Antioxidant and hepatoprotective effects of a pigment–protein complex from Chlorella vulgaris on carbon tetrachloride-induced liver damage in vivo

Abstract

The antioxidant and hepatoprotective activities of pigments in the natural form of a pigment–protein complex were investigated. Pigment–protein complex (PPC) was isolated from Chlorella vulgaris through thylakoid protein solubilization, anion exchange chromatography and gel filtration chromatography. PPC possessed a chlorophyll : lutein : protein ratio of 94 : 153 : 100 (w/w/w) according to HPLC and spectrophotometric analysis. Various antioxidant evaluation systems were used to evaluate the antioxidant activity of PPC in vitro. Results showed that PPC exhibited significant DPPH radical scavenging activity with an IC₅₀ of 313 μg mL⁻¹. Distinct Fe²⁺ ion chelating activity, reducing capacity and lipid peroxidation inhibition activity were observed at a concentration of 1 mg mL⁻¹. For the hepatoprotective effects, administration of PPC at different concentrations (50 and 100 mg kg⁻¹ BW) could significantly decrease the carbon tetrachloride-induced elevation of the hepatosomatic index. Increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were attenuated with PPC pretreatment. In addition, PPC effectively restored suppressed hepatic superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities. Moreover, PPC significantly reduced the formation of malondialdehyde (MDA). The results obtained from this study clearly verified the hepatoprotective effect of PPC in CCl₄-induced hepatotoxicity in vivo, suggesting that PPC has the potential to be exploited as a dietary supplement against free radical oxidation which could enhance resistivity against oxidative stress in the human body.

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1. Introduction

Free radicals such as the superoxide anion radical (˙O₂⁻) and hydroxyl radical (˙OH) are highly reactive species with single and unbalanced electrons, which are involved in the oxidation process of biological molecules, and will cause many adverse effects in food and biological systems.¹ In human organs, free radicals, which are inevitably produced through oxidative metabolism, induce several diseases like arteriosclerosis and cancer. Liver damage is a widespread disease which can be caused by several compounds, such as ethanol, CCl₄ and bromobenzene. The mechanism of liver injury induced by chemical compounds is thought to involve excess free radical generation and lipid peroxidation.² It has been a hot topic in the field of biochemical nutrition to search for safe antioxidants to prevent free radical-induced damage.³

Natural pigments, extracted from animals, plants and microorganisms, are one of the most important antioxidant systems that can neutralize free radicals in the cells.^4,5 It was shown that high radiant energy increased carotenoid concentration in cells, presumably to prevent photooxidation⁶ and some carotenoids strongly contributed to the protection of plants against photooxidative damage as direct quenchers of reactive oxygen species (ROS).⁷ The antioxidant activity of carotenoids such as luteins may be due to the conjugated double bonds and the phenolic hydroxyl groups at both ends of their chemical structures.⁸ Chlorophylls are also important antioxidants. They can act as lipid antioxidants in stored edible oils⁹ and prevent oxidative DNA damage and lipid peroxidation both by reducing ROS and chelating metal ions.¹⁰ However, solvent extraction is the main industrially applicable method for pigment extraction,¹¹ and consequently, the massive use of organic solvents is not environmentally friendly and destroys the natural matrix of the pigments. Actually, hydrophobic pigments in cells are found associated with the hydrophobic domains of lipid–protein complexes,¹² and they play a role in photosynthesis in the form of light-harvesting complexes (LHCs) in most plants and algae.¹³ Previous studies of LHCs mainly focused on their pigment composition, structure and energy transfer,^14,15 and by contrast, the functional activities of pigments in the natural form of the pigment–protein complex are rarely reported.

Marine algae, which have traditionally formed part of the Oriental diet, especially in China, Japan and Korea, are becoming a hot research topic because of their biological components.¹⁶ Chlorella vulgaris, which belongs to the marine microalgae, contains a large number of active substances which are beneficial to the human body, such as pigment, fat, unsaturated fatty acids, proteins, polysaccharides, squalene etc. It is usually composed of 0.2–0.5% lutein, 2–4% chlorophyll and the protein content reaches up to 50% (dry weight).¹⁷ It would be interesting to isolate the natural matrix of pigments through aqueous extraction instead of organic solvent extraction, and investigate the activities of pigments in the natural form of the pigment–protein complex (PPC). Therefore, this study aims to isolate the natural pigment–protein complex from Chlorella vulgaris and evaluate its antioxidant activities in vitro and hepatoprotective effects in CCl₄-induced hepatotoxicity in vivo. The study is of significance in suggesting that the natural protein-based pigment complex has potential for making dietary supplements against oxidation and in increasing resistivity against oxidative stress in the human body.

