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Purification, preliminary characterization and bioactivities of polysaccharides from Ostrea rivularis Gould
International Journal of Biological Macromolecules 80 (2015) 16–22
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Purification, preliminary characterization and bioactivities of polysaccharides from Ostrea rivularis Gould Shijie Li a,1 , Danyan Zhang a,1 , Jun Wu b,1 , Xia Li c,1 , Jingnian Zhang a , Mianjie Wan a , Xiaoping Lai a,∗ a
School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, PR China Shandong College of Traditional Chinese Medicine, Yantai 264199, PR China c Affiliated Huaian Hospital of Xuzhou Medical College, Huaian 223002, PR China b
a r t i c l e
i n f o
Article history: Received 15 April 2015 Received in revised form 2 June 2015 Accepted 14 June 2015 Available online 17 June 2015 Keywords: Ostrea rivularis Gould Polysaccharide Antioxidant and spermatogenesis activities
a b s t r a c t In this study, purification, preliminary characterization and biological activities of water-soluble polysaccharides from Ostrea rivularis Gould (ORP) were investigated. Firstly, crude ORP was extracted by enzyme-assisted extraction and then sequentially purified by chromatography of DEAE-52 and Sephadex G-100, producing one main purified fractions of ORPp. Furthermore, the preliminary characterization of ORPp was studied, and its antioxidant and spermatogenesis activities were evaluated. Experimental results showed that ORPp was mainly composed of glucose (76.3%) and galactose (23.7%). The average molecular weight of ORPp was 118 kDa. Besides, ORPp showed strong antioxidant activities in vitro. For the experiments of antioxidant activities in vivo, ORPp can significantly inhibited the formation of MDA in rats’ serums, and raised the activities of antioxidant enzymes and the level of total antioxidant capacity (TAOC). Furthermore, ORPp could significantly increase the weights of male rats’ sexual organs, promote sperm motility and raise epididymal sperm counts. These results suggest that ORPp could be a new source of natural antioxidants and spermatogenic agent with its potential usage in developing novel supplements and medicines. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Ostrea rivularis Gould, which is one species of oyster, is commonly found in costal areas in China and Japan [1]. The delicate taste of oyster makes it a delicacy cherished by people all around the world. Besides edible value, oyster has long been used to cure fatigue, impotence, anemia deficiency diseases, palpitations and insomnia as a traditionally Chinese medicine. In traditional Chinese medicine, oyster is used as aphrodisiacs which improve libido and male reproductive ability. Current researches have demonstrated that oyster is rich in protein, amino acid, polysaccharide, vitamins and some trace elements [2] that could contribute to some biological functions, such as anti-tumor, anti-aging [3], immunity-enhancing [4] and anti-viral [5,6]. However, despite of
Abbreviations: ORP, polysaccharides from Ostrea rivularis Gould; ORPp, the purified polysaccharide from Ostrea rivularis Gould; CAT, catalase; MDA, malondialdehyde; SOD, superoxide dismutase; TAOC, total antioxidant capacity; GSH-Px, glutathione peroxidase; FT-IR, Fourier transform-infrared spectroscopy; GC, gas chromatography; NOS, nitric oxide synthase; BW, body weight. ∗ Corresponding author. Tel.: +86 20 86361431; fax: +86 20 86361431. E-mail address: [email protected] (X. Lai). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijbiomac.2015.06.024 0141-8130/© 2015 Elsevier B.V. All rights reserved.
the potentially huge medicinal value of this kind of polysaccharide, there is so far limited literature on the purification, characterization and spermatogenesis activities of water-soluble polysaccharides from Ostrea rivularis Gould (ORP). Therefore, we report here the purification, preliminary characterization, anti-oxidative and spermatogenic activities of ORP. In preliminary experiment of our group, we have successfully extracted ORP. On this basis, ORP was separated and purified by DEAE-52 and Sephadex G-100, and then obtained purified ORP (ORPp). After these, the preliminary characterizations of ORPp were analyzed by ultraviolet (UV) spectroscopy, Fourier transforminfrared spectroscopy (FT-IR), gas chromatography (GC) and high performance liquid chromatography (HPLC). Also, the antioxidant activities in vitro and in vivo of ORPp were investigated, followed by the evaluation of spermatogenic activity of ORPp. 2. Materials and methods 2.1. Materials and reagents The oyster was purchased from Huangsha Seafood Market (Guangzhou, PR China). DEAE-52 cellulose and Sephadex G-100 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The
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Wistar rats were provided by the Experiment Animal Center of Academy of Military Medical Sciences (Beijing, China). Assay kits for malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and total antioxidant capacity (TAOC) were the products of Nanjing Jiancheng Biotechnology Co., Ltd (Nanjing, China). Other reagents were of analytical grade.
