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Astaxanthin is responsible for antiglycoxidative properties of microalga Chlorella zofingiensis
Food Chemistry 126 (2011) 1629–1635
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Astaxanthin is responsible for antiglycoxidative properties of microalga Chlorella zofingiensis Zheng Sun a, Jin Liu a, Xiaohui Zeng a, Jieqiong Huangfu a, Yue Jiang b, Mingfu Wang a,⇑, Feng Chen a,⇑ a b
School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, PR China Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China
a r t i c l e
i n f o
Article history: Received 14 September 2010 Received in revised form 13 November 2010 Accepted 5 December 2010 Available online 10 December 2010 Keywords: Astaxanthin Chlorella zofingiensis Diabetic complications Glycoxidation
a b s t r a c t The antiglycoxidative properties of microalga Chlorella zofingiensis were investigated for the first time in this study. Algal extracts containing different contents of astaxanthin were prepared. Through the comparison, it was shown that the extract rich in astaxanthin exhibited higher antioxidant abilities as well as stronger antiglycative capacities, including the inhibition of advanced glycation endproducts (AGEs) formation, glucose autoxidation as well as glycation-induced protein oxidation. The extract was further fractionated using TLC. Among all fractions obtained, the fraction of astaxanthin in diester form was found to contain the strongest inhibitory effects on the glycation cascade. Its tentative structure was subsequently identified by LC–MS analysis. These results clearly ascertained the antiglycoxidative properties of astaxanthin derived from C. zofingiensis and supported the possibility of using natural antioxidants as glycation inhibitors. The microalga C. zofingiensis, therefore, might be the beneficial food and preventive agent choice for diabetic patients. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Non-enzymatic glycation, also referred to as the Maillard reaction, is a process in which reducing sugars react spontaneously with amino groups of proteins. It occurs not only in foods but also in various biological systems, and leads to the formation of advanced glycation endproducts (AGEs). AGEs are a group of complex and heterogeneous molecules, comprising various products with different structures and characters (Ahmad & Ahmad, 2006). Once formed in living organisms, they can generate cross-links between key molecules, cause their structural modification and functional impairment by altering enzymatic activity, decreasing ligand binding, changing protein half-life and reducing the immunogenicity (Vlassara & Palace, 2002). Therefore non-enzymatic glycation and the accumulation of tissue AGEs are believed to act as major pathogenic processes in many human diseases especially diabetes and its complications, such as retinopathy, cataract, neuropathy and nephropathy (Nathan, 1993). Free radicals and oxidative steps are known to get involved in the glycation process which is called glycoxidation, and they are closely associated with the development of diabetic complications. During glycoxidation, glucose reduces molecular oxygen to generate superoxide radicals and converts itself to a dicarbonyl ketoal⇑ Corresponding authors. Tel.: +852 22990309; fax: +852 22990311 (F. Chen), Tel.: +852 22990338; fax: +852 22990340 (M. Wang). E-mail addresses: [email protected] (M. Wang), [email protected] (F. Chen). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.043
dehyde, which subsequently participates in the AGEs formation (Hunt, Bottoms, & Mitchinson, 1993). Once formed, the superoxide radicals can be converted to the highly reactive hydroxyl radicals via the Fenton reaction, which will induce further oxidative protein degradation (Wolff & Dean, 1987). Furthermore, many lines of evidence indicate that oxidative stress is the trigger that drives various biochemical pathways associated with hyperglycaemia-induced cell damage (Brownlee, 2001). Thus, regarding the key roles of oxidative stress in the development of diabetic complications, the discovery of antioxidants with inhibitory effects on the glycation cascade in recent years has received much attention. Such research is believed to offer a promising therapeutic and preventive approach (Ardstani & Yazdanparast, 2007). In fact, there is a large body of evidence that compounds with combined antioxidant and antiglycative properties are more effective in treating diabetes (Duraisamy et al., 2003; Xi et al., 2008). We are interested in screening and development of antioxidants containing significant antiglycative capacities from natural sources because compared to synthetic agents, natural products have fewer side effects and are much safer for human consumption. In microalgae, a wide range of antioxidants can be produced such as carotenoids, polyunsaturated fatty acids and polysaccharides (Chen, 1996). The green microalga Chlorella zofingiensis is known as a natural source of astaxanthin, a red ketocarotenoid that possesses potent anti-oxidative capacity. The antioxidant activity of astaxanthin is as high as 10 times more than other carotenoids such as zeaxanthin, lutein, canthaxanthin and b-carotene, and 100 times more than a-tocopherol (Miki, 1991). Although
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C. zofingiensis does not produce as much astaxanthin as another microalga Haematococcus pluvialis which accumulates the highest astaxanthin content in nature, this strain has many other advantages such as its fast growth and low sensitivity to contamination or unfavourable environments (Ip & Chen, 2005). These properties make C. zofingiensis a very suitable host for mass production of astaxanthin. However little is known about its antiglycative activity. Therefore, the aim of the present study was to investigate the effects of C. zofingiensis, a natural producer of astaxanthin in the glycoxidation process.
was monitored at 517 nm. The percentage of inhibition was calculated by: %inhibition = [1 (absorbance of the solution with extracts/absorbance of the solution without extracts)] 100%. 2.6. In vitro glycation of bovine serum albumin
2. Materials and methods
The incubation of bovine serum albumin (BSA) was performed as described by Sun et al. (2010). BSA (20 mg mL1) was incubated with glucose (800 mM) and NaN3 (0.2 mg mL1) in phosphate buffer (0.2 M, pH 7.4). All incubations were carried out at 37 °C for 2 weeks in the absence and presence of inhibitors. Aminoguanidine (AG) solution (1 mM) was used as the positive control.
2.1. Reagents and chemicals
2.7. Detection of glycated proteins by fluorescence measurement
SDS–PAGE reagents were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Unless otherwise stated, all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All analytical and HPLC grade solvents used were obtained from BDH Laboratory Supplies (Pool, UK).
The measurement of fluorescent intensity of AGEs was performed using a Hitachi F-2500 fluorescent spectrometer (Hitachi Corporation, Tokyo, Japan). The presence of AGEs was characterised by a typical fluorescence with excitation and emission maxima at 330 and 410 nm, respectively. Percent inhibition of AGE formation by each extract was calculated using the following equation, %inhibition = [1 (fluorescence of the solution with inhibitors/fluorescence of the solution without inhibitors)] 100%.
2.2. Algal strain and culture conditions The green microalga C. zofingiensis (ATCC 30412) was obtained from the American Type Culture Collection (Rockville, MD, USA). The algal cells were first grown in Kuhl medium (Kuhl, 1962) at 25 °C under continuous illumination of 25 lmol photon m2 s1. After 4 days, approximately 10% (v/v) exponentially growing algal cells were inoculated into the medium containing 30 g L1 of glucose. Cells were cultured in 250 mL Erlenmeyer flasks each containing 100 mL of medium and maintained at 25 °C with orbital shaking at 130 rpm in darkness.
2.8. Detection of glycated proteins by SDS–PAGE The formation of AGE products was also detected using SDS– PAGE. Briefly, samples were separated on a 12% polyacrylamide gel with 10 lg of protein loaded in each lane. The gels were stained with coomassie brilliant blue and photographed using the Bio-Rad Gel documentation system (Bio-Rad, Hercules, CA, USA). The intensity of protein bands was calculated using the Quantity one software (Bio-Rad, Hercules, CA, USA).
2.3. Preparation of extracts
2.9. Detection of hydroxyl radicals from sugar autoxidation
Cells were harvested at the 2nd and 14th day, which exhibited the colour of green and red, respectively. After being freeze-dried for 24 h, the algal biomass (0.1 g) was extracted with ethyl acetate (8 mL) at room temperature. The tube containing the extracts was centrifuged at 4500 g for 10 min and the supernatant was recovered. The extraction was repeated and the two supernatants were combined. Samples were purged to dryness using nitrogen and stored at 0 °C before use.
The detection of hydroxyl radicals induced by sugar autoxidation was carried out as described by Hunt, Dean, and Wolff (1988). 1 mM sodium benzoate, 500 mM glucose and 0.1 mM CuSO4 were dissolved in 100 mM potassium phosphate buffer. The mixture was incubated at 37 °C for 4 days in the absence and presence of inhibitors. The decrease in benzoate hydroxylation resulted from the scavenging of hydroxyl radical production was measured by the fluorescence intensity (excitation maxima: 308 nm; emission maxima: 410 nm).
