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Isolation of a novel lutein–protein complex from Chlorella vulgaris and its functional properties Xixi Cai, Qimin Huang and Shaoyun Wang* A novel kind of lutein–protein complex (LPC) was extracted from heterotrophic Chlorella vulgaris through aqueous extraction. The purification procedure contained solubilization of thylakoid proteins by a zwitterionic detergent CHAPS, anion exchange chromatography and gel filtration chromatography. Both wavelength scanning and HPLC analysis confirmed that lutein was the major pigment of the protein-based complex, and the mass ratio of lutein and protein was determined to be 9.72 : 100. Besides showing lipid peroxidation inhibition activity in vitro, LPC exerted significant antioxidant effects against ABTS and DPPH radicals with IC50 of 2.90 and 97. 23 μg mL−1, respectively. Meanwhile, in vivo antioxidant activity of the complex was evaluated using the mice hepatotoxicity model; LPC significantly suppressed the carbon tetrachloride-induced elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase
Received 30th November 2014, Accepted 19th April 2015 DOI: 10.1039/c4fo01096e www.rsc.org/foodfunction
1.
(AST) activities, and decreased hepatic malondialdehyde (MDA) levels and the hepatosomatic index. Moreover, LPC could effectively restore the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in the treated mice livers. Our findings further the progress in the research of natural protein-based lutein complexes, suggesting that LPC has the potential in hepatoprotection against chemical induced toxicity and in increasing the antioxidant capacity of the defense system in the human body.
Introduction
Lutein, the second most prevalent carotenoid, is widely distributed in dark green leafy plants and algae, and also accumulates in the macular region of the retina acting as an antioxidant.1 Lutein has been described as an excellent antioxidant because of its ability to quench singlet oxygen and other electronically excited molecules, and the antioxidant activity may be due to its conjugated double bonds and the phenolic hydroxyl groups on both ends of its chemical structure.2 Lutein is an essential nutrient for organisms since humans cannot synthesize lutein de novo. It is used as an important ingredient in dietary supplements and more recently in medicine, cosmetics and animal feed.3,4 However, poor solubility and emulsibility in aqueous solution severely restrict its wide application in food systems. Chlorella vulgaris, which contains 0.2–0.5% lutein, has more and more advantages of being the major source for extracting lutein for its short cycle, high yield and easy culture on a large scale when compared with plants.5–7 Solvent extraction is the main industrially applicable method for lutein extraction.3 As a result, the massive use of organic solvents such as hexane goes against environmental friendliness and destroys the natural matrix of lutein. Because of its hydro-
phobic nature, lutein in plants and animal cells is found in associated with the hydrophobic domain of lipid-protein complexes.8 Lutein is located in the thylakoid membrane in the form of light-harvesting complexes (LHCs) in plants and algae, playing the function of photosynthesis and chlorophyll photoprotection.9 LPC plays crucial roles in lutein stabilization and transfer, and the unique chromophore interacts with protein in these complexes contributing to the coloration of related tissue.10 Numerous experiments have been performed to isolate and analyze the pigment composition, structure and energy transfer of LHCs.11,12 However, the isolation of the natural lutein– protein complex and the activities of lutein in the form of a pigment–protein complex are rarely reported. It is interesting to obtain the natural matrix of lutein extract through aqueous extraction instead of using an organic solvent, and investigate the influence of protein interaction on the activities of lutein. Therefore, the objective of this study was to isolate the natural lutein–protein complex from Chlorella vulgaris and evaluate the antioxidant properties in vitro and hepatoprotective activity in CCl4-induced hepatotoxicity in mice.
2. Materials and methods 2.1.
College of Bioscience and Biotechnology, Fuzhou University, Fuzhou 350108, China. E-mail: [email protected]; Fax: +86-591-22866278; Tel: +86-591-22866375
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Materials
The algae material Chlorella vulgaris STIO02 with a relatively high lutein content was kindly provided by the Third Institute
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of Oceanography, State Oceanic Administration, P. R. China and was stored at −20 °C before use. Toyopearl DEAE-650M was purchased from TOSOH Co. (Japan). Sephadex G-50 Fine was the product of GE Healthcare (USA). 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis3-ethylbenzthiazoline-6-sulphonate (ABTS), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and lutein were obtained from Sigma Chemical Co. (USA). Bicinchoninic acid (BCA) protein kit, alanine aminotransferase (ALT) kit, aspartate aminotransferase (AST) kit, malondialdehyde (MDA) kit, superoxide dismutase (SOD) kit, catalase (CAT) kit and glutathione peroxidase (GSH-Px) kit were the products of Nanjing Jiancheng Bioengineering Institute (China). Methanol and acetonitrile used in liquid chromatography were of HPLC grade. All other chemicals and reagents used were of analytical grade and commercially available. 2.2.
Characterization of LPC
2.4.1. Absorption spectroscopy. The absorption spectrum of the lutein–protein complex was recorded on a U-2910 spectrophotometer (Hitachi Co., Japan) at room temperature. 2.4.2. Lutein determination. The lutein content of LPC was extracted from the lutein–protein complex by adding five volumes of methanol to the protein solution, followed by centrifugation to remove the precipitate. For High Performance Liquid Chromatography (HPLC) analysis, the pigment 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. 2.4.3. Protein determination. The protein content of the lutein–protein complex was separated by the method described by Maxwell11 and quantified by bicinchoninic acid.
Solubilization of thylakoid proteins
Algae were washed several times with cold distilled water and broken in phosphate buffer (20 mM, pH 7.0) by ultrasonication in an ice bath. Cell debris was removed by centrifugation (10 000 rpm for 10 min, 4 °C) and membranes were collected by 25% saturation ammonium sulfate precipitation. Then the membrane fraction was re-suspended in Tris-HCl buffer (20 mM, pH 8.0) to a concentration of chlorophyll a (Chl a) of 0.25 mg mL−1.13 Since lutein–protein complexes in Chlorella cells are intrinsic membrane-associated, it is necessary to use detergents to solubilize the thylakoid membrane proteins. Here, CHAPS, a zwitterionic detergent that can well maintain protein activity, was used. Solubilization of the membranes was carried out by the addition of 1% CHAPS for 1 h in the dark at 4 °C. Then the mixture was centrifuged at 12 500 rpm for 30 min at 4 °C. The supernatant was collected and used for further purification. 2.3.
2.4.
Isolation and purification of LPC
The solubilized thylakoid proteins were loaded onto a Toyopearl DEAE-650M column (Φ 1.6 × 20 cm), which was previously equilibrated with Tris-HCl buffer (20 mM, pH 8.0) containing 8 mM CHAPS. The column was washed with the same buffer followed by a linear gradient of 0–0.5 M NaCl. Chromatography was carried out with a flow rate of 0.5 mL min−1 and 10 min per tube. The absorbance of all fractions was monitored at 482 nm and 280 nm, respectively. At later purification stages, cation-exchange chromatography was performed on a Sephadex SP C-25 column (Φ 1.6 × 20 cm) and the elute conditions were the same as anion-exchange chromatography described above. 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 CHAPS and 50 mM NaCl. The column was run with the same buffer and the flow rate was 0.3 mL min−1. The absorbance of the fractions was monitored and the eluted protein fractions containing the lutein–protein complex were collected.
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2.5.