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120 ◦ C in an oven for 2 h. The hydrolyzed product was acetylated as described [13]. Then the acetylated samples were analyzed by a 7890N GC (Agilent Technologies, Santa Clara, CA, USA) equipped with flame ionization detector and a HP-5 fused silica capillary column (30 m × 0.32 mm × 0.25 mm) using the method reported [14]. UV spectroscopy was analyzed by the UV-2450 UV spectrophotometer (SHIMADZU, Japan) with the wavelength of 200–400 nm.
2.2. Preparation of crude ORP by enzyme assisted extraction The oyster was freeze-dried and then smashed to pass through a screen of 60 meshes. The powder was rinsed twice with 95% ethanol to remove oligosaccharides and small molecule materials. Finally, remove organic solvent to obtain pretreated dry powder. The pretreated dry powder (10.0 g) was soaked in distilled water with the ratio of water to raw material of 45: 1 (v/w, ml/g). The pH value was adjusted to 1.0. ORP was extracted with 2% (m/v) pepsin at 37 ◦ C for 180 min, with a water bath to maintain the given temperature. After the treatment, the mixture with an adjusted pH of 7.0 was centrifuged at 5000 rpm for 20 min. Then the supernatants was collected, and concentrated to a proper volume by using a vacuum rotary evaporator then mix with fourfold amount of absolute ethanol. The mixture was stirred vigorously and then kept overnight at 4 ◦ C. The precipitate was collected through centrifuging the mixture at 5000 rpm for 20 min and air-drying at 50 ◦ C to a constant weight, and the final product of which was the crude ORP. The entire procedure was then repeated with the enzyme being replaced by papain at pH 6.0 to remove the conjugate proteins [4], while dissociative proteins were removed using Sevag method [7]. 2.3. Purification of crude ORP The crude ORP was purified by DEAE-52 and Sephadex G-100 gel filtration chromatography according to the reported method with some modifications [8]. The crude ORP solution (5 ml, 20 mg/ml) was applied to a column (2.6 cm × 40 cm) of DEAE-52 cellulose which was then stepwise eluted with 0, 0.1, 0.3 and 0.5 M NaCl solutions at a flow rate of 1 ml/min. The obtained elute (10 ml/tube) was collected automatically and the polysaccharides were detected by the phenol–sulfuric acid method [9]. As a result, one main fraction of ORP was obtained. The obtained fraction was then collected, concentrated and dialyzed. Dialysis solution was mixed with fourfold amount of absolute ethanol. The mixture was stirred vigorously and then kept overnight at 4 ◦ C. The precipitate was collected through centrifuging the mixture at 5000 rpm for 20 min, and the product was freeze-dried to obtain dry powder. The dry powder was dissolved for further purification through a column (1.6 cm × 60 cm) of Sephadex G-100 at a flow rate of 15 ml/h, to gain refined fraction of ORPp which was then lyophilized for further research. 2.4. Preliminary characterization of ORPp 2.4.1. Determination of contents of carbohydrate, sulfuric radical, protein and uronic acid The contents of carbohydrate in ORPp were determined by phenol–sulfuric acid method using glucose as a standard [9]. The content of sulfate radical was determined according to the reported method [10]. The content of protein was determined by the method of Bradford using bovine serum albumin as the standard [11]. The content of uronic acid was determined according to the method of Blumenkrantz and Asboe-Hansen by using d-glucuronic acid as the standard [12]. 2.4.2. Analysis of monosaccharide composition of ORPp Gas chromatography was used for identification and quantification of monosaccharides. Firstly, the polysaccharide sample (5.0 mg) was hydrolyzed with 4 ml trifluoroacetic acid (TFA, 2 M) at
2.4.3. Molecular weight determination of ORPp The molecular weight of ORPp was characterized by highperformance liquid chromatography (HPLC) according to the reported method with slight modifications [15]. Briefly, the ORPp sample (3.6 mg) were dissolved in distilled water (2 ml), passed through a 0.45 m filter, and applied to a gel-filtration chromatographic column of TSK-GEL G3000SWxl column (7.5 mm × 300 mm, Tosoh Corp., Japan). The column was maintained at a temperature of 25 ◦ C, eluted with 0.1 M Na2 SO4 solution in PBS buffer (0.01 M, pH 6.8) at a flow rate of 0.8 ml/min. Preliminary calibration of the column was conducted by using the dextrans with various molecular weights. 