2. Materials and methods

2.1. Materials

The algae Chlorella vulgaris STIO02 was kindly provided by Third Institute of Oceanography, State Oceanic Administration, P. R. China and was stored at −20 °C before use.

Toyopearl DEAE-650M was the product of TOSOH Co. (Japan). Sephadex G-50 Fine was purchased from GE Healthcare (USA). 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and lutein were obtained from Sigma Chemical Co. (USA). The Bicinchoninic acid (BCA) protein kit and all the kits for the biochemical indexes analysis used in the animal experiment were the products of Nanjing Jiancheng Bioengineering Institute (China). Methanol and acetonitrile used in the liquid chromatography were of HPLC grade. All other chemicals and reagents used were of analytical grade and commercially available.

2.2. Solubilization of thylakoid proteins

The Chlorella vulgaris algae were lysed in phosphate buffer (20 mM, pH 7.0) by ultrasonication in an ice bath. Cell debris was removed by centrifugation at 10 000 rpm for 10 min at 4 °C. Then a 25% saturated ammonium sulfate precipitation was applied to collect the membranes. Here, CHAPS, a zwitterionic detergent that can effectively maintain protein activity, was used to solubilize the intrinsic membrane-associated pigment–protein complex. The membrane fraction was re-suspended in Tris–HCl buffer (20 mM, pH 8.0) and 1% CHAPS was added. The mixture was stirred in the dark for 1 h at 4 °C and then centrifuged at 12 500 rpm for 30 min. The supernatant was collected for further purification.

2.3. Isolation and purification of pigment–protein complex

A Toyopearl DEAE-650M column (Φ 1.6 × 20 cm) was previously equilibrated with Tris–HCl buffer (20 mM, pH 8.0) containing 8 mM of CHAPS. The solubilized thylakoid proteins were loaded onto the column and then washed with the same buffer followed by a linear gradient of 0–0.5 M NaCl with a flow rate of 0.5 mL min⁻¹ and 10 min tube⁻¹. The absorbances of all fractions were monitored at 430 nm and 280 nm. The pigment protein fractions were pooled and concentrated by ultrafiltration. The concentrate was then applied to a Sephadex G-50 column (Φ 1.6 × 100 cm) that was pre-equilibrated with Tris–HCl buffer (20 mM, pH 8.0) containing 8 mM of CHAPS and 50 mM of NaCl. The column was used with the same buffer and the flow rate was 0.3 mL min⁻¹. The absorbance of the fractions was monitored and the eluted fractions containing the pigment–protein complex were collected.

2.4. Characterization of pigment–protein complex

2.4.1. Absorption spectroscopy. The absorption spectrum of the pigment–protein complex was recorded with a U-2910 spectrophotometer (HITACHI Co., Japan) at room temperature.

2.4.2. Chlorophyll determination. Chlorophyll content was determined as described by Arnon.¹⁸ The sample was vigorously shaken with 4 volumes of acetone and kept in the dark for 20 min, then centrifugation at 12 000 rpm for 5 min was applied. The absorbance of the supernatant at 645 and 663 nm was measured. Chlorophyll content was calculated according to the following equation:

Chlorophyll (μg mL⁻¹) = 20.2A₆₄₅ + 8.02A₆₆₃.

2.4.3. Lutein analysis. Five volumes of methanol was added to the complex solution, followed by centrifugation to remove the precipitate. The pigment extraction was applied to a Shimadzu LC-20A system equipped with a Unimicro SP-120-5-C18-AP (4.6 mm × 250 mm, 3 μm) column. The mobile phase was 80% methanol and 20% acetonitrile and the absorbance was monitored at 443 nm.

2.4.4. Protein determination. The protein component of the pigment–protein complex was separated using the method described by Maxwell.¹⁹ 50 μL of the sample was mixed with 1 mL of 90% (v/v) acetone followed by centrifugation at 12 000 rpm for 5 min to removed the pigments. The pellet was re-suspended in 50 μL of 2% (w/v) SDS and then heated at 60 °C for 30 min. The protein content was quantified using a bicinchoninic acid kit according to the protocol.

