ± 1.87† |
IV – CCl4 + EESL (100 mg) | 43.44 ± 12.94† | 34.15 ± 9.22† | 93.97 ± 9.16† | 4.29 ± 1.24† |
V – CCl4 + silymarin | 39.38 ± 11.24† | 30.17 ± 10.68† | 91.62 ± 6.83† | 3.90 ± 0.08† |
A marked increase in the mean TBARS and CD level was found in the liver of Group II (CCl4-exposed) rats relative to normal (Group I) rats (Table 2); this increase was statistically significant (P < 0.01). Treatment with EESL in Group IV rats was found to result in a significant (P < 0.01) lowering of the mean TBARS and CD concentration, presumably by limiting lipid peroxidation in the hepatic tissue. CCl4 administration in Group II rats resulted in a marked decrease (relative to normal) in the level of reduced glutathione in the liver (Table 2); this decrease was statistically significant (P < 0.01). Treatment with EESL and silymarin resulted in a significantly higher concentration of GSH (P < 0.01) than that in Group II.
Group | TBARS (mM per 100 g tissue) | CD (mM per 100 g tissue) | GSH (mM per 100 g tissue) |
---|---|---|---|
a Values are mean ± SD of 8 animals in each group. Statistical analysis: ANOVA followed by Dunnett’s t-test. P < 0.01 as compared with Group I (*), Group II (†). | |||
I – Normal | 1.39 ± 0.65 | 62.24 ± 10.19 | 0.94 ± 0.02 |
II – CCl4 + LP | 1.68 ± 0.56* | 80.40 ± 15.54* | 0.45 ± 0.04* |
III – CCl4 + EESL (50 mg) | 1.53 ± 0.87† | 67.14 ± 12.53† | 0.76 ± 0.14† |
IV – CCl4 + EESL (100 mg) | 1.43 ± 0.66† | 64.02 ± 9.46† | 0.88 ± 3.29† |
V – CCl4 + silymarin | 1.40 ± 0.17† | 62.56 ± 7.07† | 0.90 ± 0.16† |
The concentrations of total proteins and bilirubin in serum are given in the Table 3. A marked elevation in the concentration of bilirubin and decreases in protein content was observed in the CCl4-treated rats compared to normal animals (P < 0.01). In rats that received EESL at a dose of 100 mg kg−1 body weight, the concentrations of bilirubin and total protein were maintained at near-normal levels.
Group | Protein (g per 100 ml serum) | Bilirubin (mg per 100 ml serum) |
---|---|---|
a Values are mean ± SD of 8 animals in each group. Statistical analysis: ANOVA followed by Dunnett’s t-test. P < 0.01 as compared with Group I (*), Group II (†). b c | ||
I – Normal | 8.65 ± 2.56 | 1.71 ± 0.09 |
II – CCl4 + LP | 6.98 ± 1.15* | 2.48 ± 0.14* |
III – CCl4 + EESL (50 mg) | 7.11 ± 1.87† | 2.02 ± 0.54† |
IV – CCl4 +EESL (100 mg) | 8.55 ± 1.32† | 1.79 ± 0.18† |
V – CCl4 + silymarin | 8.59 ± 0.16† | 1.74 ± 0.20† |
Table 4 shows the concentrations of total lipids, phospholipids, cholesterol, and triglycerides in the liver. Significant increase (P < 0.01) in the lipid profile was observed in CCl4-intoxicated rats. Co-administration of EESL significantly prevented the CCl4-induced alterations in the lipid profile.
Group | Total lipids (mg per 100 g tissue) | Phospholipids (mg per 100 g tissue) | Cholesterol (mg per 100 g tissue) | Triglycerides (mg per 100 g tissue) |
---|---|---|---|---|
a Values are mean ± SD of 8 animals in each group. Statistical analysis ANOVA followed by Dunnett t-test. P < 0.01 as compared with Group I (*), Group II (†). | ||||
I – Normal | 4375.88 ± 12.01 | 2254.64 ± 9.01 | 565.35 ± 3.16 | 447.44 ± 9.24 |
II – CCl4 + LP | 5685.72 ± 15.29* | 3017.41 ± 13.01* | 784.92 ± 4.24* | 561.23 ± 6.96* |
III – CCl4 + EESL (50 mg) | 5259.89 ± 13.52† | 2765.26 ± 11.45† | 654.36 ± 10.45† | 470.96 ± 9.63† |
IV – CCl4 + EESL (100 mg) | 4797.71 ± 9.73† | 2420.44 ± 10.12† | 571.80 ± 11.25† | 456.64 ± 14.34† |
V – CCl4 + silymarin | 4590.45 ± 12.44† | 2311.69 ± 13.58† | 569.76 ± 12.38† | 451.82 ± 10.67† |
A significant decrease in antioxidant enzymes like CAT, SOD, GPX and GST activity was observed in the liver of CCl4-administered (Group II) rats when compared to normal (Group I) rats that had received the vehicle alone (Table 5). Treatment with the EESL appeared to exert a beneficial effect, since the activities of these enzymes were significantly (P < 0.01) higher in the livers of Group III and IV rats than in Group II rats.