2.4.4. Infrared spectroscopy analysis of ORP A Nicolet 5700 spectrometer (Thermo Electron, Madison, WI, US) was applied to record FT-IR spectrum of ORPp by KBr disks method [16]. 2.5. Determination of antioxidant activities in vitro of ORP 2.5.1. Assay of superoxide radical scavenging activity The superoxide radical scavenging activity of ORPp was determined by the reported method [17] with a little modifications. Briefly, each 1.0 ml of NBT solution (145 mol/L), PMS solution (62 mol/L) and NADH solution (435 mol/L) were mixed with 1 ml sample solution with different concentrations (0.5, 1.0, 1.5, 3.0 and 5.0 mg/ml). The absorbance at 560 nm was measured against the blank (water and 0.1 M phosphate buffer instead of ORPp sample and NBT solution, respectively). The scavenging activity on superoxide radical was calculated according to the following formula: Superoxide radical scavenging activity (%) =
A0 − A1 + A2 × 100 A0 (1)
where A0 is the absorbance of control sample, A1 is the absorbance of the tested sample, and A2 is the absorbance of test sample solution without NBT solution. Ascorbic acid was used as positive control. 2.5.2. Assay of reductive potential The reductive potential of ORPp was determined according to the method of Li [18] with some modification. ORPp samples of 0.5 ml (0.5, 1.0, 1.5, 3.0 and 5.0 mg/ml), 1.0 ml pH 6.6 phosphate buffer (0.2 M) and 1.0 ml 1% (w/v) K3 Fe(CN)6 solution were incubated at 50 ◦ C for 20 min, then left to cool down. 1.0 ml trichloroacetic acid (10%, w/v) and 0.25 ml fresh 0.1% (w/v) FeCl3 were added to the mixture in sequence. The mixture was shaken and its absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicates an increase of reduction capability. 2.5.3. Ferrous metal ions chelating activity The chelating of ferrous ions by ORPp was estimated by the method of Liu [19] with some modification. Briefly, five concentrations (0.5, 1.0, 1.5, 3.0 and 5.0 mg/ml) of ORPp samples were each prepared in deionized water, 0.4 ml samples were added to a solution of 2.0 mM FeCl2 (0.05 ml). The reaction was initiated by the
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addition of 5.0 mM ferrozine (0.2 ml) and total volume was adjusted to 4 ml with water. Then, the mixture was shaken vigorously and left at room temperature for 10 min. Absorbance of the solution was then measured spectrophotometrically at 562 nm against the blank (water instead of ORPp sample and FeCl2 solution, respectively). The percentage of inhibition of ferrozine–Fe2+ complex formation was calculated according to the following formula: A0 − A1 + A2 Metal chelating effect (%) = × 100 A0
slide and then covered with a cover slip. The slide was then examined under the microscope at 400×, and the motility was scored in different fields of view. Spermatozoa showing any degree of movement were considered to be motile. All spermatozoa (motile as well as immotile) were counted with the help of a blood cell counter. Sperm motility was calculated using the following formula: Sperm motility (%) =
(2)
where A0 is the absorbance of control sample, A1 is the absorbance of the tested sample, and A2 is the absorbance of test sample solution without FeCl2 solution. 2.6. Determination of antioxidant and spermatogenic activities in vivo of ORPp 2.6.1. Animal grouping and experimental design The assay of antioxidant activities in vivo of ORPp was carried out according to the reported method [20]. Wistar male rats (8-weeksold), grade of specific pathogen free, were used in this study. The mice were maintained in separated cages, under the conditions of 21 ± 1 ◦ C and 50–60% relative humidity. They were free to access to food and water and kept on a 12 h light/dark cycles during the experiments. After adapting to their environment for 1 week, these male rats were randomly divided into 6 groups (eight for each) for experiment. Rats in Group I (normal control group) were treated with 0.9% sodium chloride (NaCl, 25 ml/kg BW) per day by hypodermic injection and gastric gavage. Rats in Group II (model control group) were treated with 9% d-galactose (d-Gal, 25 ml/kg BW) by hypodermic injection and 0.9% NaCl (25 ml/kg BW) by gastric gavage per day. Rats in Group III (positive control group) were treated with 9% d-Gal (25 ml/kg BW) by hypodermic injection and vitamin E (VE , 50 mg/kg BW) by gastric gavage each day. Mice in Group IV (low dose of ORPp), Group V (medium dose of ORPp) and Group VI (high dose of ORPp) were treated with 9% d-Gal (25 ml/kg BW) by hypodermic injection, and crude ORPp of 50, 100 and 200 mg/kg BW, respectively, by gastric gavage, was administrated once a day for 21 consecutive days. After overnight fasting following the last drug administration, the rats were weighed and killed by decapitation. 