2.5. Antioxidant activity in vitro

2.5.1. DPPH radical scavenging activity. The DPPH radical scavenging activity was tested as described²⁰ with some modifications. 1 mL of PPC was mixed with 1 mL of ethanol solution of DPPH (0.1 mM) and then kept in the dark for 30 min. The absorbance was measured at 517 nm. A control sample containing 1 mL of DPPH solution and 1 mL of distilled water was prepared. The antioxidant activity of the sample was evaluated by the scavenging rate of the DPPH radical with the following equation:

DPPH radical scavenging activity (%) = (A_control − A_sample)/A_control × 100,

where A_sample and A_control were the absorbance of the sample and control group, respectively.

2.5.2. Metal chelating activity. The metal chelating activity was determined according to the method described by Dinis²¹ with some modifications. 1 mL of PPC was mixed with 4.7 mL of H₂O and then added to 0.1 mL of 2 mM FeCl₂. Instead of PPC, the same volume of deionized water was used in a control experiment. The reaction was initiated by the addition of 0.2 mL of 5 mM ferrozine. The mixture was shaken vigorously and then kept at room temperature for 20 min and the absorbance was measured at 562 nm. The metal chelating activity of the samples was calculated by the following equation:

Metal chelating activity (%) = (A_control − A_sample)/A_control × 100, (1)

where A_sample and A_control were the absorbance of the sample and control group, respectively.

2.5.3. Reducing capacity. The reducing capacity of PPC was estimated using the method described by Oyaizu.²² 1 mL of PPC was mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50 °C for 20 min and then 2.5 mL of 10% trichloroacetic acid was added. The mixture was centrifuged at 1000 × g for 10 min and 2.5 mL of the supernatant was mixed with 2.5 mL of distilled water and 0.5 mL of 1% FeCl₃. The absorbance was then measured at 700 nm for 10 min. An increased absorbance of the reaction mixture indicated an increased reducing capacity.

2.5.4. Lipid peroxidation inhibition activity. The lipid peroxidation inhibition activity of PPC was measured in a linoleic acid emulsion system according to the method introduced by Osawa and Namiki²³ with some modification. Briefly, PPC was dissolved in distilled water to a concentration of 1 mg mL⁻¹ and then mixed with 2 mL of ethanol, 26 μL of linoleic acid and 2 mL of phosphate buffer (50 mM, pH 7.0). The mixture was incubated in a colorimetric tube with a plug at 40 °C in the dark. Here, BHT at 1 mg mL⁻¹ was used as positive control. The degree of oxidation was measured at 24 h intervals using the ferric thiocyanate (FTC) method described by Mitsuda.²⁴ 100 μL of the reaction solution was added to a solution of 4.7 mL of 75% ethanol, 0.1 mL of 30% ammonium thiocyanate and 0.1 mL of 20 mM ferrous chloride solution in 3.5% of HCl. After 3 min, the degree of color development which represented the linoleic acid oxidation was measured spectrophotometrically at 500 nm.

2.6. Effect on CCl₄-induced hepatic injury in mice

2.6.1. Animals and treatments. Fifty male Kunming mice of 29–30 g weight were purchased from Slac Laboratory Animal Center (Shanghai, China). The experiments were carried out in accordance with the guidelines issued by the Ethical Committee of Fujian Medical University (Fujian, China). Fifty mice were randomly divided into five groups with ten mice in each group. Group 1 served as a blank control. Group 2 was the CCl₄ control group in which mice were treated with CCl₄ alone. Group 3 was a positive control group in which mice were administered lutein at 20 mg kg⁻¹ body weight (BW) along with CCl₄. Groups 4 and 5 were the treatment groups in which mice were administered PPC at 100 mg kg⁻¹ BW and 50 mg kg⁻¹ BW respectively along with CCl₄. PPC suspended in deionized water and lutein suspended in peanut oil were administered to the mice at the set dose once daily by gavage for 11 days. Mice in group 1 and 2 were orally administered the same volume of deionized water. One hour after substance administration on the 10th day, all the groups were treated with single dose of 0.1% CCl₄ solution in peanut oil (10 mL kg⁻¹ BW) by gavage except for group 1 which was treated with the same volume of peanut oil. Thirty-six hours after CCl₄ treatment, mice were sacrificed by decapitation.

