Antioxidant activity and hepatoprotective potential of agaro-oligosaccharides in vitro and in vivo
© Chen et al; licensee BioMed Central Ltd. 2006
Received: 17 July 2006
Accepted: 02 December 2006
Published: 02 December 2006
Agaro-oligosaccharides derived from red seaweed polysaccharide have been reported to possess antioxidant activity. In order to assess the live protective effects of agar-oligosaccharides, we did both in vitro and in vivo studies based on own-made agaro-oligosaccharides, and the structural information of this oligosaccharide was also determined.
Structure of agaro-oligosaccharides prepared with acid hydrolysis on agar was confirmed by matrix-assisted ultraviolet laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and NMR. The antioxidant effect of agaro-oligosaccharides on intracellular reactive oxygen species (ROS) was assessed by 2', 7'-dichlorofluorescin diacetate. Carbon tetrachloride was used to induce liver injury, some index including SOD, GSH-Px, MDA, AST, ALT were examined to determine the hepatoprotective effect of agaro-oligosaccharides.
Agaro-oligosaccharides we got were composed of odd polymerizations with molecular weights ranged from 500 to 2500. Results from intracellular test indicated that agaro-oligosaccharides could significantly scavenge the level of oxidants in the hepatocytes, more beneficially, also associated with the improvement of cell viability In vivo studies of the antioxidant effects on tissue peroxidative damage induced by carbon tetrachloride in rat model indicated that agaro-oligosaccharides could elevate the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and decrease the level of malondialdehyde (MDA), glutamate oxaloacetate transaminase (AST), glutamic pyruvic transaminase (ALT) significantly. At 400 mg/kg, MDA level reduced 44 % and 21 % in liver and heart, SOD and GSH-Px increased to highest in liver and serum, while ALT level decreased 22.16 % in serum.
Overall, the results of the present study indicate that agaro-oligosaccharides can exert their in vitro and in vivo hepatoprotective effect through scavenging oxidative damage induced by ROS.
Liver is the main organ involved in the metabolism of biological toxins and medicinal agents. Such metabolism is always associated with the disturbance of hepatocyte biochemistry and generation of ROS (reactive oxygen species) . Lots of liver damages ranging from subclinical icteric hepatitis to necroinflammatory hepatitis, cirrhosis, and carcinoma have been proved to associate with the redox imbalance and OS (oxidative stress) . Therefore, a potential novel approach, namely developing antioxidant drugs to treat and protect liver injury and liver disease, has been proposed . This strategy is aimed to devise and incorporate antioxidants into the therapeutic for control of viral infections or protecting body from alcohol or other toxin damage. We think antioxidants are able to reduce hepatic inflammation and fibrosis, thus slowing or even preventing progression to cirrhosis. One of such candidates is agaro-oligosaccharides prepared from agar, which was chosen in the present study.
Agar was easily extracted from red algae and widely be used as food and gelling agent with historic record of more than a thousand years in China and Japan. In recent years, agaro-oligosaccharides which derived from agarose have been widely investigated in structures and bioactivities [4–8]. Many beneficial health properties of agaro-oligosaccharides are attributed to their antioxidant activities. For example, agaro-oligosaccharides have been proved to possess antioxidative activities in scavenging hydroxyl free radical, scavenging superoxide anion radical and inhibiting lipid peroxidation in various chemical assays [9–11]. Enoki et al.  also reported that the agarobiose shows the ability to suppress the expression of iNOS (inducible nitric oxide synthase), an enzyme associated with the production of NO. In our previous work, we also discussed the indirect attenuate effect of agaro-oligosaccharides towards oxidation of human liver cells induced by antimycin A . These reports exhibited the potential prospects of agaro-oligosaccharides as functional ingredient to prevent the ROS related diseases. However, no researches have been done about their antioxidant effect in the in vivo system. Therefore, in order to evaluate the ROS scavenging activity of agaro-oligosaccharides as well as possible liver injury protection from OS with the respects of degree of polymerization, we firstly prepared agaro-oligosaccharides with different degrees of polymerizations, then use the compounds to examine the in vitro and in vivo antioxidant effects depending on hepatocyte cellular assay of H2O2 induced damage and experimental rat model of carbon tetrachloride (CCl4) induced toxic hepatitis.