Group | SOD (U per mg protein) | CAT (U per mg protein) | GPX (U per mg protein) | GST (U per mg protein) |
---|---|---|---|---|
a Values are mean ± SD of 8 animals in each group. Statistical analysis ANOVA followed by Dunnett’s t-test. P < 0.01 as compared with Group I (*), Group II (†). | ||||
I – Normal | 5.33 ± 1.03 | 2.93 ± 0.09 | 165.96 ± 10.12 | 0.34 ± 0.17 |
II – CCl4 + LP | 4.14 ± 1.49* | 0.54 ± 0.38* | 142.12 ± 7.09* | 0.24 ± 0.04* |
III – CCl4 + EESL 50 mg | 4.48 ± 1.78† | 1.99 ± 0.69† | 155.49 ± 12.09† | 0.27 ± 0.19† |
IV – CCl4 + EESL 100 mg | 5.16 ± 0.41† | 2.78 ± 0.17† | 163.95 ± 11.43† | 0.31 ± 0.04† |
V – CCl4 + silymarin | 5.30 ± 1.19† | 2.80 ± 0.05† | 164.84 ± 5.95† | 0.33 ± 0.09† |
When compared to the histoarchitecture of the livers of Group I (normal) animals (Fig. 2A), liver cells of Group II rats (exposed to CCl4) revealed extensive damage, characterized by the disruption of the lattice nature of the hepatocyte, damaged cell membranes, degenerated nuclei, a disintegrated central vein and damaged hepatic sinusoids (Fig. 2B). In Group IV rats (exposed to CCl4 + EESL (100 mg)), only minimal disruption of the hepatic cellular structure was observed (Fig. 2C). Liver section of rats treated with CCl4 and silymarin (Group V) showed minimal inflammatory cellular infiltration (Fig. 2D).
Fig. 2 Photomicrographs of liver sections of rats stained with haematoxylin and eosin (200X). A: Normal rat showing a central vein surrounded by normal hepatocytes. B: CCl4-treated rats showing a dilated central vein and hepatocytes with fatty change and ballooning degeneration. C: CCl4 + S. laxissima (100 mg kg−1 body weight) treated rats showing the central vein and normal hepatocytes with occasional hepatocytes showing fatty change and ballooning degeneration. D: Liver section of rats treated with CCl4 and silymarin showing minimal inflammatory cellular infiltration, and near-normal liver architecture. |
Spirulina species have been used as food for thousands of years. Among blue-green algae, many species have documented biomodulatory effects. Many medicinal properties have been attributed to Spirulina species, including reduction in body weight,32reduction of cholesterol,33 increased activity of lipase,34reduction of glucose levels,35 modulation of carcinogen metabolic enzymes,36 modulation of lead toxicity37 and radical-scavenging action.38
Carbon tetrachloride (CCl4) is a well-known hepatotoxic agent. The changes associated with CCl4-induced liver damage are similar to those of acute viral hepatitis.39 CCl4-induced liver damage is a classic model used for the screening of hepatoprotective drugs. The basis of its hepatotoxicity lies in its biotransformation by the cytochrome P450 system to two radicals. The first metabolite, a trichloromethyl radical, forms covalent adducts with lipids and proteins; it can interact with O2 to form a second metabolite, a trichloromethyl peroxyl radical, or can remove hydrogen atoms to form chloroform. This sequence of events leads to lipid peroxidation of membranes and consequent liver injury. In response to this hepatocellular injury, “activated” hepatic Kupfer cells release increased quantities of active oxygen species and other bioactive agents.40
Since radicals play such an important role in CCl4-induced hepatotoxicity, it seems logical that compounds that neutralize such radicals may have a hepatoprotective effect. Indeed, various natural products have been reported to protect against CCl4-induced hepatotoxicity.41 Ample experimental and epidemiological studies support the involvement of oxidative stress in the pathogenesis and progression of several chronic diseases. In the present study it was found that EESL could effectively scavenge the radicals in a dose-dependent manner. It has been reported that CCl4 caused significant increase in hepatic lipid peroxidation due to radical injury in cirrhotic livers of rats.42 In the present study, elevated levels of TBARS and CD observed in CCl4-treated rats indicate excessive formation of radicals and activation of the lipid peroxidation system, resulting in hepatic damage. The significant decline in the concentration of these constituents in the livers of rats treated with CCl4 + EESL indicates the anti-lipid-peroxidative effect of S. laxissima.