2.4. Trolox equivalent antioxidant capacity (TEAC) assay 2.10. Detection of protein carbonyl formation The TEAC assay was performed according to Re et al. (1999) with minor modification. Briefly, a 2,20 -azinobis [3-ethylbenzothiazoline-6-sulphonate] radical anion (ABTS+) solution was prepared using 7 mM ABTS and 2.45 mM potassium persulfate. The solution was kept in the dark for 16 h before use. After being diluted with PBS, the ABTS.+ solution gave an absorbance of 0.700 ± 0.050 at 734 nm. For measuring antioxidant capacity, 10 lL of samples were mixed with 990 lL of ABTS+ solution, to give absorbance values 20–80% of that of the blank. The absorbance of mixture was measure at 734 nm after 6 min. Trolox solutions were used to create a standard curve. The results were expressed as mg Trolox g1 dried extract.
The content of protein carbonyl products generated from glycoxidation process was determined by the method of Ardstani and Yazdanparast (2007). Briefly, 1 mg glycated BSA/ original BSA was reacted with 1 mL of 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2 M HCl. After incubation at room temperature for 30 min, 1 mL of cold trichloroacetic acid (10%, w/v) was added to the mixture and centrifuged at 3000g for 10 min. The collected protein pellet was washed three times with ethanol/ethyl acetate (1:1 v/v) and re-dissolved in 1 mL of guanidine hydrochloride (6 M, pH 2.3). The absorbance of the sample was measured spectrophotometrically at 370 nm. The molar extinction coefficient of DNPH is e = 2.2 104 cm1 M1.
2.5. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical inhibition assay 2.11. Determination of thiol groups The DPPH radical scavenging assay was performed according to Blois (1958) with slight modification. Briefly, 1 mL of algal extracts was mixed with 1 mL of DPPH (0.1 mM) in ethanol solution. After 30 min incubation at room temperature, the decrease in absorbance
Thiol groups were measured according to Ellman’s assay (Ellman, 1959). Briefly, glycated BSA/ original BSA (3.5 mg mL1) was reacted with 5,50 -dithio-bis-(2-nitrobenzoic acid) (DTNB) (2.5 mM). The
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reaction was allowed to stand at room temperature for 15 min, yielding a yellow-coluored product known as 2-nitro-5-thiobenzoic acid (TNB). The absorbance was measured at 410 nm. A standard curve was prepared using L-cysteine hydrochloride monohydrate. The concentration of free sulfhydryl is calculated using the equation c = A/bE, whereas c = concentration (M), A = absorbance, b = path length in centimeters and E = 14,150 M1 cm1, the molar extinction coefficient of TNB. 2.12. Thin layer chromatography analysis The fractionation of the extract obtained from the 14th day was carried out by thin layer chromatography (TLC). The extract was evaporated under nitrogen gas and the residues were suspended in 50 lL of acetone. The stationary phase was silica gel 60 plates (Merck) whereas the mobile phase was ethyl acetate: hexane (40:60 v/v). Samples were loaded on TLC plates and separated into individual pigment classes. After the identification by HPLC analysis, each fraction was re-dissolved in 50 lL of acetone, of which 30 lL was used in following assays. 2.13. HPLC analysis To identify different fractions obtained from TLC analysis, the corresponding spots were scraped off the plate, extracted with acetone and analysed using the HPLC system, as described by Baroli, Do, Yamane, and Niyogi (2003) with minor modification. The HPLC system was equipped with a Waters 2695 separations module and a Waters 2996 photodiode array detector. After the filtration through a 0.22lm Millipore organic membrane, 20 lL of samples were analysed by HPLC on a Waters Spherisorb 5 lm ODS2 4.6 50 mm analytical column (Waters, Milford, MA, USA). Flow rate was 1.2 mL/min. Solvent A was acetonitrile: methanol:0.1 M Tris–HCl, pH 8.0 (84:2:14). Solvent B was methanol: ethyl acetate (68:32). A linear gradient was performed from 100% solvent A to 100% solvent B in 15 min, followed by 20 min of solvent B. The absorption spectra were 300–700 nm. Peaks were measured at 450 nm. The fractions were identified and quantified by using standard curves. 2.14. Liquid chromatography-mass spectrometry (LC–MS) analysis To get a clear understanding of the chemical structure of astaxanthin diester, the fraction of astaxanthin diester obtained from TLC was analysed on a LC–MS/MS instrument equipped with an electrospray ionization (ESI) source interfaced to a QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA). Liquid chromatography was run on a separation model (Agilent 1100; Agilent Technologies, Santa Clara, CA, USA) with a degasser, a quaternary pump and a thermostatted autosampler. Separation was conducted on a Sunfire™ C18 column. The mobile phases were water added with 0.05% formic acid (solvent A) and acetonitrile (solvent B), and the flow rate of mobile phase was set at 0.2 mL min1. The elution started with 90% A (water of 0.05% formic acid) for 10 min, then linear gradient to 80% B (acetonitrile) in 30 min and finally kept at 80% B till 50 min. The post running time was 20 min. The MS parameters were as follows: (experiment I) positive ion mode, spray voltage 3.5 kV, temperature 350 °C, scan range 50–1500 Da, DP 5 V; (experiment II) positive ion mode, spray voltage 4.5 kV, temperature 450 °C, scan range 50–500 Da, DP 60 V.
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3. Results and discussion Due to the significant roles of glycation in the development of diabetic complications, there has been an increasing interest in the investigation of AGE formation inhibitors in recent years. Although some pharmacological compounds showed strong inhibitory effects such as the nucleophilic hydrazine compound aminoguanidine (AG), the first AGE inhibitor explored in clinical trials, their side effects and high toxicity for diabetic patients are serious concerns for people, which hinders the ultimate approval for their commercial production (Thornalley, 2003). It is now generally accepted that using natural products such as plant extracts and purified constituents would be beneficial in the treatment of diabetes. In this study, the green microalga C. zofingiensis was evaluated for its effects on the glycoxidation process. This algal strain has been highlighted by a number of studies for its high accumulation of astaxanthin when grown with glucose as sole carbon and energy source (Ip & Chen, 2005; Sun, Wang, Li, Huang, & Chen, 2008). The alga accumulates primary carotenoids such as lutein and bcarotene to protect the cells from oxidative damage. However, if the amount of primary carotenoids is not enough, secondary carotenoids (i.e. astaxanthin, canthaxanthin and adonixanthin) will be generated to diminish the excessive oxidative stress, whereas astaxanthin acts as the major secondary carotenoids and over 90% of the astaxanthin is in the form of mono- and di-esters (Bar, Rise, Vishkautsan, & Arad, 1995). It was shown that under heterotrophic conditions, the colour of C. zofingiensis gradually changed from green to red, indicating the accumulation of astaxanthin within algal cells. The contents of total carotenoids and astaxanthin in C. zofingiensis were analysed by HPLC and shown in Fig. 1. During the cultivation, the level of astaxanthin started to increase from the 2nd day (0.159 mg g1) and achieved the peak at the 14th day (0.877 mg g1). Therefore in the present work, cells were harvested at the 2nd and 14th day for extraction, respectively, representing above two distinct stages. The two C. zofingiensis extracts were analysed for antioxidant activities at first. DPPH is a stable radical and often used for the evaluation of antioxidant ability of natural compounds. The IC50 values for DPPH inhibition by two extracts are shown in Table 1. Meanwhile, TEAC method was adopted to assess the ABTS+ radical scavenging ability of two extracts. Data from both assays clearly demonstrated that the 14th day (red) extract possessed higher antioxidant capacity than the 2nd day (green) extract, suggesting that astaxanthin might be the major component responsible for the antioxidant properties of C. zofingiensis extracts. It is known that oxidative reactions and reactive oxygen species (ROS) are involved in the nonenzymatic glycation process and greatly promote the AGE formation. Under the influence of transi-
2.15. Statistical analysis Experimental results were obtained as mean value ± SD (n = 3). Statistical analyses were performed using the SPSS statistical package (SPSS Inc., Chicago, IL, USA). Paired-samples T-test was applied. The statistical significances were achieved when p < 0.05.
Fig. 1. The accumulation of total carotenoids and astaxanthin by heterotrophic C. zofingiensis. (j) total carotenoids; (h) astaxanthin.