Preparation of agaro-oligosaccharides
Agaro-oliogsaccharides were prepared by acid hydrolysis. In order to evaluate the difference of DP of oligosaccharides on bioactivity, hydrolysis solution was fractionated by activated carbon column. After loading the hydrolysate onto column, the column was washed with 2 liters water to remove salts and monosaccharides. Followed this step, the agaro-oligosaccharides fraction was eluted sequentially with 8 %, 15 % and 25 % hydroalcoholic solution. Each fraction from the column was concentrated under reduced pressure and lyophilized.
Structural information of agaro-oligosaccharides
The average molecular weight of three fractions was measured as described by Somogyi et al. .
The nuclear magnetic resonance (NMR) spectra were acquired on an AVANCEDMX-500-NMR spectrometer. Samples were dissolved in D2O. 13C NMR spectra of 4% (w/v) solutions were recorded at 35°C under 100.69 MHz. Proton decoupled 13C NMR chemical shifts were measured in parts per million. For 1H-NMR, samples (7–10 mg) were dissolved in D2O (0.5 ml), and spectra were recorded at room temperature using a spectral width of 5.7 kHz, 90° pulse, an acquisition time of 4.4 s for 144 scans.
Mass spectrometry analysis was performed on a Bruker Reflex III MALDI-TOF-MS (Bruker-Daltonik, Germany) in the delayed extraction and positive mode. An accelerating voltage and a reflectron voltage were set at 20 kV of 22.8 kV, respectively, during the measurements. 2, 5-Dihydroxybenzoic acid was used as matrix (20 mg/ml; 3:2 water/MeCN) and approximately 10–100 pg of the DP-H agaro-oligosaccharide mixture was deposited as a mixture together with the matrix on a stainless steel target, and subsequently dried under reduced pressure. During the experiments, the laser power was adjusted to a level just above the threshold for formation of observable ions. The results from 20 to 100 laser shots were summed for sample.
Measurement of intracellular ROS generation
Intracellular oxidant stress was monitored by measuring changes in fluorescence resulting from intracellular probe oxidation.
Human hepatocyte L-02 purchased from Chinese Institute of Biochemistry and Cell Biology was cultured in RPMl-1640 medium with 20 % fetal bovine serum. Viable cells (105/ml) were plated into a 96-well for 1 day. On the day of the experiments, after removing the medium, the cells were washed with PBS for three times and then incubated with different doses of agaro-oligosaccharides in 5 % CO2 at 37°C for 2 h. After incubation, 20 μM DCFH-DA was added for another 45 min. The DCFH-DA was removed by washing the cells with PBS. 100 μM H2O2 were added into cells for 45 min and the fluorescence change was monitored by fluorescence spectorphotometer at λex = 475 nm, λem = 525 nm .
Cell viability and cytotoxicity assessment
The cell viability was quantified using MTT assay. Briefly, 1 × 104 cells were seeded in each well of microtiter plate and allowed to attach overnight. Cells were treated with various doses of agaro-oligosaccharides for different period according to the experiment purpose. For cytotoxicity test, the hepatocyte L-02 was treated for 48 h. But for the detection of protective effect of agaro-oligosaccharides on H2O2 damage, the L-02 was only treated for 2 h, and then 100 μM H2O2 was added for another 2 h. MTT in PBS was added to each well, followed by incubation for 4 h at 37°C. The formazan crystals were dissolved in DMSO. The optical density was determined with a microculture plate reader at 492 nm .
Mature Wistar rats weighing 150 ± 20 g were supplied by the animal center of Hangzhou, China. The animals were housed in a room with a 12 h light/dark cycle at about 22°C and fed on standard diet with ad libitum access to drinking water. All treatments were conducted between 9:00 am and 10:00 am to minimize variations. In this study, rats were randomly divided into six groups. Group 1 (control, n = 8): water for 10 days followed by administration of liquid paraffin only; group 2 (CCl4, n = 8): water for 10 days followed by administration of CCl4 on the final day; group 3 (positive control, n = 8): vitamin C (200 mg/kg) + CCl4; group 4 to 6 (n = 8): agaro-oligosaccharides (200, 400, 600 mg/kg, respectively) + CCl4. Rats were injected i.p. with vitamin C or agaro-oligosaccharides for ten consecutive days. On the final day, all animal except control group were administered with 20 % CCl4 in liquid paraffin at a dose 5 ml/kg to induce hepatotoxicity. Previous studies demonstrated that the OS indexes could reach a maximum at 48 h after CCl4 i.p. administration , therefore, in this work rats were sacrificed by collecting the blood from the carotid artery after 48 h of administration. Two organs (liver and heart) were excised immediately.