Antioxidant activity in vitro
2.5.1. DPPH radical scavenging activity. The DPPH radical scavenging activity assay was tested according to the method of Wu14 with some modifications. 1 mL of LPC was placed in cuvettes, and then 1 mL of ethanol solution of DPPH (0.1 mM) was added. Thirty minutes later, the absorbance was measured at 517 nm. A control sample containing 1 mL DPPH solution and 1 mL distilled water was prepared. Samples mixed with ethanol solutions without DPPH were set as blank. The antioxidant activity of the sample was evaluated by the scavenging rate of the DPPH radical with the following equation: DPPH radical scavenging activityð%Þ ¼ ½1 ðAsample Ablank Þ=Acontrol 100; where Asample, Ablank and Acontrol were the absorbance of the sample, blank and control group, respectively. The IC50 values (the concentration of the samples required to reach the scavenging rate of DPPH radicals to 50%) were calculated. 2.5.2. ABTS radical scavenging activity. The ABTS radical scavenging activity was determined according to the method of Wang.15 The ABTS radical was generated by mixing ABTS stock solution (7 mM) with an equal volume of potassium persulfate (2.45 mM) and keeping the resulting mixture in the dark at room temperature for 16 h. The ABTS radical solution was diluted in phosphate buffer (5 mM, pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm before use. 1 mL of diluted ABTS radical solution was mixed with 1 mL of LPC. The mixture was then kept in the dark for 10 min and the absorbance was measured at 734 nm. A control sample containing ABTS radical solution and distilled water without the sample was also prepared. The ABTS radical scavenging activity of samples was calculated by the following equation: ABTS radical scavenging activityð%Þ ¼ ðAcontrol Asample Þ=Acontrol 100; where Asample and Acontrol were the absorbance of the sample and control group, respectively. The IC50 values (the concen-
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tration of the samples required to reach the scavenging rate of ABTS radicals to 50%) were calculated. 2.5.3. Lipid peroxidation inhibition activity. The lipid peroxidation inhibition activity of LPC was measured in a linoleic acid emulsion system according to the method introduced by Osawa and Namiki16 with some modifications. Briefly, 1 mL of the sample was added to a solution of 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. The degree of oxidation was measured at 24 h intervals using the ferric thiocyanate (FTC) method of Mitsuda.17 100 μL of the reaction solution was mixed with 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% HCl. After 3 min, the degree of color development that represented the linoleic acid oxidation was measured spectrophotometrically at 500 nm.
4 °C, the supernatant was collected and the activities of SOD, CAT, GSH-Px and MDA were determined with the corresponding diagnostic kits. The total protein content of liver homogenate was measured according to the method of Bradford, using bovine serum albumin as the standard.18
2.6.
3.1.
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). Mice were randomly divided into five groups with ten mice in each group. Group I was untreated with CCl4 and served as vehicle control. Group II was the CCl4 control group in which mice were treated with CCl4, without LPC. Group III was a positive control group in which mice were administrated lutein at 10 mg kg−1 per body weight (BW) along with CCl4. Groups IV and V were the treatment groups in which mice were administrated LPC at 10 mg lutein kg−1 BW and 5 mg lutein kg−1 BW respectively along with CCl4. LPC suspended in deionized water and lutein suspended in peanut oil were given to the mice at the set dose once daily by gavage for 11 days. Mice in groups I and II were orally given the same volume of deionized water. One hour after substance administration on the 10th day, all the groups were treated with a single dose of 0.1% CCl4 solution in peanut oil (10 mL kg−1 BW) by gavage except for group I which was treated with the same volume of peanut oil. Thirty-six hours after CCl4 treatment, mice were sacrificed after being decapitated. 2.6.2. Determination of hepatosomatic index. Mice were weighed and then tenderly 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 of each mouse were taken in EDTA-anticoagulant tubes from the orbit before they were sacrificed. After centrifugation, the serum was collected and activities of serum ALT and AST were analyzed according to the protocols of relevant diagnostic kits. 2.6.4. Analysis of hepatic biochemical indexes. Liver tissues were taken, weighed and then homogenized in cold normal saline. After centrifugation at 3000 rpm for 10 min at
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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 by one-way analysis of variance (ANOVA) with Duncan’s test for post hoc analysis. p < 0.05 values were considered as statistically significant.
3. Results Isolation and characterization of LPC
A crude pigment–protein extract of Chlorella vulgaris was obtained by aqueous extraction. The absorption spectrum of the crude extract was measured. An absorption peak was generated at 482 nm, which might be attributed to carotenoids including lutein (data not shown). Chromatography was employed to improve the purity of the lutein–protein complex. As shown in Fig. 1A, the crude extract of pigment protein was divided into four fractions by DEAE anion-exchange chromatography and fractions P1 and P2 exhibited obvious absorbance at both 280 nm and 482 nm. However, fraction P1 was yellow colored while P2 was dark green, indicating that there was a great amount of chlorophyll contained in P2. Therefore, fraction P2 was not considered further. The yellow fraction P1 which did not bind to the anion-exchange column was pooled and loaded onto a SP cation-exchange column under the same elution conditions. However, fraction P1 was still not absorbed (data not shown). That the solubilized protein did not bind to moderately charged cation- or anion-exchange columns indicated high relative hydrophobicity in a CHAPS detergent environment.1 For further purification, the concentrated fraction P1 was applied to a Sephadex G-50 column. Four fractions were obtained as shown in Fig. 1B. Pigment proteins were mainly contained in fraction P1-2 while the other fractions were only protein contained. In the absorbance spectrum of P1-2, shown in Fig. 2, the fraction exhibited 280 nm protein absorbance and a prominent triple peak vibronic structure typical of most luteins centered at 454 nm, indicating that P1-2 was the fraction of interest. Besides, there was a bathochromic shift of the absorbance peaks of LPC (429, 454 and 482 nm) relative to lutein (421, 443 and 472 nm), which could be explained by refractive index effects associated with dissolution of lutein in the protein components of the lipoproteins.19 The relative absorbance of A454/A280 was enhanced to 1.12 by gel filtration chromatography compared to 0.54 of DEAE anion-exchange chromatography. HPLC analysis of the most purified fraction confirmed that lutein was the major pigment of P1-2 (Fig. 3), and the mass ratio of lutein and
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Fig. 3
HPLC analysis of LPC.
Table 1
Free radical scavenging activity of LPC
IC50 (μg mL−1)a
LPC Lutein a
Fig. 1 Purification of LPC. (A) DEAE anion-exchange chromatography, (B) Sephadex G-50 gel filtration chromatography.
Fig. 2
ABTS
DPPH
2.90 ± 0.15 17.81 ± 0.06
97.23 ± 1.20 360.03 ± 0.82
Calculation was based on the content of lutein.
3.2.
Antioxidant activity in vitro
To evaluate the antioxidant activities of lutein in the form of LPC in vitro, both free radical scavenging effects and lipid peroxidation inhibition activity were investigated. Results indicated that LPC could scavenge the ABTS and DPPH radicals effectively in a dose-dependent manner (data not shown). As shown in Table 1, the IC50 values of LPC against ABTS and DPPH radicals were 2.90 and 97.23 μg mL−1, which were approximately six and four times lower than those of lutein respectively, suggesting significantly stronger free radical scavenging effects of lutein in the form of LPC. The antioxidant activity of LPC against the peroxidation of linoleic acid was investigated. As shown in Fig. 4, the control had the highest absorbance at 500 nm, indicating the highest degree of oxidation, while the samples with LPC and lutein had lower absorbance. However, lutein showed stronger prevention of linoleic acid peroxidation than LPC under the same content of lutein (100 μg mL−1), which may be due to the improvement in hydrophilicity of lutein interaction with protein.