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Z. Sun et al. / Food Chemistry 126 (2011) 1629–1635 Table 2 Effects of the 2nd day (green) and the 14th day (red) C. zofingiensis extracts (1.0 mg mL1) as well as fractions separated from the 14th day (red) C. zofingiensis extract on protein carbonyl formation and thiol group content of glucose-modified BSA.
Table 1 Antioxidant activity of the 2nd day (green) and the 14th day (red) C. zofingiensis extracts determined by DPPH and TEAC methods. Sample
TEACa
DPPH (IC50)b
The 2nd day (green) extract The 14th day (red) extract
3.04 ± 0.21 3.85 ± 0.27
2.96 ± 0.05 1.04 ± 0.02
Each value represents the mean ± SD (n = 3). a TEAC was expressed as mg Trolox equivalent g1 dried extract. b IC50 was expressed as mg mL1.
tion metal ions, especially copper and iron ions, the glucose autoxidation occurs, producing numerous hydroxyl radicals which in turn contribute to protein impairments (Wolff & Dean, 1987). To investigate whether C. zofingiensis extracts could inhibit glucose autoxidation, they were reacted with sodium benzoate with the presence of glucose and Cu2+. The amount of benzoate hydroxylation induced by glucose autoxidation was detected by measuring its characteristic fluorescence. As shown in Fig. 2A, both green and red extracts exhibited significant inhibitory effects on the formation of benzoate hydroxylation. Increasing the concentration (500–1000 lg mL1) was associated with lower values of hydroxylated benzoate. At the concentration of 1000 lg mL1, algal extracts suppressed the formation of benzoate hydroxylation by 62.33% and 52.95%, respectively. These results suggested that C.
A
Sample
Protein carbonyl content (nmol mg1 protein)
Thiol group (pmol mg1 protein)
Controla Controlb The 2nd day (green) extract The 14th day (red) extract Neoxanthin Free astaxanthin Free adonixanthin Astaxanthin monoester Astaxanthin diester Adonixanthin ester Lutein + zeaxanthin AG (1 mM)
0.79 ± 0.17 5.75 ± 0.28 3.85 ± 0.18*
14.27 ± 0.78 7.64 ± 0.37 8.05 ± 0.35*
3.37 ± 0.14*
8.96 ± 0.25*
5.63 ± 0.16 4.59 ± 0.16* 5.15 ± 0.13 3.61 ± 0.13*
7.45 ± 0.51 7.56 ± 0.43 7.82 ± 0.33 8.43 ± 0.31*
2.87 ± 0.12* 4.14 ± 0.08* 4.70 ± 0.22* 2.58 ± 0.14*
9.76 ± 0.33** 9.25 ± 0.30** 8.78 ± 0.35* 9.34 ± 0.48*
Each value represents the mean ± SD (n = 3). a Reaction mixture without glucose. b Reaction mixture in the presence of glucose. * p < 0.05 significant differences with controlb ** p < 0.01 significant differences with controlb
% Inhibition of benzoate hydroxylation
80 ** **
60
40
** **
**
20 *
0 200
500 Concentration (µg mL-1) The 14th day (Red)
80 **
60 **
40
*
*
*
20
*
r
th ax an
in
+z e in te Lu
A
do n
ix
nt h sta xa A
th xa n A
sta
an th
in
on m in
do ea Fr e
es te
r ste di -e
oe
an t ni x
xa nt sta ea Fr e
ste r
n hi
n hi
n hi xa nt N
in
0
eo
B
% Inhibition of benzoate hydroxylation
The 2nd day (Green)
1000
Fig. 2. Inhibitory effects of the 2nd day (green) and the 14th day (red) C. zofingiensis extracts (Panel A) as well as fractions separated from the 14th day (red) C. zofingiensis extract (Panel B) on the generation of benzoate hydroxylation induced by glucose autoxidation. Each value represents the mean ± SD (n = 3). Fluorescent intensities of solutions with inhibitors were significantly different from that of the control solution, as marked with ⁄p < 0.05 or ⁄⁄p < 0.01.
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detected. Results showed that the original BSA maintained a very low level of carbonyl content (0.79 nmol mg1). However, after the incubation with glucose for 14 days, the carbonyl content significantly increased to 5.75 nmol mg1 in the glycated BSA. The 1.0 mg mL1 red and green extracts significantly decreased it to 3.37 nmol mg1 and 3.85 nmol mg1, respectively. On the other hand, thiol groups were measured to determine the protein modification by excessive free radicals. Results showed that due to the glycation-induced protein oxidation, a sharp decrease of thiol group content occurred in BSA, reducing from 14.27 to 7.64 pmol mg1. However, both C. zofingiensis extracts displayed significant effects to stop the loss of thiol groups at the concentration of 1.0 mg mL1, whereas the green extract displayed relatively weaker effects. In both assays, 1 mM AG solution, a commonly used antiglycative agent was used as a positive control, which exhibited good anti-protein oxidation capacities. To assess the effects of C. zofingiensis extracts on the formation of AGEs, a commonly used in vitro model, BSA-glucose system was adopted. The sugar-mediated fluorescence intensity, which is the characteristic of AGEs was measured at excitation and emission of
% Inhibition of AGE formation
zofingiensis extracts especially the red one might possess capacities to scavenge hydroxyl radical production, and/or chelate transition metals, resulting in the decrease of benzoate hydroxylation. In addition to glucose autoxidation, ROS may also lead to the modification of proteins, which is known as the oxidative modification. It takes place when protein side chains are attacked by free radicals accompanying by the generation of carbonyl groups (aldehydes and ketones). Some protein carbonyl derivatives (ketoaldehydes and ketoamines) could also be generated during the glycoxidation process (Dalle-Donne, Rossi, Giustarini, Milzani, & Colombo, 2003). Carbonyl groups are chemically stable and therefore regarded as useful biomarkers of oxidative damage (Stadtman & Levin, 2000). Another commonly used indicator for protein oxidation is the loss of thiol groups, which is often employed to quantify the modification of cysteine residues (Bourdon, Loreau, & Blache, 1999). To determine the effects of C. zofingiensis extracts on glycation-induced protein oxidation, the extents of protein carbonyl formation and thiol groups were measured (Table 2). Carbonyl groups could be derivatised with 2,4dinitrophenylhydrazine (DNPH), forming stable 2,4-dinitrophenyl (DNP) hydrazone products, which were spectrophotometrically
100
**
**
**
80 60
**
40
*
*
20
**
0
100
200
500
1mM AG
Concentration (µg mL-1) The 14th day (Red)
100
**
**
80 60 *
40 *
*
*
20
AG M
ea x in +z Lu
te
xa do ni
A
A
1m
an th in
te es n hi nt
in nt h
sta xa
3
r
r -e di
on om A
hi n xa nt
2
sta
ste
r es te
n hi xa ni
ad o
Fr ee
Fr ee
as ta xa
eo xa N
nt
nt h
hi
in
n
0 nt
% Inhibition of AGE formation
The 2nd day (Green)
1
4
5
6
7
Fig. 3. Inhibitory effects of the 2nd day (green) and the 14th day (red) C. zofingiensis extracts (Panel A) as well as fractions separated from the 14th day (red) C. zofingiensis extract (Panel B) on the formation of AGEs in BSA-glucose system. Each value represents the mean ± SD (n = 3). AG solution (1 mM) was used as a positive control. Fluorescent intensities of solutions with inhibitors were significantly different from that of the control solution, as marked with ⁄p < 0.05 or ⁄⁄p < 0.01. Panel C: SDS–PAGE profile of glycated protein. BSA (20 mg mL1) was incubated with glucose (800 mM) and NaN3 (0.2 mg mL1) in phosphate buffer (0.2 M, pH 7.4). All incubations were carried out at 37 °C for 2 weeks. 1. Mw marker; 2. glycated BSA; 3. AG (1 mM); 4. BSA; 5. the 14th day (red) C. zofingiensis extract (500 lg mL1); 6. the 2nd day (green) C. zofingiensis extract (500 lg mL1); 7. astaxanthin diester.