Serum was separated by centrifugation at 1000 × g at 4°C for 10 min. 10 % organ homogenates including liver and heart were prepared in ice-cold isotonic physiological saline. The GSH-Px, MDA, SOD, AST and ALT levels of tissue and serum were measured by spectrophotometric methods as described in the assay kits.
All data are expressed as mean ± SD. In cell based assay, the control and agaro-oligosaccharides treated cells were compared by student t-test. In animal assay, the statistical tests were one-way ANOVA followed by post-hoc Newman-Keuls multiple comparisons test. A probability level of 0.05 was considered statistically significant.
Preparation and structure analysis of agaro-oligosaccharides
Activated charcoal column has been performed as saccharide isolation tool for decades. Depending on this technology, we successfully achieved to isolate three fractions of agaro-oligosaccharides with average molecular weight of 619, 1126 and 1631, respectively, eluted by 8 %, 15 % and 25 % aqueous alcoholic solution. We use these three fractions for the following experiments, designated as DP-L, DP-M and DP-H, according to their differences in molecular weight.
Chemical shift assignments for 1H-NMR and 13C-NMR spectra of agaro-oligosaccharides
Chemical shifts (ppm)
3.63 c/3.67 d
3.84 e/4.06 f
The antioxidant action of agaro-oligosaccharides in cell based assay
We firstly investigated the antioxidant activities of agaro-oligosaccharides in the cellular system. DCFH-DA, which can be conversed from non-fluorescence into fluorescence through oxidation, was used as fluorescent probe to monitor the changes of oxidative stress in hepatocyte L-02 induced by addition of H2O2. In our experiment, all the measurements were carried out at the steady stage (incubation time, 60 min) in order to minimize variations, because it has been reported that treatment of H2O2 will lead to the abruption of ROS in few minutes, and then decrease to a steady stage .
Protective effect of agaro-oligosaccharides on oxidative stress injury
Effect of agaro-oligosaccharides on an acute CCl4oxidative damage
We further studied the in vivo antioxidant effects of agaro-oligosaccharides. It was not uncommon that compounds possessing in vitro activity, however, fail to maintain the activity when administrated into body. We established an oxidative animal model by CCl4 injection. Considering the proliferation and antioxidant effects of agaro-oligosaccharides on hepatocyte, we used the mixture of DP-M and DP-H as our sample for animal test. The effects of agaro-oligosaccharides on oxidative stress in rats were estimated by determining the activities of MDA, SOD, GSH-Px, ALT and AST in serum and tissues.
Effect of agaro-oligosaccharides on MDA activity in different organs of CCl4 induced rats a
Liver (nmol/mg prot)
Heart (nmol/mg prot)
2.75 ± 0.51
0.56 ± 0.08
4.20 ± 0.22
4.62 ± 0.77#
0.68 ± 0.05
4.44 ± 0.64
2.59 ± 0.02*
0.67 ± 0.11
3.53 ± 0.74
G4 (200 mg/kg)
3.45 ± 0.77
0.54 ± 0.11
3.33 ± 0.11
G5 (400 mg/kg)
2.71 ± 0.18*
0.53 ± 0.14
3.36 ± 0.63
G6 (600 mg/kg)
2.99 ± 0.47
0.45 ± 0.02
2.82 ± 0.66
Effect of agaro-oligosaccharides on SOD activity in different organs of CCl4 induced rats a
Liver (U/mg prot)
34.18 ± 2.45
46.80 ± 2.84
313.77 ± 24.01
26.97 ± 6.69#
27.71 ± 2.26#
306.89 ± 19.29
33.11 ± 2.79*
32.75 ± 1.73
318.35 ± 16.39
G4 (200 mg/kg)
33.45 ± 2.87*
34.37 ± 1.34
361.08 ± 9.37*#
G5 (400 mg/kg)
38.64 ± 8.44*
37.33 ± 2.45
365.53 ± 21.13*#
G6 (600 mg/kg)
35.42 ± 2.86*
40.49 ± 2.21*
364.92 ± 14.21*#
Effect of agaro-oligosaccharides on GSH-Px activity in different organs of CCl4 induced rats a
Serum (× 103 NU)
159.17 ± 6.97
200.20 ± 15.46
12.50 ± 1.44
91.60 ± 3.97#
191.81 ± 36.90
10.38 ± 1.48#
119.41 ± 9.86
204.86 ± 17.11
11.02 ± 0.66
G4 (200 mg/kg)
120.50 ± 17.05
203.63 ± 25.01
12.18 ± 1.95
G5 (400 mg/kg)
127.19 ± 12.17*
217.40 ± 10.82
13.13 ± 1.21
G6 (600 mg/kg)
118.92 ± 17.56
248.47 ± 39.28
12.26 ± 1.30
Among therapeutics for liver diseases, protective drugs have been attracted more and more attentions, such as antioxidant prevention approaches. In this paper, we focused on the in vitro and in vivo antixoidative activities of agaro-oligosaccharides with the model related with liver disease.