2.6.2. Assay of antioxidant activity in serums Blood samples were harvested immediately in centrifuge tube. After 1 h, the blood samples were centrifuged at 4000 g for 10 min to afford the serums required. All above treatments were done at 4 ◦ C. Activities of SOD, CAT and GSH-Px, level of MDA and TAOC were measured by using commercial reagent kits guided by the instruction manuals. 2.6.3. Effect on the weight of sexual organs The testis, seminal vesicles, epididymis, and prostate glands were carefully removed and weighed. Then calculate the proportion of above organs. 2.6.4. Sperm analysis The motility, viability, and number of spermatozoa in the cauda epididymis were assessed. The cauda epididymis was transected at the point of origin of the vas deferens at the distal end and placed in a watch glass containing 0.5 ml of normal saline (0.9% NaCl) maintained at 37 ◦ C. The tissue was minced carefully with the help of fine forceps and scissors to ensure the extrusion of spermatozoa. The tissue fractions were removed using the fine forceps and a needle, and the suspension was used for sperm analysis. 2.6.5. Assay of sperm motility Sperm motility was tested by observing the movement under microscope. A drop of sperm suspension was placed on a clear glass
n1 × 100 n2
(3)
where n1 was number of motile spermatozoa, n2 was total number of spermatozoa. 2.6.6. Assay of sperm viability Sperm viability was assessed by a supravital staining technique based on the principle that cells with damaged plasma membrane take up the stain, whereas viable ones do not. A drop of sperm suspension and a drop of 1% Congo red were placed on a clear glass slide and mixed thoroughly. A portion of the mixture was transferred to a second slide, and a thin film was prepared. The slide was then examined under the microscope (400×), and about 100 spermatozoa (viable and dead) were counted from different fields of the slide. Spermatozoa appearing pinkish (stained) were considered to be dead, whereas those appearing colorless (unstained) were counted as being viable. Sperm viability was calculated in percent by using the following formula: Sperm viability (%) =
n1 × 100 n2
(4)
where n1 was the number of viable spermatozoa and n2 was the total number of spermatozoa. 2.6.7. Assay of sperm count Sperm count was investigated according to the reported method [21]. A hemocytometer with improved Neubauer ruling was employed for counting the spermatozoa. The sperm suspension was diluted to 20 times with normal saline. The preparation was then thoroughly mixed, and one drop was added to both sides of the hemocytometer. The number of spermatozoa was counted in the four corner squares of the hemocytometer under a microscope at 400×. Spermatozoa on both sides of the hemocytometer were counted, and the average number was recorded. The number of spermatozoa per cauda epididymis was expressed as follows: Sperm number = N × 0.2 × 106
(5)
where N was averaged number of spermatozoa counted. 2.7. Statistical analysis Data were statistically analyzed, using the SPSS 18.0 software package (SPSS, Chicago, IL, USA), by one-way ANOVA. Significant differences between two means were determined by Tukey’s test. P-values < 0.05 were regarded as significant. 3. Results and discussion 3.1. Separation and purification of ORP The crude ORP was obtained by enzyme-assisted extraction and it was then separated through DEAE-52 cellulose. As the result, one main independent elution peak (F1 ) was obtained as shown in Fig. 1A. This fraction was collected, concentrated, dialyzed, freezedried and loaded into a column of Sephadex G-100. The column was eluted with deioned water, and the resulting elute was collected. As showed in Fig. 1B, the fraction generated one single elution peak, representing of ORPp. The fraction was collected, concentrated,
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Fig. 1. Elution profiles of crude ORP on DEAE-52 cellulose anion-exchange chromatography column with 0–0.5 M NaCl elute (A) and elution curve of polysaccharide fraction from DEAE-52 cellulose on size-exclusion chromatography column of Sephadex G-100 (B).