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Astaxanthin diester Degraded Chlorophyll Astaxanthin monoester Adonixanthin ester Chlorophyll a
Chlorophyll b Free astaxanthin Free adonixanthin Lutein + zeaxanthin
Neoxanthin Fig. 4. Fractions separated by thin layer chromatography (TLC) from the 14th day (red) C. zofingiensis extract.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
330 and 410, respectively (Cervantes-Laurean, Jacobson, & Jacobson, 1996). In Fig. 3A, at different concentrations (100– 500 lg mL1), both extracts significantly suppressed the fluorescence intensity in a dose-dependent manner, whereas the red one, i.e. the astaxanthin-rich extract, exhibited much stronger inhibitory effects. Its inhibitory rate was 80.64% at the concentration of 500 lg mL1, higher than the effect of the green extract (inhibitory rate: 67.26%) and comparable to 1 mM AG solution (inhibitory rate: 81.23%). Meanwhile, the formation of AGE products was detected using SDS–PAGE (Fig. 3C). After incubation at 37 °C for 14 days, BSA was glycated and displayed a larger molecular weight (90 kDa) than the original one (66 kDa), suggesting the occurrence of cross-linking reactions. The intensity of protein bands was calculated. Results showed that at the concentration of 500 lg mL1, both green and red extracts markedly suppressed the formation of glycation products. Based on these evidences, the potential antiglycoxidative effects of C. zofingiensis have been strongly supported. The algal extracts with antioxidant activity can significantly reduce the glucose autoxidation, diminish glycation-induced protein oxidation and inhibit the formation of AGEs. These data supported the view that antioxidant agents could be used as powerful glycation inhibitors. Through the comparison between two extracts of C. zofingiensis, it was shown that the higher the astaxanthin content, the stronger the antiglycoxidative capacity. These evidences suggested that antiglycoxidative properties of C. zofingiensis might be due to the presence of astaxanthin. To confirm whether astaxanthin is the main compound responsible for observed activities, and to get a clear understanding of its and/ or other components’ contributions in glycoxidation reactions, the astaxanthin-rich extract was fractionated by TLC. Seven major fractions were obtained. HPLC results revealed that they were astaxanthin diester (containing tiny amounts of beta-carotene), astaxanthin monoester, adonixanthin
ester, free astaxanthin, free adonixanthin, lutein + zeaxanthin and neoxanthin, respectively (Fig. 4). These fractions were subsequently re-dissolved in 50 lL of acetone, of which 30 lL were used in following assays. In the BSA-glucose system, fractions showed very diverse abilities to suppress the fluorescence intensity (Fig. 3B). The astaxanthin diester was found to have the strongest antiglycative capacity (inhibitory rate: 83.59%), higher than the effect of 1 mM AG solution (inhibitory rate: 81.23%). Such results were also confirmed on the SDS–PAGE profile (Fig. 3C). Inhibitory rates of remaining fractions decreased in the order of astaxanthin monoester (29.48%) > free astaxanthin (16.89%) > lutein + zeaxanthin (15.76%) > adonixanthin ester (13.63%), whereas neoxanthin and free adonixanthin showed no effects. In the glucose autoxidation assay, except for the fraction of neoxanthin showing no activity, other fractions significantly inhibited the generation of benzoate hydroxylation (Fig. 2B). Astaxanthin diester was found to exhibit the highest inhibitory rate (62.27%). Inhibitory rates of remaining fractions decreased in the order of astaxanthin monoester (35.07%) > lutein + zeaxanthin (20.90%) > adonixanthin ester (14.81%) > free astaxanthin (14.34%) > free adonixanthin (9.24%). In the protein carbonyl assay, the fraction of astaxanthin diester exhibited the highest inhibitory activity (Table 2). It decreased the carbonyl content from 5.75 to 2.87 nmol mg1, followed by astaxanthin monoester (3.61 nmol mg1) and adonixanthin ester (4.14 nmol mg1). Other fractions showed relatively weaker effects in which neoxanthin failed to diminish the carbonylation. In the thiol group assay, expect for fractions of neoxanthin, free adonixanthin and free astaxanthin, all other fractions displayed significant effects to stop the loss of thiol groups. astaxanthin diester was found to exhibit the highest activity (thiol group content: 9.76 pmol mg1), followed by adonixanthin ester (9.25 pmol mg1), lutein + zeaxanthin (8.78 pmol mg1) and astaxanthin monoester (8.43 pmol mg1). These results, taken together, clearly demonstrated the hypothesised roles of astaxanthin in antiglycoxidative activities of C. zofingiensis extracts. Although several compounds e.g. adonixanthin and lutein + zeaxanthin were also involved in the blockage of glycation cascade, astaxanthin diester was the major contributor, providing the most significant protective effects. The esterified form of astaxanthin in C. zofingiensis is believed to be a more stabilised structure to maintain the high antioxidant ability (Kobayashi & Sakamoto, 1999). LC–MS was adopted to identify the fatty acid composition of astaxanthin diester. According to the LC–MS results, the detected fragment of 862.4 was presumably identified as astaxanthin-octadecanoic acid (C18:0), 282.4 as oleic acid (C18:1), and 257.0 as palmitic acid (C16:0). These LC–MS results suggested that the fatty acids in these astaxanthin diesters are highly possible to be octadecanoic acid, oleic acid and palmitic acid, which were also in accordant to the GC–MS detection and library searching results (Grynbaum et al., 2005).
4. Conclusions The green microalga C. zofingiensis was systematically evaluated for its antiglycoxidative properties for the first time in this study. Results showed that the C. zofingiensis extracts possessed antioxidant capacities and significant inhibitory effects on the glycation cascade in various model systems. Further studies revealed that astaxanthin was the major component contributing to observed activities. Taken together, these findings supported the possibility of using natural antioxidants as glycation inhibitors and suggested the beneficial roles of astaxanthin, which is an essential nutritional component for human health in diabetes. The astaxanthin-rich microalga C. zofingiensis therefore might be regarded as a potential therapeutic and preventive agent for diabetes and its complica-
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tions. In future, detailed cell model studies and in vivo investigations are needed to provide more evidence. Acknowledgement This work was supported by the GRF Grand of the Research Grants Council of Hong Kong. References Ahmad, M. S., & Ahmad, N. (2006). Antiglycation properties of aged garlic extract: Possible role in prevention of diabetic complications. Journal of Nutrition, 136, 796–799. Ardstani, A., & Yazdanparast, R. (2007). Inhibitory effects of ethyl acetate extract of Teucrium polium on in vitro protein glycoxidation. Food and Chemical Toxicology, 45, 2402–2411. Bar, E., Rise, M., Vishkautsan, M., & Arad, S. (1995). Pigments and structural changes in Chlorella zofingiensis upon light and nitrogen stress. Journal of Plant Pysiology, 146, 527–534. Baroli, I., Do, A. D., Yamane, T., & Niyogi, K. K. (2003). Zeaxanthin accumulation in the absence of a functional xanthophyll cycle protects Chlamydomonas reinhardtii from photooxidative stress. Plant Cell, 15, 992–1008. Blois, M. S. (1958). Antioxidant determination by the use of a stable free radical. Nature, 181, 1190–1200. Bourdon, E., Loreau, N., & Blache, D. (1999). Glucose and free radicals impair the antioxidant properties of serum albumin. The FASEB Journal, 13, 233–244. Brownlee, M. (2001). Biochemistry and molecular cell biology of diabetic complications. Nature, 414, 813–820. Cervantes-Laurean, D., Jacobson, E. L., & Jacobson, M. K. (1996). Glycation and glycoxidation of histones by ADP-ribose. Journal of Biological Chemistry, 217, 10461–10469. Chen, F. (1996). High cell density culture of microalgae in heterotrophic growth. Trends in Biotechnology, 14, 421–426. Dalle-Donne, I., Rossi, R., Giustarini, D., Milzani, A., & Colombo, R. (2003). Protein carbonyl groups as biomarkers of oxidative stress. Clinica Chemica Acta, 329, 23–38. Duraisamy, Y., Gaffney, J., Slevin, M., Smith, C. A., Williamson, K., & Ahmed, N. (2003). Aminosalicylic acid reduces the antiproliferative effect of hyperglycaemia, advanced glycation endproducts and glycated basic fibroblast growth factor in cultured bovine aortic endothelial cells: Comparison with aminoguanidine. Molecular and Cellular Biochemistry, 246, 143–153. Ellman, G. L. (1959). Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics, 82, 70–77.