Agaro-oligosaccharides are linear oligomers cleaved from agar which is built of 1, 4-linked 3, 6-anhydro-α-L-galactose alternating with 1, 3-linked β-D-galactopyranose. When agar is attacked by degradation reagents, such as hydrolysis enzyme, acid or alkali, numerous possibilities for combination, viz., the repetition of AG, GA, AGA, or GAG, etc will exist. In this research, depending on NMR and MALDI-TOF-MS analysis, we detected the precise structural features of our hydrolysate. NMR results give us information that our product is agarose structure, furthermore, there was no signal of A at reducing end. In the spectrum of MALDI-TOF-MS, the first high intensity peak observed at m/z 509 was assigned to (Mtri+Na)+ containing two galactopyranose (Galp) residues and one 3,6-anhydrogalactopyranose (AnGalp) residues, followed by a series of agaro-oligosaccharides: agaropentaose, agaroheptanose, agarononaose, and so forth. In our case, the agaro-oligosaccharides with odd polymerization degree were dominant.
For the in vitro antioxidant studies, we noticed that agaro-oligosaccharides expressed different antioxidant abilities with different ranges of DPs. In them, the fraction of DP-H with average MW of 1631 showed highest free radical scavenging activity which agrees well with the result obtained by Zhao et al. . However, Enoki et al.  found, in a different assay system, that agarobiose possessed the highest ability to inhibit the expression of iNOS. Therefore, comparison of structure-bioactivity in vitro for different studies should be careful bearing different assays in mind.
It is quite significant that the in vivo animal experiment for agaro-oligosaccharides is quite consistent with the in vitro assays. Besides successful protection of liver damage by efficiently inhibiting MDA formation and decreasing AST and ALT, agaro-oligosaccharides enhance the activities of antioxidant enzyme system of the host, including SOD, GSH-Px. We also notice that vitamin C only slightly reduced AST and ALT level in rats in our experiment, although it prevented MDA formation effectively (Fig. 7). The result indicates that agaro-oligosaccharides have better impact to improve the hepatoprotective ability. Since antioxidant enzymes such as SOD and GSH-Px are considered to be a primary defense system for oxidative damage prevention, agaro-oligosaccharides exert antioxidant not only through its own radical scavenging activity, but also, by boost the host antioxidant enzyme system. On the other hand, we found that when the sample concentration increased from 400 mg/kg to 600 mg/kg, several indexes showed a different change. At concentration of 600 mg/kg, the MDA level increased slightly and SOD, GSH-Px and AST activities reduced a little. This result implied that excessive administration of agaro-oligosaccharides will decrease their antioxidant ability with unknown reasons.
In conclusion, by carefully examining the antioxidant protective effects of agaro-oligosaccharides both in vitro and in vivo, the agaro-oligosaccharides prepared via solid acid hydrolysis showed consistent and concentration-dependent antioxidation activities, as well as significant protection against liver injury.
These results support a beneficial relationship between antioxidant activity and hepatoprotective effect of agaro-oligosaccharides which belong to agaro-series with odd numbers of sugar unit as their dominant composition.
- A :
- DCFH-DA :
2', 7'-dichlorodihydrofluorescein diacetate
- DP :
degree of polymerization
- DP-H :
Degree of Polymerization-High, representing the experiment group of the agaro-oligosaccharides with average molecular weight of 1631, eluted by 25 % ethanol from the charcoal column
- DP-L :
Degree of Polymerization-Low, representing the experiment group of agaro-oligosaccharides with average molecular weight of 619, eluted by 8 % ethanol from the charcoal column
- DP-M :
Degree of Polymerization-Middle, representing the experiment group of agaro-oligosaccharides with average molecular weight of 1126, eluted by 15 % ethanol from the charcoal column
- G :
- MDA :
- MTT :
3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide
- MW :
- OS :
- ROS :
reactive oxygen species
This work was supported by grants from Zhejiang Provincial Science and Education Projects (2003C32030, 20051693), and Ningbo Science and Technology Project362 (2004830).
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