Absorption spectrum of LPC.
3.3. protein was determined to be 9.72 : 100. On the basis of its absorption spectrum and HPLC profile, fraction P1-2 can be assigned to be LPC.
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Effect on CCl4-induced hepatic injury in mice
Many studies have shown that the hepatoprotective effects may be associated with antioxidant capacity.20 To study the antioxidant effects of LPC in vivo, the well-described CCl4-induced mice hepatotoxicity model was used.
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Fig. 4 Lipid peroxidation inhibition activity of LPC. The activity was measured in a linoleic acid emulsion system with a lutein concentration of 100 μg mL−1.
3.3.1. Effect of LPC on mice hepatosomatic index. Consumption of CCl4 induced liver swelling and reduced weight gain, and as a result the hepatosomatic index was elevated. As shown in Fig. 5, the hepatosomatic index of the mice significantly increased after CCl4 treatment (group II) when compared to the vehicle control ( p < 0.05), which is the symptom of fatty liver. The CCl4-induced increase in the hepatosomatic index could be mitigated by pretreatment with LPC at different doses (5, 10 mg lutein kg−1 BW, groups V and IV). Moreover, pretreatment with LPC containing 10 mg lutein kg−1 BW (group IV) could significantly attenuate the elevation by 15.6%, stronger than the alleviated effect of lutein alone at the same level, which was observed at 12.8% (group III). 3.3.2. Effect of LPC on serum ALT and AST. Serum transaminases such as ALT and AST were used as biochemical markers for hepatic injury. The hepatoprotective effect of LPC on the serum ALT and AST is shown in Fig. 6. Mice treated
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Fig. 5 Effect of LPC on the mice hepatosomatic index. Group I, vehicle control; group II, CCl4; group III, lutein (10 mg kg−1 BW) + CCl4; group IV, LPC (10 mg lutein kg−1 BW) + CCl4; group V, LPC (5 mg lutein kg−1 BW) + CCl4. Different lower case letters correspond to significant differences at p < 0.05.
with CCl4 alone showed moderate hepatic injury (AST/ALT < 1.0) as evidenced by a significant rise of the activities of ALT and AST in serum ( p < 0.05), which indicate the hepatocyte damage and loss of membrane integrity. Pretreatment with LPC containing 5, 10 mg lutein kg−1 BW (groups V and IV) and lutein (10 mg kg−1 BW, group III) could depress the elevated activities of serum ALT by 22.4%, 27.2%, and 26.2% respectively (Fig. 6A), and it could at the same time depress the elevated activities of serum AST by 33.28%, 42.65%, and 35.45% respectively compared with the CCl4 model group (Fig. 6B). Therefore, LPC could show a stronger depression activity on serum ALT and AST when compared to lutein alone at the level of 10 mg kg−1 BW (Fig. 6A and B). 3.3.3. Effect of LPC on hepatic SOD, CAT, GSH-Px and MDA. The effects of LPC on the hepatic biochemical indexes
Fig. 6 Effect of LPC on mice serum ALT (A) and AST (B). Group I, vehicle control; group II, CCl4; group III, lutein (10 mg kg−1 BW) + CCl4; group IV, LPC (10 mg lutein kg−1 BW) + CCl4; group V, LPC (5 mg lutein kg−1 BW) + CCl4. Different lower case letters correspond to significant differences at p < 0.05.
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Group
MDA (nM mg−1 protein)
SOD (U mg−1 protein)
CAT (U mg−1 protein)
GSH-Px (U mg−1 protein)
I II III IV V
2.36 ± 0.17b 3.03 ± 0.10a 2.48 ± 0.14b 2.53 ± 0.33b 2.57 ± 0.55b
193.72 ± 5.99a 168.25 ± 11.60b 201.10 ± 3.73a 191.09 ± 9.13a 186.75 ± 15.54a
55.09 ± 5.38a 37.43 ± 4.33c 43.41 ± 5.46bc 46.50 ± 1.14b 41.37 ± 7.22bc
821.81 ± 17.89a 679.54 ± 41.30b 800.63 ± 11.13a 772.69 ± 21.56a 776.11 ± 1.25a
Group I, vehicle control; group II, CCl4; group III, lutein (10 mg kg−1 BW) + CCl4; group IV, LPC (10 mg lutein kg−1 BW) + CCl4; group V, LPC (5 mg lutein kg−1 BW) + CCl4; Values are expressed as mean ± SD for 10 replications. Mean values within a row with different lower case letters correspond to significant differences at p < 0.05.
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a
are summarized in Table 2. The results showed that the activities of SOD, CAT and GSH-Px were decreased by 13.1%, 32.1% and 17.3% respectively in the livers of mice while a 28.4% increase of the MDA content was observed when exposed to CCl4 ( p < 0.05). Pretreatment with LPC (5, 10 mg lutein kg−1 BW) could restore the activities of hepatic SOD and GSH-Px effectively ( p < 0.05) and also increase the activities of hepatic CAT. Besides, LPC could significantly attenuate CCl4-induced elevated levels of hepatic MDA dose-dependently ( p < 0.05). The positive control lutein (group III) also showed obvious precaution to CCl4-induced hepatic injury at these parameters. However, LPC showed comparative activities ( p > 0.05) on the hepatic biochemical indexes summarized in Table 2 when compared to lutein alone at the same level of 10 mg kg−1 BW (group III and group IV).
4.
Discussion
Lutein, a well-known commercial dietary antioxidant, may reduce oxidative damage in cells and human tissues because of its ability to quench singlet oxygen and other electronically excited molecules. In the present study, the natural lutein– protein complex from Chlorella vulgaris was first isolated and the antioxidant properties in vitro and hepatoprotective activity in CCl4-induced hepatotoxicity in mice were evaluated in detail. Scavenging of ABTS and DPPH radicals is rapid and efficient using antioxidant assays. DPPH is a stable radical which has an unpaired valence electron at one atom of the nitrogen bridge and it exhibits a dark purple color and shows maximum absorbance at 517 nm in an ethanol solution.21 Oxidized ABTS generates a bluish-green colored cation-radical and shows strong absorbance at 734 nm. The antioxidant activity of LPC on DPPH and ABTS radicals may be attributed to a direct role in trapping free radicals by accepting an electron or a hydrogen radical.15,22 Lipid peroxidation is a complicated process involving the interaction of free radicals with polyunsaturated fatty acids, resulting in various highly reactive electrophilic aldehydes, such as peroxyl and alkoxy radicals that can form pre-existing lipid peroxides to initiate lipid peroxidation.23 LPC and lutein exhibited moderate inhibition of lipid peroxidation in the linoleic acid emulsion system, indi-
Food Funct.