2.6.2. Determination of the hepatosomatic index. Mice were weighed and then carefully sacrificed. The liver tissues were taken, washed and weighed. The hepatosomatic index was defined as the ratio of wet liver weight to body weight.

2.6.3. Analysis of serum biochemical indexes. Blood samples from each mouse were collected in EDTA-anticoagulant tubes from the orbit before sacrifice. After centrifugation, the serum was collected and the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed according to the protocols of the relevant diagnostic kits.

2.6.4. Analysis of hepatic biochemical indexes. Liver tissues were homogenized in cold normal saline. The supernatant was collected after centrifugation at 3000 rpm for 10 min at 4 °C, and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were determined using the corresponding diagnostic kits. The total protein content of the liver homogenate was measured according to the method described by Bradford,²⁵ using bovine serum albumin as the standard.

2.7. Statistical analysis

All data are presented as means ± standard deviation (SD). Statistical evaluation was carried out with IBM SPSS 19.0 software. Comparisons of multiple treatment conditions were analyzed using one-way analysis of variance (ANOVA) with Duncan’s test for post hoc analysis and p < 0.05 values were considered as statistically significant.

3. Results and discussion

3.1. Isolation of pigment–protein complex from Chlorella

A crude pigment–protein complex extract of Chlorella was obtained by aqueous extraction with the addition of CHAPS and chromatography was employed to improve the purity of the pigment–protein complex. Through DEAE anion-exchange chromatography, the crude extract was divided into four fractions (Fig. 1a). Fraction P2 exhibited an obvious absorbance at both 280 nm and 430 nm and was dark green. For further purification, fraction P2 was concentrated by ultrafiltration and applied to a Sephadex G-50 column. Three fractions were obtained as shown in Fig. 1b. The pigment–protein complex was mainly present in fraction P2-2 while the other fractions contained only proteins. The absorbance spectrum of P2-2 is shown in Fig. 2. The profile was slightly similar to that of the crude extract, indicating the reasonable recovery achieved after isolation through chromatography. The profile included obvious chlorophyll absorbance at 430 and 672 nm. The peak at 482 nm indicated the presence of lutein when compared with the standard marker pigments (data not shown).

Fig. 1 Purification of the pigment–protein complex from Chlorella. (a) DEAE anion-exchange chromatography, (b) Sephadex G-50 gel filtration chromatography.

Fig. 2 Absorption spectrum of the pigment–protein complex.

At the same time, increased A₄₃₀/A₂₈₀ and A₄₈₂/A₂₈₀ purity ratios for PPC could be observed. Through two-step chromatography, the relative absorbance of A₄₃₀/A₂₈₀ increased almost 3 fold, and the content of chlorophyll was determined to be 0.94 g g⁻¹ protein by spectrophotometry (Table 1). There was also a 2.6 fold increase in purity ratio of A₄₈₂/A₂₈₀ through the isolation procedure. The major carotenoid existing in the fraction was confirmed to be lutein by HPLC and was quantified to be 1.53 g g⁻¹ protein for PPC (Table 2).

Table 1 Chlorophyll content and purity ratio of the pigment–protein complex from Chlorella vulgaris

	Chlorophyll (g g^−1 protein)	Purity ratio (A430/A280)
Crude extract	0.40	1.15
PPC	0.94	3.17

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Table 2 Lutein content and purity ratio of the pigment–protein complex from Chlorella vulgaris

	Lutein (g g^−1 protein)	Purity ratio (A482/A280)
Crude extract	0.69	0.37
PPC	1.53	0.95

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3.2. Antioxidant activity of the pigment–protein complex in vitro

To evaluate the antioxidant activity of the pigment–protein complex from Chlorella vulgaris, different antioxidant parameters were determined in vitro.

3.2.1. Free radical scavenging activity. The free radical scavenging activity of PPC was determined by the scavenging rate of the DPPH radical. The DPPH radical has an unpaired valence electron at one atom of a nitrogen bridge and it can accept a hydrogen radical or electron to become stable with the color changing from dark purple to colorless and the maximum absorbance at 517 nm being reduced.²⁶ As shown in Fig. 3a, PPC could scavenge the DPPH radical effectively in a dose-dependent manner. The IC₅₀ value of PPC against the DPPH radical was determined to be 313 μg mL⁻¹. Previous studies showed that pigments such as lutein and chlorophyll could provide hydrogen or electrons to reduce free radicals such as DPPH.¹⁰ PPC, which possesses a high content of lutein and chlorophyll, presumably trapped the DPPH radical as an electron or hydrogen donor.