freeze-dried and the purified products were obtained. The yield of ORPp was 32.8%. 3.2. Preliminary characterization of ORPp 3.2.1. Contents of carbohydrate, sulfate, protein and uronic acid in ORPp As shown in Fig. 2A, there was a weak optical absorption of ORPp at 280 nm manifesting the existence of protein or nucleic acid in ORPp. The contents of carbohydrate, sulfate, protein and uronic acid in ORPp were 68.2%, 7.64%, 4.33% and 9.48%, respectively. The existence of protein was consistent with the UV spectrum analysis, there was a weak absorption at 260–280 nm in the UV spectrum. ORPp was characterized by FT-IR spectroscopy as showed in Fig. 2B. There was a strong and broad peak around the 3415 cm−1 of ORPp infrared spectroscopy, which was belonged to the hydroxyl groups stretching vibration [22]. The weak peaks around 2930 and 1320 cm−1 were assigned to the C-H asymmetric stretching vibration [23]. The bands at 1633.7 and 1337.2 cm−1 were characteristic signals for the deprotonated carboxylic group (COO− ), indicating ORPp being acidic polysaccharides. The weak peak around 1240 cm−1 was assigned to the O SO3 − . A strong extensive absorption in the region of 1000–1200 cm−1 for stretching vibrations of C OH side groups and the C O C glycosidic band vibrations were observed in the spectra [24]. Furthermore, absorption around 1071.9 cm−1 was the furan glycosides characteristic absorption peaks [25]. All these were characteristic absorptions of polysaccharides. GC analysis showed that ORPp was composed of galactose [26] and glucose (Glu) in a molar percent of 23.7% and 76.3%, respectively (Fig. 2C). The average molecular weight of ORPp was
Fig. 2. UV (A), FT-IR spectra (B), and GC chromatogram of monosaccharide (C) of ORPp.
calculated as 118 kDa by HPLC according to the calibration curve with standard dextran–glucose (D-Glc). 3.3. Antioxidant activity in vitro of ORPp 3.3.1. Superoxide radical scavenging activity of ORPp In reactive oxygen species (ROS), superoxide radical is usually formed as a precursor, which could form stronger ROS, such as singlet oxygen and hydroxyl. Although superoxide radical is a relatively weak oxidant, it can cause lipid peroxidation triggered. Beside these, superoxide can also form the hydrogen peroxide, precursor of hydroxyl and so on, then indirectly lead to lipid peroxidation triggered [27]. Taking these reasons into account, it is important to remove superoxide radicals in the process of organism antioxidant. As it is shown in Fig. 3A, the scavenging effects of ORPp were the most evident at all of the tested concentrations and even stronger than ascorbic acid. Also, the superoxide radical scavenging effects of ORPp and ascorbic acid were positively correlated well with
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3.3.3. Chelating ability of ferrous ion Chelation is an important parameter in the sense that iron is essential for oxygen transport, respiration and activity of many enzymes, which are required for life. However, iron is an extremely reactive metal and will catalyze oxidative changes in lipids, proteins and other cellular components. Among the transition metals, iron is known as the most important lipid oxidation pro-oxidant due to its high reactivity. The ferrous state of iron accelerates lipid oxidation by breaking down hydrogen and lipid peroxides to reactive free radicals via the Fenton reaction. Fe3+ ion also produces radicals from peroxides although the rate is ten times slower than that of Fe2+ ion. Fe2+ ion is the most powerful pro-oxidant among the various species of metal ions. Ferrozine can quantitatively form complexes with Fe2+ . In the presence of chelating agents, the complex formation can be disrupted, resulting in a decreasingly intense red color of the complex. Therefore, measuring the level of color reduction allows the estimation of metal chelating activity in terms of the co-existing chelator [29,30]. Fig. 3C showed chelating activities of ORPp for Fe2+ . At a concentration of 0.5–5 mg/ml, ferrous metalions chelating activity of ORPp was significantly stronger than Vc. The results indicated that ORPp has strong chelating ability of ferrous ion. 3.4. Antioxidant activities of ORPp in vivo
Fig. 3. Scavenging affects on superoxide radical (A), reductive potential (B) and metal chelating activity (C) of ORPp.