cating that they could interfere with the propagation cycle of lipid radicals and then slow linoleic acid oxidation. CCl4 is a widely known hepatotoxicity revulsant. A trichloromethyl radical (CCl3•), the biotransformed metabolite of CCl4 stimulated by cytochrome P-450, has been demonstrated to block the synthesis of protein and disorder the lipid metabolism, which results in accumulation of triglycerides (TG) in liver and the development of fatty liver.24 Moreover, CCl3• can further transform into a more active trichloromethyl peroxyl radical (CCl3O3•) rapidly and then initiate lipid peroxidation, leading to cell membrane damage, intracellular enzyme leakage and even cell necrosis.20 MDA is the end-product of lipid peroxidation and the levels can reflect the extent of cellular damage indirectly. It is widely used as an index of lipid peroxidation mediated by free radicals.25 The present results verify the effects of CCl4-induced oxidative stress in liver as evidenced by the elevated levels of serum ALT and AST that normally exist in the cytoplasm and hepatic MDA after CCl4 treatment. The suppression of the CCl4-induced elevation of serum ALT, AST and the hepatic MDA levels brought about by LPC indicates stabilization of cell membrane as well as mitigation of hepatic damage. To counteract the chain reaction of free radicals, well-integrated antioxidant systems are endowed to the organisms, which include enzymes such as SOD, CAT and GSH-Px, acting as the first line of defense against free radical induced oxidative stress. SOD is an effective defense enzyme that transforms super anions into H2O2 and O2, and CAT existing in all aerobic cells can further convert H2O2 into H2O and O2. GSH-Px is another kind of enzyme that can metabolize H2O2 and alkyl hydroperoxides by the reducing action of glutathione.26 Excess free radicals can be obliterated immediately by an enzymatic scavenger to maintain an oxidation–reduction balance under normal conditions. Our study showed that the activities of SOD, CAT and GSH-Px were suppressed after CCl4 challenge, indicating oxidative damage to the livers. However, the pretreatment with LPC could notably restore the activities of the antioxidant enzymes in the livers of CCl4-treated mice, suggesting a protective effect of LPC against CCl4-induced hepatotoxicity in mice. The hepatoprotective effect of LPC may be attributed to the presence of protein-based lutein. The present study clearly revealed the protective effect of lutein against CCl4-induced
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hepatotoxicity in mice. Previous studies had also showed that carotenoid lutein could significantly reduce the increased levels of serum ALT, AST and alkaline phosphatases (ALP) on paracetamol-, CCl4- and ethanol-induced hepatic damage in rats, and the action may be due to lutein’s ability to scavenge reactive oxygen radicals.27 Lutein in the form of LPC may be released and absorbed by the digestive system and plays an important role in hepatoprotection. The effects of LPC might be a consequence of the free radical scavenging effect and inhibitory action on lipid peroxidation of lutein, and protein provided the lutein with good stability and emulsibility in aqueous solution.
5. Conclusions The natural lutein–protein complex was first isolated and purified from Chlorella vulgaris. LPC showed significant scavenging effects towards ABTS and DPPH radicals and inhibitory activity on lipid peroxidation in vitro. LPC could reduce CCl4-induced hepatic injury in vivo. The results suggest that the natural lutein protein complex, with good stability and emulsibility in aqueous solution, has the potential for protecting the liver against chemical induced toxicity and increasing the antioxidant capacity of the defense system in the human body.
Acknowledgements This work was supported by the National Marine public welfare scientific research funding of China (no. 201305022).
References 1 P. Bhosale, B. Li, M. Sharifzadeh, W. Gellermann, J. M. Frederick, K. Tsuchida and P. S. Bernstein, Biochemistry, 2009, 48, 4798–4807. 2 E. R. Sindhu, K. C. Preethi and R. Kuttan, Indian J. Exp. Biol., 2010, 48, 843–848. 3 E. Christaki, E. Bonos, I. Giannenas and P. Florou-Paneri, J. Sci. Food Agric., 2012, 93, 5–11. 4 K. Frede, A. Henze, M. Khalil, S. Baldermann, F. J. Schweigert and H. Rawel, J. Funct. Foods, 2014, 8, 118– 127. 5 M. C. Chan, S. H. Ho, D. J. Lee, C. Y. Chen, C. C. Huang and J. S. Chang, Biochem. Eng. J., 2013, 78, 24–31.
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6 X. M. Shi, F. Chen, J. P. Yuan and H. Chen, J. Appl. Phycol., 1997, 9, 445–450. 7 J. Zhang, Z. Sun, P. Sun, T. Chen and F. Chen, Food Funct., 2014, 5, 413–425. 8 M. Vishnevetsky, M. Ovadis and A. Vainstein, Trends Plant Sci., 1999, 4, 232–235. 9 F. G. Xiao, L. Shen and H. F. Ji, Biochem. Biophys. Res. Commun., 2011, 414, 1–4. 10 Z. E. Jouni and M. A. Wells, J. Biol. Chem., 1996, 271, 14722–14726. 11 D. P. Maxwell, S. Falk and N. P. A. Huner, Plant Physiol., 1995, 107, 687–694. 12 R. Fujii, M. Kita, Y. Iinuma, N. Oka, Y. Takaesu, T. Taira, M. Iha, R. J. Cogdell and H. Hashimoto, Photosynth. Res., 2012, 111, 157–163. 13 D. I. Arnon, Plant Physiol., 1949, 24, 1–15. 14 H. C. Wu, H. M. Chen and C. Y. Shiau, Food Res. Int., 2003, 36, 949–957. 15 B. Wang, Z. R. Li, C. F. Chi, Q. H. Zhang and H. Y. Luo, Peptides, 2012, 36, 240–250. 16 T. Osawa and M. Namiki, Agric. Biol. Chem., 1981, 45, 735– 739. 17 H. Mitsuda, K. Yuasumoto and K. Iwami, Eiyo to Shokuryo, 1966, 19, 210–214. 18 M. M. Bradford, Anal. Biochem., 1976, 72, 248–254. 19 P. F. Zagalsky, Pure Appl. Chem., 1976, 47, 103– 120. 20 Q. Liu, G. Tian, H. Yan, X. Geng, Q. Cao, H. Wang and T. B. Ng, J. Agric. Food Chem., 2014, 62, 8858– 8866. 21 O. P. Sharma and T. K. Bhat, Food Chem., 2009, 113, 1202– 1205. 22 A. Zulueta, M. J. Esteve and A. Frígola, Food Chem., 2009, 114, 310–316. 23 I. Andreu, D. Neshchadin, E. Rico, M. Griesser, A. Samadi, I. M. Morera, G. Gescheidt and M. A. Miranda, Chemistry, 2011, 17, 10089–10096. 24 A. Vanitha, K. N. Murthy, V. Kumar, G. Sakthivelu, J. M. Veigas, P. Saibaba and G. A. Ravishankar, Int. J. Toxicol., 2007, 26, 159–167. 25 N. Cheng, B. Du, Y. Wang, H. Gao, W. Cao, J. B. Zheng and F. Feng, Food Funct., 2014, 5, 900–908. 26 Q. Liu, B. H. Kong, G. X. Li, N. Liu and X. F. Xia, Food Chem. Toxicol., 2011, 49, 1316–1321. 27 E. R. Sindhu, A. P. Firdous, K. C. Preethi and R. Kuttan, J. Pharm. Pharmacol., 2010, 62, 1054–1060.