Fig. 3 Antioxidant activity of PPC in vitro. (a) DPPH radical scavenging activity, (b) metal chelating activity, (c) reducing capacity, (d) lipid peroxidation inhibition activity.

3.2.2. Metal chelating activity. Some transition metals can trigger the formation of free radicals via a chain reaction through electron transport. Fe²⁺ is one of the most important pro-oxidants since it can catalyze the Fenton reaction to stimulate lipid peroxidation and also accelerate peroxidation by decomposing lipid hydroperoxides into peroxyl and alkoxyl radicals that can themselves abstract hydrogen and perpetuate the chain reaction of lipid peroxidation.²⁷ Thus, the chelation of Fe²⁺ can retard metal-catalyzed oxidation. The results showed that PPC could chelate Fe²⁺ and block the formation of the Fe²⁺–ferrozine complex dose-dependently (Fig. 3b). The percentage of Fe²⁺ chelated by PPC reached up to 45% at a concentration of 1 mg mL⁻¹. The PPC chelation was considered to be attributed to the chlorophyll in the complex. Chlorophylls are Mg²⁺–porphyrin derivatives and porphyrin was found to be the essential structure for the antioxidant activity of chlorophyll derivatives. Chlorophylls can chelate Fe²⁺ by virtue of the substitution of the Mg²⁺ in the porphyrin for the Fe²⁺.¹⁰

3.2.3. Reducing capacity. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. For the measurement of the reducing capacity of PPC, the transformation of Fe³⁺ to Fe²⁺ in the presence of the complex was investigated. As shown in Fig. 3c, the reducing capacity of PPC increased with increasing concentration. At a concentration of 1 mg mL⁻¹, PPC exhibited a reducing capacity of 0.33. The results indicated that PPC has the potential to react with free radicals and block radical chain reactions.

3.2.4. Lipid peroxidation inhibition activity. Lipid peroxidation is a complicated process, which is thought to proceed via radical mediated abstraction of hydrogen atoms from methylene carbons, resulting in various highly reactive electrophilic aldehydes, such as peroxyl and alkoxyl radicals that can form pre-existing lipid peroxide to initiate lipid peroxidation.²⁸ The inhibitory effect of PPC on lipid peroxidation was investigated in a linoleic acid emulsion system. The results showed that PPC could almost completely suppress lipid peroxidation at 1 mg mL⁻¹, which was comparable to the effect of BHT at the same concentration (Fig. 3d). The antioxidant effects of chlorophyll and lutein on the autoxidation of oils were verified previously.^8,9 The present study showed that PPC exhibited multiple antioxidant activities, assuming the possibility that the action of PPC as a lipid peroxidation protector may be related to its radical scavenging activity, iron binding capacity and reducing capacity.

3.3. Effect on CCl₄-induced hepatic injury in mice

CCl₄ is a widely known hepatotoxic revulsant, the metabolism of which results in the rapid generation of reactive radicals such as the trichloromethyl peroxyl radical (CCl₃O₃˙) and then initiates lipid peroxidation, leading to cell membrane damage, intracellular enzyme leakage and even cell necrosis. It has been demonstrated that hepatoprotective effects may be associated with antioxidant capacity.²⁹ The natural pigment, carotenoid lutein, which has been verified to have significant hepatoprotective effects in CCl₄-induced liver damage, was used at 20 mg kg⁻¹ BW as a positive control.³⁰

3.3.1. Effect of PPC on the hepatosomatic index in mice. As shown in Fig. 4, the hepatosomatic index of the mice treated with CCl₄ increased markedly when compared to the control group (p < 0.05), which was the symptom of fatty liver. The biotransformed metabolites of CCl₄, including the trichloromethyl radical (CCl₃˙) can result in the accumulation of triglycerides (TG) in the liver and the development of fatty liver through blocking protein synthesis and causing a lipid metabolism disorder.³¹ Pretreatment with PPC at different doses (50, 100 mg kg⁻¹ BW) could significantly decrease the CCl₄-induced increase in the hepatosomatic index and the effects were comparable to the positive control (Fig. 4).