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Grynbaum, M. D., Hentschel, P., Putzbach, K., Rehbein, J., Krucker, M., Nicholson, G., et al. (2005). Unambiguous detection of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superba Dana). Journal of Separation Science, 28, 1685–1693. Hunt, J. V., Bottoms, M. A., & Mitchinson, M. J. (1993). Oxidative alterations in the experimental glycation model of diabetes mellitus are due to protein-glucose adduct oxidation. The Biochemical Journal, 291, 529–535. Hunt, J. V., Dean, R. T., & Wolff, S. P. (1988). Hydroxyl radical production and autoxidative glycosylation. Glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes mellitus and aging. Biochemical Journal, 256, 205–212. Ip, P. F., & Chen, F. (2005). Production of astaxanthin by the green microalga Chlorella zofingiensis in the dark. Process Biochemistry, 40, 733–738. Kobayashi, M., & Sakamoto, Y. (1999). Singlet oxygen quenching ability of astaxanthin esters from the green alga Haematococcus pluvialis. Biotechnology Letters, 21, 265–269. Kuhl, A. (1962). Zur physiologie der speicherung kondensetem organischer phosphate in Chlorella. In: Beiträge zur Physiologie und Morphologie der Algen. Gustav Fischer Verlage, Stuttgart, West Germany. Miki, W. (1991). Biological functions and activities of animal carotenoids. Pure and Applied Chemistry, 63, 141–146. Nathan, D. M. (1993). Long-term complications of diabetes mellitus. The New England Journal of Medicine, 328, 1676–1685. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26, 1231–1237. Stadtman, E. R., & Levin, R. L. (2000). Protein oxidation. Annals of the New York Academy of Sciences, 899, 191–208. Sun, Z., Peng, X. F., Liu, J., Fan, K. W., Wang, M. F., & Chen, F. (2010). Inhibitory effects of microalgal extracts on the formation of advanced glycation endproducts (AGEs). Food Chemistry, 120, 261–267. Sun, N., Wang, Y., Li, Y. T., Huang, J. C., & Chen, F. (2008). Sugar-based growth, astaxanthin accumulation and carotenogenic transcription of heterotrophic Chlorella zofingiensis (Chlorophyta). Process Biochemistry, 43, 1288–1292. Thornalley, P. J. (2003). Use of aminoguanidine (pimagedine) to prevent the formation of advanced glycation endproducts. Archives of Biochemistry and Biophysics, 419, 31–40. Vlassara, H., & Palace, M. R. (2002). Diabetes and advanced glycation endproducts. Journal of Internal Medicine, 251, 87–101. Wolff, S. P., & Dean, R. T. (1987). Glucose autoxidation and protein modification: The potential role of ‘autoxidative glycosylation’ in diabetes mellitus. The Biochemical Journal, 245, 243–250. Xi, M., Hai, C., Tang, H., Chen, M., Fang, K., & Liang, X. (2008). Antioxidant and antiglycation properties of total saponins extracted from traditional Chinese medicine used to treat diabetes mellitus. Phytotherapy Research, 22, 228–237.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Astaxanthin is responsible for antiglycoxidative properties of microalga Chlorella zofingiensis Zheng Sun a, Jin Liu a, Xiaohui Zeng a, Jieqiong Huangfu a, Yue Jiang b, Mingfu Wang a,⇑, Feng Chen a,⇑ a b
School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, PR China Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China
a r t i c l e
i n f o
Article history: Received 14 September 2010 Received in revised form 13 November 2010 Accepted 5 December 2010 Available online 10 December 2010 Keywords: Astaxanthin Chlorella zofingiensis Diabetic complications Glycoxidation
a b s t r a c t The antiglycoxidative properties of microalga Chlorella zofingiensis were investigated for the first time in this study. Algal extracts containing different contents of astaxanthin were prepared. Through the comparison, it was shown that the extract rich in astaxanthin exhibited higher antioxidant abilities as well as stronger antiglycative capacities, including the inhibition of advanced glycation endproducts (AGEs) formation, glucose autoxidation as well as glycation-induced protein oxidation. The extract was further fractionated using TLC. Among all fractions obtained, the fraction of astaxanthin in diester form was found to contain the strongest inhibitory effects on the glycation cascade. Its tentative structure was subsequently identified by LC–MS analysis. These results clearly ascertained the antiglycoxidative properties of astaxanthin derived from C. zofingiensis and supported the possibility of using natural antioxidants as glycation inhibitors. The microalga C. zofingiensis, therefore, might be the beneficial food and preventive agent choice for diabetic patients. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Non-enzymatic glycation, also referred to as the Maillard reaction, is a process in which reducing sugars react spontaneously with amino groups of proteins. It occurs not only in foods but also in various biological systems, and leads to the formation of advanced glycation endproducts (AGEs). AGEs are a group of complex and heterogeneous molecules, comprising various products with different structures and characters (Ahmad & Ahmad, 2006). Once formed in living organisms, they can generate cross-links between key molecules, cause their structural modification and functional impairment by altering enzymatic activity, decreasing ligand binding, changing protein half-life and reducing the immunogenicity (Vlassara & Palace, 2002). Therefore non-enzymatic glycation and the accumulation of tissue AGEs are believed to act as major pathogenic processes in many human diseases especially diabetes and its complications, such as retinopathy, cataract, neuropathy and nephropathy (Nathan, 1993). Free radicals and oxidative steps are known to get involved in the glycation process which is called glycoxidation, and they are closely associated with the development of diabetic complications. During glycoxidation, glucose reduces molecular oxygen to generate superoxide radicals and converts itself to a dicarbonyl ketoal⇑ Corresponding authors. Tel.: +852 22990309; fax: +852 22990311 (F. Chen), Tel.: +852 22990338; fax: +852 22990340 (M. Wang). E-mail addresses: [email protected] (M. Wang), [email protected] (F. Chen). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.043
dehyde, which subsequently participates in the AGEs formation (Hunt, Bottoms, & Mitchinson, 1993). Once formed, the superoxide radicals can be converted to the highly reactive hydroxyl radicals via the Fenton reaction, which will induce further oxidative protein degradation (Wolff & Dean, 1987). Furthermore, many lines of evidence indicate that oxidative stress is the trigger that drives various biochemical pathways associated with hyperglycaemia-induced cell damage (Brownlee, 2001). Thus, regarding the key roles of oxidative stress in the development of diabetic complications, the discovery of antioxidants with inhibitory effects on the glycation cascade in recent years has received much attention. Such research is believed to offer a promising therapeutic and preventive approach (Ardstani & Yazdanparast, 2007). In fact, there is a large body of evidence that compounds with combined antioxidant and antiglycative properties are more effective in treating diabetes (Duraisamy et al., 2003; Xi et al., 2008). We are interested in screening and development of antioxidants containing significant antiglycative capacities from natural sources because compared to synthetic agents, natural products have fewer side effects and are much safer for human consumption. In microalgae, a wide range of antioxidants can be produced such as carotenoids, polyunsaturated fatty acids and polysaccharides (Chen, 1996). The green microalga Chlorella zofingiensis is known as a natural source of astaxanthin, a red ketocarotenoid that possesses potent anti-oxidative capacity. The antioxidant activity of astaxanthin is as high as 10 times more than other carotenoids such as zeaxanthin, lutein, canthaxanthin and b-carotene, and 100 times more than a-tocopherol (Miki, 1991). Although
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C. zofingiensis does not produce as much astaxanthin as another microalga Haematococcus pluvialis which accumulates the highest astaxanthin content in nature, this strain has many other advantages such as its fast growth and low sensitivity to contamination or unfavourable environments (Ip & Chen, 2005). These properties make C. zofingiensis a very suitable host for mass production of astaxanthin. However little is known about its antiglycative activity. Therefore, the aim of the present study was to investigate the effects of C. zofingiensis, a natural producer of astaxanthin in the glycoxidation process.
was monitored at 517 nm. The percentage of inhibition was calculated by: %inhibition = [1 (absorbance of the solution with extracts/absorbance of the solution without extracts)] 100%. 2.6. In vitro glycation of bovine serum albumin
2. Materials and methods
The incubation of bovine serum albumin (BSA) was performed as described by Sun et al. (2010). BSA (20 mg mL1) was incubated with glucose (800 mM) and NaN3 (0.2 mg mL1) in phosphate buffer (0.2 M, pH 7.4). All incubations were carried out at 37 °C for 2 weeks in the absence and presence of inhibitors. Aminoguanidine (AG) solution (1 mM) was used as the positive control.