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Cite this: DOI: 10.1039/c4fo01096e
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Isolation of a novel lutein–protein complex from Chlorella vulgaris and its functional properties Xixi Cai, Qimin Huang and Shaoyun Wang* A novel kind of lutein–protein complex (LPC) was extracted from heterotrophic Chlorella vulgaris through aqueous extraction. The purification procedure contained solubilization of thylakoid proteins by a zwitterionic detergent CHAPS, anion exchange chromatography and gel filtration chromatography. Both wavelength scanning and HPLC analysis confirmed that lutein was the major pigment of the protein-based complex, and the mass ratio of lutein and protein was determined to be 9.72 : 100. Besides showing lipid peroxidation inhibition activity in vitro, LPC exerted significant antioxidant effects against ABTS and DPPH radicals with IC50 of 2.90 and 97. 23 μg mL−1, respectively. Meanwhile, in vivo antioxidant activity of the complex was evaluated using the mice hepatotoxicity model; LPC significantly suppressed the carbon tetrachloride-induced elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase
Received 30th November 2014, Accepted 19th April 2015 DOI: 10.1039/c4fo01096e www.rsc.org/foodfunction
1.
(AST) activities, and decreased hepatic malondialdehyde (MDA) levels and the hepatosomatic index. Moreover, LPC could effectively restore the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in the treated mice livers. Our findings further the progress in the research of natural protein-based lutein complexes, suggesting that LPC has the potential in hepatoprotection against chemical induced toxicity and in increasing the antioxidant capacity of the defense system in the human body.
Introduction
Lutein, the second most prevalent carotenoid, is widely distributed in dark green leafy plants and algae, and also accumulates in the macular region of the retina acting as an antioxidant.1 Lutein has been described as an excellent antioxidant because of its ability to quench singlet oxygen and other electronically excited molecules, and the antioxidant activity may be due to its conjugated double bonds and the phenolic hydroxyl groups on both ends of its chemical structure.2 Lutein is an essential nutrient for organisms since humans cannot synthesize lutein de novo. It is used as an important ingredient in dietary supplements and more recently in medicine, cosmetics and animal feed.3,4 However, poor solubility and emulsibility in aqueous solution severely restrict its wide application in food systems. Chlorella vulgaris, which contains 0.2–0.5% lutein, has more and more advantages of being the major source for extracting lutein for its short cycle, high yield and easy culture on a large scale when compared with plants.5–7 Solvent extraction is the main industrially applicable method for lutein extraction.3 As a result, the massive use of organic solvents such as hexane goes against environmental friendliness and destroys the natural matrix of lutein. Because of its hydro-
phobic nature, lutein in plants and animal cells is found in associated with the hydrophobic domain of lipid-protein complexes.8 Lutein is located in the thylakoid membrane in the form of light-harvesting complexes (LHCs) in plants and algae, playing the function of photosynthesis and chlorophyll photoprotection.9 LPC plays crucial roles in lutein stabilization and transfer, and the unique chromophore interacts with protein in these complexes contributing to the coloration of related tissue.10 Numerous experiments have been performed to isolate and analyze the pigment composition, structure and energy transfer of LHCs.11,12 However, the isolation of the natural lutein– protein complex and the activities of lutein in the form of a pigment–protein complex are rarely reported. It is interesting to obtain the natural matrix of lutein extract through aqueous extraction instead of using an organic solvent, and investigate the influence of protein interaction on the activities of lutein. Therefore, the objective of this study was to isolate the natural lutein–protein complex from Chlorella vulgaris and evaluate the antioxidant properties in vitro and hepatoprotective activity in CCl4-induced hepatotoxicity in mice.
2. Materials and methods 2.1.
College of Bioscience and Biotechnology, Fuzhou University, Fuzhou 350108, China. E-mail: [email protected]; Fax: +86-591-22866278; Tel: +86-591-22866375
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Materials
The algae material Chlorella vulgaris STIO02 with a relatively high lutein content was kindly provided by the Third Institute
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of Oceanography, State Oceanic Administration, P. R. China and was stored at −20 °C before use. Toyopearl DEAE-650M was purchased from TOSOH Co. (Japan). Sephadex G-50 Fine was the product of GE Healthcare (USA). 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis3-ethylbenzthiazoline-6-sulphonate (ABTS), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and lutein were obtained from Sigma Chemical Co. (USA). Bicinchoninic acid (BCA) protein kit, alanine aminotransferase (ALT) kit, aspartate aminotransferase (AST) kit, malondialdehyde (MDA) kit, superoxide dismutase (SOD) kit, catalase (CAT) kit and glutathione peroxidase (GSH-Px) kit were the products of Nanjing Jiancheng Bioengineering Institute (China). Methanol and acetonitrile used in liquid chromatography were of HPLC grade. All other chemicals and reagents used were of analytical grade and commercially available. 2.2.
Characterization of LPC
2.4.1. Absorption spectroscopy. The absorption spectrum of the lutein–protein complex was recorded on a U-2910 spectrophotometer (Hitachi Co., Japan) at room temperature. 2.4.2. Lutein determination. The lutein content of LPC was extracted from the lutein–protein complex by adding five volumes of methanol to the protein solution, followed by centrifugation to remove the precipitate. For High Performance Liquid Chromatography (HPLC) analysis, the pigment 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. 2.4.3. Protein determination. The protein content of the lutein–protein complex was separated by the method described by Maxwell11 and quantified by bicinchoninic acid.
Solubilization of thylakoid proteins
Algae were washed several times with cold distilled water and broken in phosphate buffer (20 mM, pH 7.0) by ultrasonication in an ice bath. Cell debris was removed by centrifugation (10 000 rpm for 10 min, 4 °C) and membranes were collected by 25% saturation ammonium sulfate precipitation. Then the membrane fraction was re-suspended in Tris-HCl buffer (20 mM, pH 8.0) to a concentration of chlorophyll a (Chl a) of 0.25 mg mL−1.13 Since lutein–protein complexes in Chlorella cells are intrinsic membrane-associated, it is necessary to use detergents to solubilize the thylakoid membrane proteins. Here, CHAPS, a zwitterionic detergent that can well maintain protein activity, was used. Solubilization of the membranes was carried out by the addition of 1% CHAPS for 1 h in the dark at 4 °C. Then the mixture was centrifuged at 12 500 rpm for 30 min at 4 °C. The supernatant was collected and used for further purification. 2.3.
2.4.
Isolation and purification of LPC
The solubilized thylakoid proteins were loaded onto a Toyopearl DEAE-650M column (Φ 1.6 × 20 cm), which was previously equilibrated with Tris-HCl buffer (20 mM, pH 8.0) containing 8 mM CHAPS. The column was washed with the same buffer followed by a linear gradient of 0–0.5 M NaCl. Chromatography was carried out with a flow rate of 0.5 mL min−1 and 10 min per tube. The absorbance of all fractions was monitored at 482 nm and 280 nm, respectively. At later purification stages, cation-exchange chromatography was performed on a Sephadex SP C-25 column (Φ 1.6 × 20 cm) and the elute conditions were the same as anion-exchange chromatography described above. 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 CHAPS and 50 mM NaCl. The column was run with the same buffer and the flow rate was 0.3 mL min−1. The absorbance of the fractions was monitored and the eluted protein fractions containing the lutein–protein complex were collected.