the increase of concentration from 0.5 to 5.0 mg/ml. The results indicated that ORPp has strong scavenging activity on superoxide radical.
3.3.2. Reductive potential of ORPp Reducing capacity of compounds is a symbol of potential antioxidant capacity. Various mechanisms, including reducing capacity, decomposition of peroxides, prevention of chain initiation, prevention of continued hydrogen abstraction binding of transition metal ion catalysts, and radical scavenging have been claimed to explain the antioxidant activities [28]. Fig. 3B showed the reductive activity of ORPp using the potassium ferricyanide reduction method. The reducing power of ORPp increased significantly with increasing concentration of samples. When the concentration exceeded 5 mg/ml, reductive power of ORPp was as high as 49%. This indicated that the reduction potential of ORPp was strong.
Rats injected with d-Gal have been used as an aging animal model in some previous studies. d-Gal can cause accumulation of ROS, or stimulate free radical production indirectly by forming advanced glycation end-product (AGE) in vivo. On the other hand, VE has been applied in early studies as a positive control agent because of its highly efficient antioxidation and prevention of lipid peroxidation [31]. Thus, in this study, mice treated with d-Gal were used as an aging animal model while VE was used as positive control medicine. Effects of ORPp on the activities of SOD, CAT, GSH-Px and the levels of MDA and TAOC in serums in aging mice are presented in Table 1. Apparently, a marked increase in MDA and significant decreases (P < 0.05) of antioxidant enzymes activities (SOD, GSH-Px, and CAT) and TAOC were observed in serums between the treatments of Group I (normal control group) and Group II (model control group). ORPp and VE treatments inhibited significantly (P < 0.05) the formation of MDA in mice serums and raised the activities of antioxidant enzymes and the level of TAOC in a dose-dependent manner (Groups IV–VI). The results shown that, injection of d-Gal can cause oxidative injury of the body, and VE and ORPp could overcome this damage. The mechanism of this damage-preventing function may be a result of the enhanced activity of endogenous antioxidant enzyme (SOD, GSH-Px, and CAT), promoted TOAC and decreased the content of MDA. 3.5. Effect on sexual organ weight The structure and functional degradation of gonad is closely related to aging process. Gonad atrophy is the most obvious degradation of endocrine system during aging, resulting in an impaired immune function and inducing aging [32]. Therefore, the research of aging of gonad helps to reveal the aging mechanism. As showed in Table 2, the sexual organs of aging rats with the treatment of d-Gal were markedly atrophic. Administration of the ORPp at the doses of 50, 100 and 200 mg/kg BW resulted in a significantly dose-dependent increase in weights of all sexual organs. Then weight increase caused by the injection of middle and high dose of ORPp was even higher than that of VE . These indicated that ORPp can prevent the gonad atrophy resulted from aging, the mechanism of which may be (or partially) related to the antioxidant activity of ORPp.