2.1. Reagents and chemicals
2.7. Detection of glycated proteins by fluorescence measurement
SDS–PAGE reagents were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Unless otherwise stated, all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All analytical and HPLC grade solvents used were obtained from BDH Laboratory Supplies (Pool, UK).
The measurement of fluorescent intensity of AGEs was performed using a Hitachi F-2500 fluorescent spectrometer (Hitachi Corporation, Tokyo, Japan). The presence of AGEs was characterised by a typical fluorescence with excitation and emission maxima at 330 and 410 nm, respectively. Percent inhibition of AGE formation by each extract was calculated using the following equation, %inhibition = [1 (fluorescence of the solution with inhibitors/fluorescence of the solution without inhibitors)] 100%.
2.2. Algal strain and culture conditions The green microalga C. zofingiensis (ATCC 30412) was obtained from the American Type Culture Collection (Rockville, MD, USA). The algal cells were first grown in Kuhl medium (Kuhl, 1962) at 25 °C under continuous illumination of 25 lmol photon m2 s1. After 4 days, approximately 10% (v/v) exponentially growing algal cells were inoculated into the medium containing 30 g L1 of glucose. Cells were cultured in 250 mL Erlenmeyer flasks each containing 100 mL of medium and maintained at 25 °C with orbital shaking at 130 rpm in darkness.
2.8. Detection of glycated proteins by SDS–PAGE The formation of AGE products was also detected using SDS– PAGE. Briefly, samples were separated on a 12% polyacrylamide gel with 10 lg of protein loaded in each lane. The gels were stained with coomassie brilliant blue and photographed using the Bio-Rad Gel documentation system (Bio-Rad, Hercules, CA, USA). The intensity of protein bands was calculated using the Quantity one software (Bio-Rad, Hercules, CA, USA).
2.3. Preparation of extracts
2.9. Detection of hydroxyl radicals from sugar autoxidation
Cells were harvested at the 2nd and 14th day, which exhibited the colour of green and red, respectively. After being freeze-dried for 24 h, the algal biomass (0.1 g) was extracted with ethyl acetate (8 mL) at room temperature. The tube containing the extracts was centrifuged at 4500 g for 10 min and the supernatant was recovered. The extraction was repeated and the two supernatants were combined. Samples were purged to dryness using nitrogen and stored at 0 °C before use.
The detection of hydroxyl radicals induced by sugar autoxidation was carried out as described by Hunt, Dean, and Wolff (1988). 1 mM sodium benzoate, 500 mM glucose and 0.1 mM CuSO4 were dissolved in 100 mM potassium phosphate buffer. The mixture was incubated at 37 °C for 4 days in the absence and presence of inhibitors. The decrease in benzoate hydroxylation resulted from the scavenging of hydroxyl radical production was measured by the fluorescence intensity (excitation maxima: 308 nm; emission maxima: 410 nm).
2.4. Trolox equivalent antioxidant capacity (TEAC) assay 2.10. Detection of protein carbonyl formation The TEAC assay was performed according to Re et al. (1999) with minor modification. Briefly, a 2,20 -azinobis [3-ethylbenzothiazoline-6-sulphonate] radical anion (ABTS+) solution was prepared using 7 mM ABTS and 2.45 mM potassium persulfate. The solution was kept in the dark for 16 h before use. After being diluted with PBS, the ABTS.+ solution gave an absorbance of 0.700 ± 0.050 at 734 nm. For measuring antioxidant capacity, 10 lL of samples were mixed with 990 lL of ABTS+ solution, to give absorbance values 20–80% of that of the blank. The absorbance of mixture was measure at 734 nm after 6 min. Trolox solutions were used to create a standard curve. The results were expressed as mg Trolox g1 dried extract.
The content of protein carbonyl products generated from glycoxidation process was determined by the method of Ardstani and Yazdanparast (2007). Briefly, 1 mg glycated BSA/ original BSA was reacted with 1 mL of 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2 M HCl. After incubation at room temperature for 30 min, 1 mL of cold trichloroacetic acid (10%, w/v) was added to the mixture and centrifuged at 3000g for 10 min. The collected protein pellet was washed three times with ethanol/ethyl acetate (1:1 v/v) and re-dissolved in 1 mL of guanidine hydrochloride (6 M, pH 2.3). The absorbance of the sample was measured spectrophotometrically at 370 nm. The molar extinction coefficient of DNPH is e = 2.2 104 cm1 M1.
2.5. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical inhibition assay 2.11. Determination of thiol groups The DPPH radical scavenging assay was performed according to Blois (1958) with slight modification. Briefly, 1 mL of algal extracts was mixed with 1 mL of DPPH (0.1 mM) in ethanol solution. After 30 min incubation at room temperature, the decrease in absorbance
Thiol groups were measured according to Ellman’s assay (Ellman, 1959). Briefly, glycated BSA/ original BSA (3.5 mg mL1) was reacted with 5,50 -dithio-bis-(2-nitrobenzoic acid) (DTNB) (2.5 mM). The
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reaction was allowed to stand at room temperature for 15 min, yielding a yellow-coluored product known as 2-nitro-5-thiobenzoic acid (TNB). The absorbance was measured at 410 nm. A standard curve was prepared using L-cysteine hydrochloride monohydrate. The concentration of free sulfhydryl is calculated using the equation c = A/bE, whereas c = concentration (M), A = absorbance, b = path length in centimeters and E = 14,150 M1 cm1, the molar extinction coefficient of TNB. 2.12. Thin layer chromatography analysis The fractionation of the extract obtained from the 14th day was carried out by thin layer chromatography (TLC). The extract was evaporated under nitrogen gas and the residues were suspended in 50 lL of acetone. The stationary phase was silica gel 60 plates (Merck) whereas the mobile phase was ethyl acetate: hexane (40:60 v/v). Samples were loaded on TLC plates and separated into individual pigment classes. After the identification by HPLC analysis, each fraction was re-dissolved in 50 lL of acetone, of which 30 lL was used in following assays. 2.13. HPLC analysis To identify different fractions obtained from TLC analysis, the corresponding spots were scraped off the plate, extracted with acetone and analysed using the HPLC system, as described by Baroli, Do, Yamane, and Niyogi (2003) with minor modification. The HPLC system was equipped with a Waters 2695 separations module and a Waters 2996 photodiode array detector. After the filtration through a 0.22lm Millipore organic membrane, 20 lL of samples were analysed by HPLC on a Waters Spherisorb 5 lm ODS2 4.6 50 mm analytical column (Waters, Milford, MA, USA). Flow rate was 1.2 mL/min. Solvent A was acetonitrile: methanol:0.1 M Tris–HCl, pH 8.0 (84:2:14). Solvent B was methanol: ethyl acetate (68:32). A linear gradient was performed from 100% solvent A to 100% solvent B in 15 min, followed by 20 min of solvent B. The absorption spectra were 300–700 nm. Peaks were measured at 450 nm. The fractions were identified and quantified by using standard curves. 2.14. Liquid chromatography-mass spectrometry (LC–MS) analysis To get a clear understanding of the chemical structure of astaxanthin diester, the fraction of astaxanthin diester obtained from TLC was analysed on a LC–MS/MS instrument equipped with an electrospray ionization (ESI) source interfaced to a QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA). Liquid chromatography was run on a separation model (Agilent 1100; Agilent Technologies, Santa Clara, CA, USA) with a degasser, a quaternary pump and a thermostatted autosampler. Separation was conducted on a Sunfire™ C18 column. The mobile phases were water added with 0.05% formic acid (solvent A) and acetonitrile (solvent B), and the flow rate of mobile phase was set at 0.2 mL min1. The elution started with 90% A (water of 0.05% formic acid) for 10 min, then linear gradient to 80% B (acetonitrile) in 30 min and finally kept at 80% B till 50 min. The post running time was 20 min. The MS parameters were as follows: (experiment I) positive ion mode, spray voltage 3.5 kV, temperature 350 °C, scan range 50–1500 Da, DP 5 V; (experiment II) positive ion mode, spray voltage 4.5 kV, temperature 450 °C, scan range 50–500 Da, DP 60 V.