Food Funct.
2.5.
Antioxidant activity in vitro
2.5.1. DPPH radical scavenging activity. The DPPH radical scavenging activity assay was tested according to the method of Wu14 with some modifications. 1 mL of LPC was placed in cuvettes, and then 1 mL of ethanol solution of DPPH (0.1 mM) was added. Thirty minutes later, the absorbance was measured at 517 nm. A control sample containing 1 mL DPPH solution and 1 mL distilled water was prepared. Samples mixed with ethanol solutions without DPPH were set as blank. The antioxidant activity of the sample was evaluated by the scavenging rate of the DPPH radical with the following equation: DPPH radical scavenging activityð%Þ ¼ ½1 ðAsample Ablank Þ=Acontrol 100; where Asample, Ablank and Acontrol were the absorbance of the sample, blank and control group, respectively. The IC50 values (the concentration of the samples required to reach the scavenging rate of DPPH radicals to 50%) were calculated. 2.5.2. ABTS radical scavenging activity. The ABTS radical scavenging activity was determined according to the method of Wang.15 The ABTS radical was generated by mixing ABTS stock solution (7 mM) with an equal volume of potassium persulfate (2.45 mM) and keeping the resulting mixture in the dark at room temperature for 16 h. The ABTS radical solution was diluted in phosphate buffer (5 mM, pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm before use. 1 mL of diluted ABTS radical solution was mixed with 1 mL of LPC. The mixture was then kept in the dark for 10 min and the absorbance was measured at 734 nm. A control sample containing ABTS radical solution and distilled water without the sample was also prepared. The ABTS radical scavenging activity of samples was calculated by the following equation: ABTS radical scavenging activityð%Þ ¼ ðAcontrol Asample Þ=Acontrol 100; where Asample and Acontrol were the absorbance of the sample and control group, respectively. The IC50 values (the concen-
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tration of the samples required to reach the scavenging rate of ABTS radicals to 50%) were calculated. 2.5.3. Lipid peroxidation inhibition activity. The lipid peroxidation inhibition activity of LPC was measured in a linoleic acid emulsion system according to the method introduced by Osawa and Namiki16 with some modifications. Briefly, 1 mL of the sample was added to a solution of 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. The degree of oxidation was measured at 24 h intervals using the ferric thiocyanate (FTC) method of Mitsuda.17 100 μL of the reaction solution was mixed with 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% HCl. After 3 min, the degree of color development that represented the linoleic acid oxidation was measured spectrophotometrically at 500 nm.
4 °C, the supernatant was collected and the activities of SOD, CAT, GSH-Px and MDA were determined with the corresponding diagnostic kits. The total protein content of liver homogenate was measured according to the method of Bradford, using bovine serum albumin as the standard.18
2.6.
3.1.
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). Mice were randomly divided into five groups with ten mice in each group. Group I was untreated with CCl4 and served as vehicle control. Group II was the CCl4 control group in which mice were treated with CCl4, without LPC. Group III was a positive control group in which mice were administrated lutein at 10 mg kg−1 per body weight (BW) along with CCl4. Groups IV and V were the treatment groups in which mice were administrated LPC at 10 mg lutein kg−1 BW and 5 mg lutein kg−1 BW respectively along with CCl4. LPC suspended in deionized water and lutein suspended in peanut oil were given to the mice at the set dose once daily by gavage for 11 days. Mice in groups I and II were orally given the same volume of deionized water. One hour after substance administration on the 10th day, all the groups were treated with a single dose of 0.1% CCl4 solution in peanut oil (10 mL kg−1 BW) by gavage except for group I which was treated with the same volume of peanut oil. Thirty-six hours after CCl4 treatment, mice were sacrificed after being decapitated. 2.6.2. Determination of hepatosomatic index. Mice were weighed and then tenderly 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 of each mouse were taken in EDTA-anticoagulant tubes from the orbit before they were sacrificed. After centrifugation, the serum was collected and activities of serum ALT and AST were analyzed according to the protocols of relevant diagnostic kits. 2.6.4. Analysis of hepatic biochemical indexes. Liver tissues were taken, weighed and then homogenized in cold normal saline. After centrifugation at 3000 rpm for 10 min at
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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 by one-way analysis of variance (ANOVA) with Duncan’s test for post hoc analysis. p < 0.05 values were considered as statistically significant.
3. Results Isolation and characterization of LPC
A crude pigment–protein extract of Chlorella vulgaris was obtained by aqueous extraction. The absorption spectrum of the crude extract was measured. An absorption peak was generated at 482 nm, which might be attributed to carotenoids including lutein (data not shown). Chromatography was employed to improve the purity of the lutein–protein complex. As shown in Fig. 1A, the crude extract of pigment protein was divided into four fractions by DEAE anion-exchange chromatography and fractions P1 and P2 exhibited obvious absorbance at both 280 nm and 482 nm. However, fraction P1 was yellow colored while P2 was dark green, indicating that there was a great amount of chlorophyll contained in P2. Therefore, fraction P2 was not considered further. The yellow fraction P1 which did not bind to the anion-exchange column was pooled and loaded onto a SP cation-exchange column under the same elution conditions. However, fraction P1 was still not absorbed (data not shown). That the solubilized protein did not bind to moderately charged cation- or anion-exchange columns indicated high relative hydrophobicity in a CHAPS detergent environment.1 For further purification, the concentrated fraction P1 was applied to a Sephadex G-50 column. Four fractions were obtained as shown in Fig. 1B. Pigment proteins were mainly contained in fraction P1-2 while the other fractions were only protein contained. In the absorbance spectrum of P1-2, shown in Fig. 2, the fraction exhibited 280 nm protein absorbance and a prominent triple peak vibronic structure typical of most luteins centered at 454 nm, indicating that P1-2 was the fraction of interest. Besides, there was a bathochromic shift of the absorbance peaks of LPC (429, 454 and 482 nm) relative to lutein (421, 443 and 472 nm), which could be explained by refractive index effects associated with dissolution of lutein in the protein components of the lipoproteins.19 The relative absorbance of A454/A280 was enhanced to 1.12 by gel filtration chromatography compared to 0.54 of DEAE anion-exchange chromatography. HPLC analysis of the most purified fraction confirmed that lutein was the major pigment of P1-2 (Fig. 3), and the mass ratio of lutein and
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Fig. 3
HPLC analysis of LPC.
Table 1
Free radical scavenging activity of LPC
IC50 (μg mL−1)a
LPC Lutein a
Fig. 1 Purification of LPC. (A) DEAE anion-exchange chromatography, (B) Sephadex G-50 gel filtration chromatography.
Fig. 2
ABTS
DPPH
2.90 ± 0.15 17.81 ± 0.06
97.23 ± 1.20 360.03 ± 0.82
Calculation was based on the content of lutein.
3.2.
Antioxidant activity in vitro
To evaluate the antioxidant activities of lutein in the form of LPC in vitro, both free radical scavenging effects and lipid peroxidation inhibition activity were investigated. Results indicated that LPC could scavenge the ABTS and DPPH radicals effectively in a dose-dependent manner (data not shown). As shown in Table 1, the IC50 values of LPC against ABTS and DPPH radicals were 2.90 and 97.23 μg mL−1, which were approximately six and four times lower than those of lutein respectively, suggesting significantly stronger free radical scavenging effects of lutein in the form of LPC. The antioxidant activity of LPC against the peroxidation of linoleic acid was investigated. As shown in Fig. 4, the control had the highest absorbance at 500 nm, indicating the highest degree of oxidation, while the samples with LPC and lutein had lower absorbance. However, lutein showed stronger prevention of linoleic acid peroxidation than LPC under the same content of lutein (100 μg mL−1), which may be due to the improvement in hydrophilicity of lutein interaction with protein.