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Table 1 Effects of ORPp on the activities of SOD, CAT, GSH-Px and levels of MDA and TAOC in serums. Group
SOD (U/ml)
I II III IV V VI
134 122 158 151 162 168
± ± ± ± ± ±
CAT (U/ml)
3 5* 4# 6# 4# 5#
71 63 75 72 85 87
± ± ± ± ± ±
3 4* 2# 3# 5# 4#
GSH-Px (U/ml) 7389 7046 7834 7537 7763 7712
± ± ± ± ± ±
128 158* 101# 116# 142# 135#
MDA (nmol/ml) 13.8 16.4 10.4 11.4 10.7 9.6
± ± ± ± ± ±
TAOC (U/ml)
0.52 0.26* 0.37# 0.53# 0.59# 0.75#
19.3 16.3 24.4 23.0 26.4 29.6
± ± ± ± ± ±
1.28 0.69* 0.89# 1.39# 0.95# 1.17#
The data were presented as mean ± SD (n = 8). * Compared with normal control group (Group I), P < 0.05. # Compared with model control group (Group II), P < 0.05. Table 2 Effect of ORPp on body and sexual organ weights. Group
Weight of testis (mg/100 g BW)
I II III IV V VI
870 790 845 837 856 867
± ± ± ± ± ±
17.3 14.8* 21.9# 19.7# 15.9# 22.4#
Weight of prostate (mg/100 g BW) 261 228 257 244 266 271
± ± ± ± ± ±
10.4 13.2* 13.4# 16.4# 11.2# 15.6#
Weight of seminal (mg/100 g BW) 315 274 329 307 325 322
± ± ± ± ± ±
19.4 11.7* 16.4# 14.8# 15.5# 18.3#
Weight of epididymis (mg/100 g BW) 635 551 619 612 644 651
± ± ± ± ± ±
22.7 24.3* 27.4# 23.3# 28.9# 28.2#
The data were presented as mean ± SD (n = 8). * Compared with normal control group (Group I), P < 0.05. # Compared with model control group (Group II), P < 0.05. Table 3 Effect of ORPp on male rats’ sperm motility and number of spermatozoa in cauda epididymis. Group
Sperm motility (%)
I II III IV V VI
80.4 69.3 82.4 77.2 81.9 83.3
± ± ± ± ± ±
2.5 1.9* 2.9# 3.8# 4.2# 3.2#
Sperm viability (%) 76.4 63.9 75.9 73.5 79.3 78.7
± ± ± ± ± ±
1.88 2.16* 1.64# 3.24# 2.93# 2.37#
Sperm number (106 /mL) 6.3 5.1 6.2 5.5 5.8 6.2
± ± ± ± ± ±
0.13 0.08* 0.09# 0.15# 0.11# 0.16#
The data were presented as mean ± SD (n = 8). * Compared with normal control group (Group I), P < 0.05. # Compared with model control group (Group II), P < 0.05.
3.6. Sperm analysis With the development of aging, different degrees of degradations can appear in terms of the structure and functions of testis, such as the decrease of spermatogenesis function and sperm activity, which finally lead to senescence of testis. Increased oxidative stress is closely related to aging, whereas anti-aging medicine can function against the oxidative damage of oxidative stress on the body, through which it can effectively prolong the life span of the body in normal range, so that the aging process can be delayed [33]. Therefore, the research of spermatogenesis activity is highly useful for the investigation of anti-aging effects. The motility of caudal epididymal spermatozoa was decreased significantly (P < 0.05) in model control group compared with the normal control group. Sperm motility in the Group IV-VI was 77.2%, 81.9%, and 83.3%, respectively with no significant difference compared with 80.4% in the Group I. The motility of spermatozoa in Group V and VI were higher than Group III. Sperm viability of model control group was significantly lower than normal control group, indicating that sperm viability was related to the aging process. ORPp and VE potentially have a favorable effect on sperm viability. The amounts of epididymal sperm counts were significantly increased in the treated groups: 5.5, 5.8 and 6.2 × 106 /ml in Group IV, V and VI, respectively, compared with 5.1 × 106 /ml in the Group II (Table 3).