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3. Results and discussion Due to the significant roles of glycation in the development of diabetic complications, there has been an increasing interest in the investigation of AGE formation inhibitors in recent years. Although some pharmacological compounds showed strong inhibitory effects such as the nucleophilic hydrazine compound aminoguanidine (AG), the first AGE inhibitor explored in clinical trials, their side effects and high toxicity for diabetic patients are serious concerns for people, which hinders the ultimate approval for their commercial production (Thornalley, 2003). It is now generally accepted that using natural products such as plant extracts and purified constituents would be beneficial in the treatment of diabetes. In this study, the green microalga C. zofingiensis was evaluated for its effects on the glycoxidation process. This algal strain has been highlighted by a number of studies for its high accumulation of astaxanthin when grown with glucose as sole carbon and energy source (Ip & Chen, 2005; Sun, Wang, Li, Huang, & Chen, 2008). The alga accumulates primary carotenoids such as lutein and bcarotene to protect the cells from oxidative damage. However, if the amount of primary carotenoids is not enough, secondary carotenoids (i.e. astaxanthin, canthaxanthin and adonixanthin) will be generated to diminish the excessive oxidative stress, whereas astaxanthin acts as the major secondary carotenoids and over 90% of the astaxanthin is in the form of mono- and di-esters (Bar, Rise, Vishkautsan, & Arad, 1995). It was shown that under heterotrophic conditions, the colour of C. zofingiensis gradually changed from green to red, indicating the accumulation of astaxanthin within algal cells. The contents of total carotenoids and astaxanthin in C. zofingiensis were analysed by HPLC and shown in Fig. 1. During the cultivation, the level of astaxanthin started to increase from the 2nd day (0.159 mg g1) and achieved the peak at the 14th day (0.877 mg g1). Therefore in the present work, cells were harvested at the 2nd and 14th day for extraction, respectively, representing above two distinct stages. The two C. zofingiensis extracts were analysed for antioxidant activities at first. DPPH is a stable radical and often used for the evaluation of antioxidant ability of natural compounds. The IC50 values for DPPH inhibition by two extracts are shown in Table 1. Meanwhile, TEAC method was adopted to assess the ABTS+ radical scavenging ability of two extracts. Data from both assays clearly demonstrated that the 14th day (red) extract possessed higher antioxidant capacity than the 2nd day (green) extract, suggesting that astaxanthin might be the major component responsible for the antioxidant properties of C. zofingiensis extracts. It is known that oxidative reactions and reactive oxygen species (ROS) are involved in the nonenzymatic glycation process and greatly promote the AGE formation. Under the influence of transi-
2.15. Statistical analysis Experimental results were obtained as mean value ± SD (n = 3). Statistical analyses were performed using the SPSS statistical package (SPSS Inc., Chicago, IL, USA). Paired-samples T-test was applied. The statistical significances were achieved when p < 0.05.
Fig. 1. The accumulation of total carotenoids and astaxanthin by heterotrophic C. zofingiensis. (j) total carotenoids; (h) astaxanthin.
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Z. Sun et al. / Food Chemistry 126 (2011) 1629–1635 Table 2 Effects of the 2nd day (green) and the 14th day (red) C. zofingiensis extracts (1.0 mg mL1) as well as fractions separated from the 14th day (red) C. zofingiensis extract on protein carbonyl formation and thiol group content of glucose-modified BSA.
Table 1 Antioxidant activity of the 2nd day (green) and the 14th day (red) C. zofingiensis extracts determined by DPPH and TEAC methods. Sample
TEACa
DPPH (IC50)b
The 2nd day (green) extract The 14th day (red) extract
3.04 ± 0.21 3.85 ± 0.27
2.96 ± 0.05 1.04 ± 0.02
Each value represents the mean ± SD (n = 3). a TEAC was expressed as mg Trolox equivalent g1 dried extract. b IC50 was expressed as mg mL1.
tion metal ions, especially copper and iron ions, the glucose autoxidation occurs, producing numerous hydroxyl radicals which in turn contribute to protein impairments (Wolff & Dean, 1987). To investigate whether C. zofingiensis extracts could inhibit glucose autoxidation, they were reacted with sodium benzoate with the presence of glucose and Cu2+. The amount of benzoate hydroxylation induced by glucose autoxidation was detected by measuring its characteristic fluorescence. As shown in Fig. 2A, both green and red extracts exhibited significant inhibitory effects on the formation of benzoate hydroxylation. Increasing the concentration (500–1000 lg mL1) was associated with lower values of hydroxylated benzoate. At the concentration of 1000 lg mL1, algal extracts suppressed the formation of benzoate hydroxylation by 62.33% and 52.95%, respectively. These results suggested that C.
A
Sample
Protein carbonyl content (nmol mg1 protein)
Thiol group (pmol mg1 protein)
Controla Controlb The 2nd day (green) extract The 14th day (red) extract Neoxanthin Free astaxanthin Free adonixanthin Astaxanthin monoester Astaxanthin diester Adonixanthin ester Lutein + zeaxanthin AG (1 mM)
0.79 ± 0.17 5.75 ± 0.28 3.85 ± 0.18*
14.27 ± 0.78 7.64 ± 0.37 8.05 ± 0.35*
3.37 ± 0.14*
8.96 ± 0.25*
5.63 ± 0.16 4.59 ± 0.16* 5.15 ± 0.13 3.61 ± 0.13*
7.45 ± 0.51 7.56 ± 0.43 7.82 ± 0.33 8.43 ± 0.31*
2.87 ± 0.12* 4.14 ± 0.08* 4.70 ± 0.22* 2.58 ± 0.14*
9.76 ± 0.33** 9.25 ± 0.30** 8.78 ± 0.35* 9.34 ± 0.48*
Each value represents the mean ± SD (n = 3). a Reaction mixture without glucose. b Reaction mixture in the presence of glucose. * p < 0.05 significant differences with controlb ** p < 0.01 significant differences with controlb
% Inhibition of benzoate hydroxylation
80 ** **
60
40
** **
**
20 *
0 200
500 Concentration (µg mL-1) The 14th day (Red)
80 **
60 **
40
*
*
*
20
*
r
th ax an
in
+z e in te Lu
A
do n
ix
nt h sta xa A
th xa n A
sta
an th
in
on m in
do ea Fr e
es te
r ste di -e
oe
an t ni x
xa nt sta ea Fr e
ste r
n hi
n hi
n hi xa nt N
in
0
eo
B
% Inhibition of benzoate hydroxylation
The 2nd day (Green)
1000
Fig. 2. Inhibitory effects of the 2nd day (green) and the 14th day (red) C. zofingiensis extracts (Panel A) as well as fractions separated from the 14th day (red) C. zofingiensis extract (Panel B) on the generation of benzoate hydroxylation induced by glucose autoxidation. Each value represents the mean ± SD (n = 3). Fluorescent intensities of solutions with inhibitors were significantly different from that of the control solution, as marked with ⁄p < 0.05 or ⁄⁄p < 0.01.