Absorption spectrum of LPC.
3.3. protein was determined to be 9.72 : 100. On the basis of its absorption spectrum and HPLC profile, fraction P1-2 can be assigned to be LPC.
Food Funct.
Effect on CCl4-induced hepatic injury in mice
Many studies have shown that the hepatoprotective effects may be associated with antioxidant capacity.20 To study the antioxidant effects of LPC in vivo, the well-described CCl4-induced mice hepatotoxicity model was used.
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Fig. 4 Lipid peroxidation inhibition activity of LPC. The activity was measured in a linoleic acid emulsion system with a lutein concentration of 100 μg mL−1.
3.3.1. Effect of LPC on mice hepatosomatic index. Consumption of CCl4 induced liver swelling and reduced weight gain, and as a result the hepatosomatic index was elevated. As shown in Fig. 5, the hepatosomatic index of the mice significantly increased after CCl4 treatment (group II) when compared to the vehicle control ( p < 0.05), which is the symptom of fatty liver. The CCl4-induced increase in the hepatosomatic index could be mitigated by pretreatment with LPC at different doses (5, 10 mg lutein kg−1 BW, groups V and IV). Moreover, pretreatment with LPC containing 10 mg lutein kg−1 BW (group IV) could significantly attenuate the elevation by 15.6%, stronger than the alleviated effect of lutein alone at the same level, which was observed at 12.8% (group III). 3.3.2. Effect of LPC on serum ALT and AST. Serum transaminases such as ALT and AST were used as biochemical markers for hepatic injury. The hepatoprotective effect of LPC on the serum ALT and AST is shown in Fig. 6. Mice treated
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Fig. 5 Effect of LPC on the mice hepatosomatic index. Group I, vehicle control; group II, CCl4; group III, lutein (10 mg kg−1 BW) + CCl4; group IV, LPC (10 mg lutein kg−1 BW) + CCl4; group V, LPC (5 mg lutein kg−1 BW) + CCl4. Different lower case letters correspond to significant differences at p < 0.05.
with CCl4 alone showed moderate hepatic injury (AST/ALT < 1.0) as evidenced by a significant rise of the activities of ALT and AST in serum ( p < 0.05), which indicate the hepatocyte damage and loss of membrane integrity. Pretreatment with LPC containing 5, 10 mg lutein kg−1 BW (groups V and IV) and lutein (10 mg kg−1 BW, group III) could depress the elevated activities of serum ALT by 22.4%, 27.2%, and 26.2% respectively (Fig. 6A), and it could at the same time depress the elevated activities of serum AST by 33.28%, 42.65%, and 35.45% respectively compared with the CCl4 model group (Fig. 6B). Therefore, LPC could show a stronger depression activity on serum ALT and AST when compared to lutein alone at the level of 10 mg kg−1 BW (Fig. 6A and B). 3.3.3. Effect of LPC on hepatic SOD, CAT, GSH-Px and MDA. The effects of LPC on the hepatic biochemical indexes
Fig. 6 Effect of LPC on mice serum ALT (A) and AST (B). Group I, vehicle control; group II, CCl4; group III, lutein (10 mg kg−1 BW) + CCl4; group IV, LPC (10 mg lutein kg−1 BW) + CCl4; group V, LPC (5 mg lutein kg−1 BW) + CCl4. Different lower case letters correspond to significant differences at p < 0.05.
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Group
MDA (nM mg−1 protein)
SOD (U mg−1 protein)
CAT (U mg−1 protein)
GSH-Px (U mg−1 protein)
I II III IV V
2.36 ± 0.17b 3.03 ± 0.10a 2.48 ± 0.14b 2.53 ± 0.33b 2.57 ± 0.55b
193.72 ± 5.99a 168.25 ± 11.60b 201.10 ± 3.73a 191.09 ± 9.13a 186.75 ± 15.54a
55.09 ± 5.38a 37.43 ± 4.33c 43.41 ± 5.46bc 46.50 ± 1.14b 41.37 ± 7.22bc
821.81 ± 17.89a 679.54 ± 41.30b 800.63 ± 11.13a 772.69 ± 21.56a 776.11 ± 1.25a
Group I, vehicle control; group II, CCl4; group III, lutein (10 mg kg−1 BW) + CCl4; group IV, LPC (10 mg lutein kg−1 BW) + CCl4; group V, LPC (5 mg lutein kg−1 BW) + CCl4; Values are expressed as mean ± SD for 10 replications. Mean values within a row with different lower case letters correspond to significant differences at p < 0.05.
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a
are summarized in Table 2. The results showed that the activities of SOD, CAT and GSH-Px were decreased by 13.1%, 32.1% and 17.3% respectively in the livers of mice while a 28.4% increase of the MDA content was observed when exposed to CCl4 ( p < 0.05). Pretreatment with LPC (5, 10 mg lutein kg−1 BW) could restore the activities of hepatic SOD and GSH-Px effectively ( p < 0.05) and also increase the activities of hepatic CAT. Besides, LPC could significantly attenuate CCl4-induced elevated levels of hepatic MDA dose-dependently ( p < 0.05). The positive control lutein (group III) also showed obvious precaution to CCl4-induced hepatic injury at these parameters. However, LPC showed comparative activities ( p > 0.05) on the hepatic biochemical indexes summarized in Table 2 when compared to lutein alone at the same level of 10 mg kg−1 BW (group III and group IV).
4.
Discussion
Lutein, a well-known commercial dietary antioxidant, may reduce oxidative damage in cells and human tissues because of its ability to quench singlet oxygen and other electronically excited molecules. In the present study, the natural lutein– protein complex from Chlorella vulgaris was first isolated and the antioxidant properties in vitro and hepatoprotective activity in CCl4-induced hepatotoxicity in mice were evaluated in detail. Scavenging of ABTS and DPPH radicals is rapid and efficient using antioxidant assays. DPPH is a stable radical which has an unpaired valence electron at one atom of the nitrogen bridge and it exhibits a dark purple color and shows maximum absorbance at 517 nm in an ethanol solution.21 Oxidized ABTS generates a bluish-green colored cation-radical and shows strong absorbance at 734 nm. The antioxidant activity of LPC on DPPH and ABTS radicals may be attributed to a direct role in trapping free radicals by accepting an electron or a hydrogen radical.15,22 Lipid peroxidation is a complicated process involving the interaction of free radicals with polyunsaturated fatty acids, resulting in various highly reactive electrophilic aldehydes, such as peroxyl and alkoxy radicals that can form pre-existing lipid peroxides to initiate lipid peroxidation.23 LPC and lutein exhibited moderate inhibition of lipid peroxidation in the linoleic acid emulsion system, indi-
Food Funct.