Fig. 4 Effect of PPC on the hepatosomatic index in mice. * denotes statistical significance compared with the CCl₄-treated group, where p < 0.05. △ denotes statistical significance compared with the control group, where p < 0.05.

3.3.2. Effect of PPC on serum ALT and AST. Fig. 5 presents the effects of PPC on serum ALT and AST. Mice treated with CCl₄ alone showed serious acute liver damage, as revealed by a significant increase in serum ALT and AST (p < 0.05), which indicated the loss of hepatocyte membrane integrity and the release of transaminase from the cytoplasm to the serum. Treatment with PPC at 100 and 50 mg kg⁻¹ BW attenuated the increase in ALT activity by 29.3% and 13.1% respectively, and produced a 35.5% and 24.11% suppression in AST activity, indicating stabilization of the cell membrane and mitigation of the hepatic damage caused by CCl₄. In addition, the effects of PPC at 100 mg kg⁻¹ BW on the serum ALT and AST were superior to the positive control.

Fig. 5 Effect of PPC on the activities of serum (a) ALT and (b) AST. * denotes statistical significance compared with the CCl₄-treated group, where p < 0.05. △ denotes statistical significance compared with the control group, where p < 0.05.

3.3.3. Effect of PPC on hepatic MDA, SOD, CAT and GSH-Px. Treatment with CCl₄ promoted lipid peroxidation in the liver, the extent of which was specified by the level of MDA. MDA is the end-product of lipid peroxidation and is widely used as a marker of lipid peroxidation mediated by free radicals.² An enhanced hepatic MDA level could be observed after CCl₄ treatment (Fig. 6a). Pretreatment with PPC at 50, 100 mg kg⁻¹ BW could reduce the formation of MDA dose-dependently, indicating that PPC could inhibit lipid peroxidation induced by CCl₄ in the liver.

Fig. 6 Effect of PPC on the activities of hepatic (a) MDA, (b) SOD, (c) CAT and (d) GSH-Px. * denotes statistical significance compared with the CCl₄-treated group, where p < 0.05. △ denotes statistical significance compared with the control group, where p < 0.05.

Antioxidant enzymes such as SOD, CAT and GSH-Px act as the first line of defense against free radical induced oxidative stress. SOD is an effective defense enzyme that catalyzes the reduction of super anions into H₂O₂ and O₂, and H₂O₂ can be further converted into H₂O and O₂ by CAT and GSH-Px.³² The status of these enzymes is an appropriate indirect assessment of the pro-oxidant-antioxidant status in tissues.²⁹ The results showed that the activities of SOD, CAT and GSH-Px were remarkably decreased after exposure to CCl₄ by 13.1%, 32.1% and 17.3% respectively (Fig. 6b–d), indicating oxidative damage to the liver. However, the suppressed enzyme activities could be restored by PPC pretreatment and the effects were significant at a concentration of 100 mg kg⁻¹ BW (p < 0.05), implying that the hepatoprotective effects of PPC in CCl₄-induced liver damage result from the stabilization of intracellular antioxidant defense systems.

The antioxidant activity of an antioxidant compound has been attributed to various mechanisms, among which are radical scavenging, binding of transition metal ion catalysts, reductive capacity, prevention of chain reaction initiation, decomposition of peroxides and prevention of continued hydrogen abstraction.²⁷ The results obtained from this study clearly indicate that PPC had powerful antioxidant activity via various antioxidant mechanisms in vitro. Protein provided the pigments with good stability and solubility in aqueous solution, and the hepatoprotective effects of PPC might be attributed to the various antioxidant effects of the pigments.

4. Conclusions

The antioxidant and hepatoprotective activities of pigments in the form of a pigment–protein complex were investigated in the present study. The results showed that the natural pigment–protein complex isolated from Chlorella vulgaris exhibited significant antioxidant activities in vitro, including DPPH radical scavenging, metal chelation, reducing capacity and lipid peroxidation inhibition. Moreover, PPC manifested discernible protective action in CCl₄-induced hepatotoxicity in vivo, indicating that PPC could be a promising candidate for use in dietary supplements against free radical-induced damage.

Acknowledgements

This work was supported by National Natural Science Foundation of China (No. 31571779), High & New project of Fujian Marine Fisheries Department (No. [2015]20), the National Marine Public Welfare Project (No. 201305022), Regional Demonstration of Marine Economy Innovative Development Project (No. 12PYY001SF08).

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