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detected. Results showed that the original BSA maintained a very low level of carbonyl content (0.79 nmol mg1). However, after the incubation with glucose for 14 days, the carbonyl content significantly increased to 5.75 nmol mg1 in the glycated BSA. The 1.0 mg mL1 red and green extracts significantly decreased it to 3.37 nmol mg1 and 3.85 nmol mg1, respectively. On the other hand, thiol groups were measured to determine the protein modification by excessive free radicals. Results showed that due to the glycation-induced protein oxidation, a sharp decrease of thiol group content occurred in BSA, reducing from 14.27 to 7.64 pmol mg1. However, both C. zofingiensis extracts displayed significant effects to stop the loss of thiol groups at the concentration of 1.0 mg mL1, whereas the green extract displayed relatively weaker effects. In both assays, 1 mM AG solution, a commonly used antiglycative agent was used as a positive control, which exhibited good anti-protein oxidation capacities. To assess the effects of C. zofingiensis extracts on the formation of AGEs, a commonly used in vitro model, BSA-glucose system was adopted. The sugar-mediated fluorescence intensity, which is the characteristic of AGEs was measured at excitation and emission of
% Inhibition of AGE formation
zofingiensis extracts especially the red one might possess capacities to scavenge hydroxyl radical production, and/or chelate transition metals, resulting in the decrease of benzoate hydroxylation. In addition to glucose autoxidation, ROS may also lead to the modification of proteins, which is known as the oxidative modification. It takes place when protein side chains are attacked by free radicals accompanying by the generation of carbonyl groups (aldehydes and ketones). Some protein carbonyl derivatives (ketoaldehydes and ketoamines) could also be generated during the glycoxidation process (Dalle-Donne, Rossi, Giustarini, Milzani, & Colombo, 2003). Carbonyl groups are chemically stable and therefore regarded as useful biomarkers of oxidative damage (Stadtman & Levin, 2000). Another commonly used indicator for protein oxidation is the loss of thiol groups, which is often employed to quantify the modification of cysteine residues (Bourdon, Loreau, & Blache, 1999). To determine the effects of C. zofingiensis extracts on glycation-induced protein oxidation, the extents of protein carbonyl formation and thiol groups were measured (Table 2). Carbonyl groups could be derivatised with 2,4dinitrophenylhydrazine (DNPH), forming stable 2,4-dinitrophenyl (DNP) hydrazone products, which were spectrophotometrically
100
**
**
**
80 60
**
40
*
*
20
**
0
100
200
500
1mM AG
Concentration (µg mL-1) The 14th day (Red)
100
**
**
80 60 *
40 *
*
*
20
AG M
ea x in +z Lu
te
xa do ni
A
A
1m
an th in
te es n hi nt
in nt h
sta xa
3
r
r -e di
on om A
hi n xa nt
2
sta
ste
r es te
n hi xa ni
ad o
Fr ee
Fr ee
as ta xa
eo xa N
nt
nt h
hi
in
n
0 nt
% Inhibition of AGE formation
The 2nd day (Green)
1
4
5
6
7
Fig. 3. Inhibitory effects of the 2nd day (green) and the 14th day (red) C. zofingiensis extracts (Panel A) as well as fractions separated from the 14th day (red) C. zofingiensis extract (Panel B) on the formation of AGEs in BSA-glucose system. Each value represents the mean ± SD (n = 3). AG solution (1 mM) was used as a positive control. Fluorescent intensities of solutions with inhibitors were significantly different from that of the control solution, as marked with ⁄p < 0.05 or ⁄⁄p < 0.01. Panel C: SDS–PAGE profile of glycated protein. BSA (20 mg mL1) was incubated with glucose (800 mM) and NaN3 (0.2 mg mL1) in phosphate buffer (0.2 M, pH 7.4). All incubations were carried out at 37 °C for 2 weeks. 1. Mw marker; 2. glycated BSA; 3. AG (1 mM); 4. BSA; 5. the 14th day (red) C. zofingiensis extract (500 lg mL1); 6. the 2nd day (green) C. zofingiensis extract (500 lg mL1); 7. astaxanthin diester.
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Astaxanthin diester Degraded Chlorophyll Astaxanthin monoester Adonixanthin ester Chlorophyll a
Chlorophyll b Free astaxanthin Free adonixanthin Lutein + zeaxanthin
Neoxanthin Fig. 4. Fractions separated by thin layer chromatography (TLC) from the 14th day (red) C. zofingiensis extract.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
330 and 410, respectively (Cervantes-Laurean, Jacobson, & Jacobson, 1996). In Fig. 3A, at different concentrations (100– 500 lg mL1), both extracts significantly suppressed the fluorescence intensity in a dose-dependent manner, whereas the red one, i.e. the astaxanthin-rich extract, exhibited much stronger inhibitory effects. Its inhibitory rate was 80.64% at the concentration of 500 lg mL1, higher than the effect of the green extract (inhibitory rate: 67.26%) and comparable to 1 mM AG solution (inhibitory rate: 81.23%). Meanwhile, the formation of AGE products was detected using SDS–PAGE (Fig. 3C). After incubation at 37 °C for 14 days, BSA was glycated and displayed a larger molecular weight (90 kDa) than the original one (66 kDa), suggesting the occurrence of cross-linking reactions. The intensity of protein bands was calculated. Results showed that at the concentration of 500 lg mL1, both green and red extracts markedly suppressed the formation of glycation products. Based on these evidences, the potential antiglycoxidative effects of C. zofingiensis have been strongly supported. The algal extracts with antioxidant activity can significantly reduce the glucose autoxidation, diminish glycation-induced protein oxidation and inhibit the formation of AGEs. These data supported the view that antioxidant agents could be used as powerful glycation inhibitors. Through the comparison between two extracts of C. zofingiensis, it was shown that the higher the astaxanthin content, the stronger the antiglycoxidative capacity. These evidences suggested that antiglycoxidative properties of C. zofingiensis might be due to the presence of astaxanthin. To confirm whether astaxanthin is the main compound responsible for observed activities, and to get a clear understanding of its and/ or other components’ contributions in glycoxidation reactions, the astaxanthin-rich extract was fractionated by TLC. Seven major fractions were obtained. HPLC results revealed that they were astaxanthin diester (containing tiny amounts of beta-carotene), astaxanthin monoester, adonixanthin
ester, free astaxanthin, free adonixanthin, lutein + zeaxanthin and neoxanthin, respectively (Fig. 4). These fractions were subsequently re-dissolved in 50 lL of acetone, of which 30 lL were used in following assays. In the BSA-glucose system, fractions showed very diverse abilities to suppress the fluorescence intensity (Fig. 3B). The astaxanthin diester was found to have the strongest antiglycative capacity (inhibitory rate: 83.59%), higher than the effect of 1 mM AG solution (inhibitory rate: 81.23%). Such results were also confirmed on the SDS–PAGE profile (Fig. 3C). Inhibitory rates of remaining fractions decreased in the order of astaxanthin monoester (29.48%) > free astaxanthin (16.89%) > lutein + zeaxanthin (15.76%) > adonixanthin ester (13.63%), whereas neoxanthin and free adonixanthin showed no effects. In the glucose autoxidation assay, except for the fraction of neoxanthin showing no activity, other fractions significantly inhibited the generation of benzoate hydroxylation (Fig. 2B). Astaxanthin diester was found to exhibit the highest inhibitory rate (62.27%). Inhibitory rates of remaining fractions decreased in the order of astaxanthin monoester (35.07%) > lutein + zeaxanthin (20.90%) > adonixanthin ester (14.81%) > free astaxanthin (14.34%) > free adonixanthin (9.24%). In the protein carbonyl assay, the fraction of astaxanthin diester exhibited the highest inhibitory activity (Table 2). It decreased the carbonyl content from 5.75 to 2.87 nmol mg1, followed by astaxanthin monoester (3.61 nmol mg1) and adonixanthin ester (4.14 nmol mg1). Other fractions showed relatively weaker effects in which neoxanthin failed to diminish the carbonylation. In the thiol group assay, expect for fractions of neoxanthin, free adonixanthin and free astaxanthin, all other fractions displayed significant effects to stop the loss of thiol groups. astaxanthin diester was found to exhibit the highest activity (thiol group content: 9.76 pmol mg1), followed by adonixanthin ester (9.25 pmol mg1), lutein + zeaxanthin (8.78 pmol mg1) and astaxanthin monoester (8.43 pmol mg1). These results, taken together, clearly demonstrated the hypothesised roles of astaxanthin in antiglycoxidative activities of C. zofingiensis extracts. Although several compounds e.g. adonixanthin and lutein + zeaxanthin were also involved in the blockage of glycation cascade, astaxanthin diester was the major contributor, providing the most significant protective effects. The esterified form of astaxanthin in C. zofingiensis is believed to be a more stabilised structure to maintain the high antioxidant ability (Kobayashi & Sakamoto, 1999). LC–MS was adopted to identify the fatty acid composition of astaxanthin diester. According to the LC–MS results, the detected fragment of 862.4 was presumably identified as astaxanthin-octadecanoic acid (C18:0), 282.4 as oleic acid (C18:1), and 257.0 as palmitic acid (C16:0). These LC–MS results suggested that the fatty acids in these astaxanthin diesters are highly possible to be octadecanoic acid, oleic acid and palmitic acid, which were also in accordant to the GC–MS detection and library searching results (Grynbaum et al., 2005).
4. Conclusions The green microalga C. zofingiensis was systematically evaluated for its antiglycoxidative properties for the first time in this study. Results showed that the C. zofingiensis extracts possessed antioxidant capacities and significant inhibitory effects on the glycation cascade in various model systems. Further studies revealed that astaxanthin was the major component contributing to observed activities. Taken together, these findings supported the possibility of using natural antioxidants as glycation inhibitors and suggested the beneficial roles of astaxanthin, which is an essential nutritional component for human health in diabetes. The astaxanthin-rich microalga C. zofingiensis therefore might be regarded as a potential therapeutic and preventive agent for diabetes and its complica-
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