cating that they could interfere with the propagation cycle of lipid radicals and then slow linoleic acid oxidation. CCl4 is a widely known hepatotoxicity revulsant. A trichloromethyl radical (CCl3•), the biotransformed metabolite of CCl4 stimulated by cytochrome P-450, has been demonstrated to block the synthesis of protein and disorder the lipid metabolism, which results in accumulation of triglycerides (TG) in liver and the development of fatty liver.24 Moreover, CCl3• can further transform into a more active trichloromethyl peroxyl radical (CCl3O3•) rapidly and then initiate lipid peroxidation, leading to cell membrane damage, intracellular enzyme leakage and even cell necrosis.20 MDA is the end-product of lipid peroxidation and the levels can reflect the extent of cellular damage indirectly. It is widely used as an index of lipid peroxidation mediated by free radicals.25 The present results verify the effects of CCl4-induced oxidative stress in liver as evidenced by the elevated levels of serum ALT and AST that normally exist in the cytoplasm and hepatic MDA after CCl4 treatment. The suppression of the CCl4-induced elevation of serum ALT, AST and the hepatic MDA levels brought about by LPC indicates stabilization of cell membrane as well as mitigation of hepatic damage. To counteract the chain reaction of free radicals, well-integrated antioxidant systems are endowed to the organisms, which include enzymes such as SOD, CAT and GSH-Px, acting as the first line of defense against free radical induced oxidative stress. SOD is an effective defense enzyme that transforms super anions into H2O2 and O2, and CAT existing in all aerobic cells can further convert H2O2 into H2O and O2. GSH-Px is another kind of enzyme that can metabolize H2O2 and alkyl hydroperoxides by the reducing action of glutathione.26 Excess free radicals can be obliterated immediately by an enzymatic scavenger to maintain an oxidation–reduction balance under normal conditions. Our study showed that the activities of SOD, CAT and GSH-Px were suppressed after CCl4 challenge, indicating oxidative damage to the livers. However, the pretreatment with LPC could notably restore the activities of the antioxidant enzymes in the livers of CCl4-treated mice, suggesting a protective effect of LPC against CCl4-induced hepatotoxicity in mice. The hepatoprotective effect of LPC may be attributed to the presence of protein-based lutein. The present study clearly revealed the protective effect of lutein against CCl4-induced
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hepatotoxicity in mice. Previous studies had also showed that carotenoid lutein could significantly reduce the increased levels of serum ALT, AST and alkaline phosphatases (ALP) on paracetamol-, CCl4- and ethanol-induced hepatic damage in rats, and the action may be due to lutein’s ability to scavenge reactive oxygen radicals.27 Lutein in the form of LPC may be released and absorbed by the digestive system and plays an important role in hepatoprotection. The effects of LPC might be a consequence of the free radical scavenging effect and inhibitory action on lipid peroxidation of lutein, and protein provided the lutein with good stability and emulsibility in aqueous solution.
5. Conclusions The natural lutein–protein complex was first isolated and purified from Chlorella vulgaris. LPC showed significant scavenging effects towards ABTS and DPPH radicals and inhibitory activity on lipid peroxidation in vitro. LPC could reduce CCl4-induced hepatic injury in vivo. The results suggest that the natural lutein protein complex, with good stability and emulsibility in aqueous solution, has the potential for protecting the liver against chemical induced toxicity and increasing the antioxidant capacity of the defense system in the human body.
Acknowledgements This work was supported by the National Marine public welfare scientific research funding of China (no. 201305022).
References 1 P. Bhosale, B. Li, M. Sharifzadeh, W. Gellermann, J. M. Frederick, K. Tsuchida and P. S. Bernstein, Biochemistry, 2009, 48, 4798–4807. 2 E. R. Sindhu, K. C. Preethi and R. Kuttan, Indian J. Exp. Biol., 2010, 48, 843–848. 3 E. Christaki, E. Bonos, I. Giannenas and P. Florou-Paneri, J. Sci. Food Agric., 2012, 93, 5–11. 4 K. Frede, A. Henze, M. Khalil, S. Baldermann, F. J. Schweigert and H. Rawel, J. Funct. Foods, 2014, 8, 118– 127. 5 M. C. Chan, S. H. Ho, D. J. Lee, C. Y. Chen, C. C. Huang and J. S. Chang, Biochem. Eng. J., 2013, 78, 24–31.
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Paper
6 X. M. Shi, F. Chen, J. P. Yuan and H. Chen, J. Appl. Phycol., 1997, 9, 445–450. 7 J. Zhang, Z. Sun, P. Sun, T. Chen and F. Chen, Food Funct., 2014, 5, 413–425. 8 M. Vishnevetsky, M. Ovadis and A. Vainstein, Trends Plant Sci., 1999, 4, 232–235. 9 F. G. Xiao, L. Shen and H. F. Ji, Biochem. Biophys. Res. Commun., 2011, 414, 1–4. 10 Z. E. Jouni and M. A. Wells, J. Biol. Chem., 1996, 271, 14722–14726. 11 D. P. Maxwell, S. Falk and N. P. A. Huner, Plant Physiol., 1995, 107, 687–694. 12 R. Fujii, M. Kita, Y. Iinuma, N. Oka, Y. Takaesu, T. Taira, M. Iha, R. J. Cogdell and H. Hashimoto, Photosynth. Res., 2012, 111, 157–163. 13 D. I. Arnon, Plant Physiol., 1949, 24, 1–15. 14 H. C. Wu, H. M. Chen and C. Y. Shiau, Food Res. Int., 2003, 36, 949–957. 15 B. Wang, Z. R. Li, C. F. Chi, Q. H. Zhang and H. Y. Luo, Peptides, 2012, 36, 240–250. 16 T. Osawa and M. Namiki, Agric. Biol. Chem., 1981, 45, 735– 739. 17 H. Mitsuda, K. Yuasumoto and K. Iwami, Eiyo to Shokuryo, 1966, 19, 210–214. 18 M. M. Bradford, Anal. Biochem., 1976, 72, 248–254. 19 P. F. Zagalsky, Pure Appl. Chem., 1976, 47, 103– 120. 20 Q. Liu, G. Tian, H. Yan, X. Geng, Q. Cao, H. Wang and T. B. Ng, J. Agric. Food Chem., 2014, 62, 8858– 8866. 21 O. P. Sharma and T. K. Bhat, Food Chem., 2009, 113, 1202– 1205. 22 A. Zulueta, M. J. Esteve and A. Frígola, Food Chem., 2009, 114, 310–316. 23 I. Andreu, D. Neshchadin, E. Rico, M. Griesser, A. Samadi, I. M. Morera, G. Gescheidt and M. A. Miranda, Chemistry, 2011, 17, 10089–10096. 24 A. Vanitha, K. N. Murthy, V. Kumar, G. Sakthivelu, J. M. Veigas, P. Saibaba and G. A. Ravishankar, Int. J. Toxicol., 2007, 26, 159–167. 25 N. Cheng, B. Du, Y. Wang, H. Gao, W. Cao, J. B. Zheng and F. Feng, Food Funct., 2014, 5, 900–908. 26 Q. Liu, B. H. Kong, G. X. Li, N. Liu and X. F. Xia, Food Chem. Toxicol., 2011, 49, 1316–1321. 27 E. R. Sindhu, A. P. Firdous, K. C. Preethi and R. Kuttan, J. Pharm. Pharmacol., 2010, 62, 1054–1060.
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