The antioxidant properties of Nostoc sphaeroides Kützing (Nostocaceae) and Aulosira fertilisima Ghose (Nostocaceae) on CCl4-induced hepatic damage in rats had been reported earlier from our laboratory.43 It has been reported that C-phycocyanin from Spirulina platensis effectively inhibited CCl4-induced lipid peroxidation in rat liver in vivo.44GSH is a major non-protein thiol in living organisms which plays a central role in coordinating the body's antioxidant defense processes.45 Decline in the GSH content in the liver of CCl4-intoxicated rats, and its subsequent return towards the near-normality in the group administered with CCl4 + EESL, also reveal the anti-lipid-peroxidative effect of S. laxissima.
Estimating the activities of serum marker enzymes such as AST, ALT, ALP and GGT, can enable assessment of liver function. When the liver cell plasma membrane is damaged, a variety of enzymes normally located in the cytosol are released into the bloodstream. Their amount in the serum is a useful quantitative marker of the extent and type of hepatocellular damage.46 The normalization of the above enzyme levels seen in rats treated with the cyanobacterial formulation (100 mg kg−1 body weight) indicates the possibility of EESL being able to inhibit liver cell injury and reducing the leakage of the above enzymes in to the blood. It has been reported that serum transaminases return to normal levels with the healing of liver parenchyma and regeneration of liver cells.47 It was also reported that the alcohol extract of Spirulina maxima Geitler (Pseudanabaenaceae) inhibited lipid peroxidation more significantly than the chemical antioxidants like α-tocopherol and β-carotene.48Phycocyanin significantly reduced the hepatotoxicity caused by CCl4, which induces the formation of radicals. The hepatoprotective effect of phycocyanin was therefore attributed to the inhibition of reactions involved in the formation of reactive metabolites, and possibly due to its radical-scavenging activity.49
The site-specific oxidative damage of some of the susceptible amino acids of protein is now regarded as the major cause of metabolic dysfunction during pathogenesis. The capacity of liver synthesize albumins is adversely affected by hepatotoxins.50 Administration of hepatotoxins like CCl4 causes depression in protein biosynthesis, which is due to the disruption and disassociation of polyribosomes from the endoplasmic reticulum. The lowered levels of total proteins recorded in the serum of CCl4-treated rats can be attributed to these features. Attainment of near-normality in protein content of serum in rats treated with CCl4 + LP + EESL further confirmed the anti-hepatotoxic effect of S. laxissima.
The body has an effective mechanism to prevent and neutralize radical-induced damage. This is accomplished by a set of endogenous antioxidant enzymes, such as SOD, CAT, GPX and GST. When the balance between ROS production and antioxidant defenses is lost, oxidative stress results, which through a series of events deregulates the cellular functions, leading to various pathological conditions.51 Any compound, natural or synthetic, with antioxidant properties may contribute towards the partial or total alleviation of this type of damage. In the present study, decline in the level of antioxidant enzymes like SOD, CAT, GPX and GST observed in CCl4-treated rats is a clear manifestation of the excessive formation of radicals and activation of lipid peroxidation system, resulting in tissue damage. The significant increases in the concentration of these constituents in liver tissues of animals treated with CCl4 + EESL indicate the antioxidant effect of EESL. It was reported that Dunaliella salina Teodoresco (Dunaliellaceae), a green marine alga, has the ability to protect against oxidative stress in vivo using animal models.52 It has been established that carotenoids from microalgae exert their action against CCl4 liver injury by lipid peroxidation, either through decreased production of radical derivatives or due to the antioxidant activity of the protective agent itself.53 The antioxidant activity of Laminaria japonica Areschoug (Laminariaceae) and Ecklonia stolonifera Okamura (Lessoniaceae) against CCl4-induced hepatotoxicity has been reported.54
During attack by toxins, the lipid profile of serum and tissues increases. CCl4-poisoned rats appear to have a deranged hepatic triglyceride secretory mechanism. Accumulation of triglycerides in the liver during CCl4 poisoning results not from a defect in the release of triglycerides into plasma, but perhaps from an increase in hepatic synthesis of triglycerides.55 Treatment of rats with CCl4 induces centrilobular necrosis, which results in the accumulation of fat in the liver. Fat from the peripheral adipose tissue is translocated to the liver and kidney, leading to its accumulation during toxicity. CCl4 also interferes with hepatic phospholipid synthesis. Intoxication of experimental animals with CCl4 altered the membrane structure and function, as shown by the increases in cholesterol and phospholipid concentrations, and hence an increased cholesterol-to-phospholipid ratio.56 Pre-treatment of experimental animals with EESL extract prevented the alterations of membrane fluidity, with a decrease in the cholesterol-to-phospholipid ratio, which was elevated by animals treated just with CCl4 alone. Thus S. laxissima plays a role in peroxidation by inhibiting the radical attack on biomembranes. It has been reported that C-phycocyanin from Spirulina platensis effectively inhibits marked CCl4-induced changes in the lipid profile of the rat liver.57
Footnote |
† Present address: Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India. |
This journal is © The Royal Society of Chemistry 2011 |