All these showed that ORPp has a novel spermatogenesis activity, which might be related to the antioxidant activity of ORPp. Generally, the excess free radicals is a factor that leads to damaged organ, declined immunity, the onset of various diseases and the process of aging [34]. However, the strong antioxidant activity of ORPp could scavenge excess free radicals so that the normal functions and operations of organs can be maintained and thus the aging process can be delayed. 4. Conclusion Polysaccharide was extracted from Ostrea rivularis Gould, and one purified fraction ORPp was obtained from the crude ORP through sequential purification by chromatography of DEAE-52 and Sephadex G-100. Then, the ORPp was characterized by chemical analysis, GC, HPLC and FT-IR. Experimental results showed that ORPp was mainly composed of glucose (76.3%) and galactose (23.7%). The average molecular weight of ORPp was determined to be 118 kDa. And there was infrared characteristic absorption peak of polysaccharides in the FT-IR spectroscopy of ORPp. Beside these, the antioxidant and spermatogenic activities of ORPp were evaluated. The results demonstrated that ORPp possessed strong scavenging activities on superoxide radical, reductive potential and metal chelating activities. For antioxidant activity in vivo, ORPp significantly raised the level of TAOC, reduced the formation of MDA and enhanced the activities of SOD and GSH-Px. Furthermore, ORPp could significant increased the weights of all sexual organs of rats, promoted sperm motility and raised epididymal sperm counts. Further work on detailed characterization of ORPp is in progress. Acknowledgement This research is supported by the Special Science and Technology Cooperation Fund of Hongkong, Macao and Taiwan in China (2014DFH30010). References [1] H. Que, X. Liu, H. Wang, S. Zhang, G. Zhang, F. Zhang, Chin. J. Zool. 4 (2003) 110–113. [2] Z. Duan, X. Liu, Meat Res. 7 (2011) 29–31.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Purification, preliminary characterization and bioactivities of polysaccharides from Ostrea rivularis Gould Shijie Li a,1 , Danyan Zhang a,1 , Jun Wu b,1 , Xia Li c,1 , Jingnian Zhang a , Mianjie Wan a , Xiaoping Lai a,∗ a
School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, PR China Shandong College of Traditional Chinese Medicine, Yantai 264199, PR China c Affiliated Huaian Hospital of Xuzhou Medical College, Huaian 223002, PR China b
a r t i c l e
i n f o
Article history: Received 15 April 2015 Received in revised form 2 June 2015 Accepted 14 June 2015 Available online 17 June 2015 Keywords: Ostrea rivularis Gould Polysaccharide Antioxidant and spermatogenesis activities
a b s t r a c t In this study, purification, preliminary characterization and biological activities of water-soluble polysaccharides from Ostrea rivularis Gould (ORP) were investigated. Firstly, crude ORP was extracted by enzyme-assisted extraction and then sequentially purified by chromatography of DEAE-52 and Sephadex G-100, producing one main purified fractions of ORPp. Furthermore, the preliminary characterization of ORPp was studied, and its antioxidant and spermatogenesis activities were evaluated. Experimental results showed that ORPp was mainly composed of glucose (76.3%) and galactose (23.7%). The average molecular weight of ORPp was 118 kDa. Besides, ORPp showed strong antioxidant activities in vitro. For the experiments of antioxidant activities in vivo, ORPp can significantly inhibited the formation of MDA in rats’ serums, and raised the activities of antioxidant enzymes and the level of total antioxidant capacity (TAOC). Furthermore, ORPp could significantly increase the weights of male rats’ sexual organs, promote sperm motility and raise epididymal sperm counts. These results suggest that ORPp could be a new source of natural antioxidants and spermatogenic agent with its potential usage in developing novel supplements and medicines. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Ostrea rivularis Gould, which is one species of oyster, is commonly found in costal areas in China and Japan [1]. The delicate taste of oyster makes it a delicacy cherished by people all around the world. Besides edible value, oyster has long been used to cure fatigue, impotence, anemia deficiency diseases, palpitations and insomnia as a traditionally Chinese medicine. In traditional Chinese medicine, oyster is used as aphrodisiacs which improve libido and male reproductive ability. Current researches have demonstrated that oyster is rich in protein, amino acid, polysaccharide, vitamins and some trace elements [2] that could contribute to some biological functions, such as anti-tumor, anti-aging [3], immunity-enhancing [4] and anti-viral [5,6]. However, despite of
Abbreviations: ORP, polysaccharides from Ostrea rivularis Gould; ORPp, the purified polysaccharide from Ostrea rivularis Gould; CAT, catalase; MDA, malondialdehyde; SOD, superoxide dismutase; TAOC, total antioxidant capacity; GSH-Px, glutathione peroxidase; FT-IR, Fourier transform-infrared spectroscopy; GC, gas chromatography; NOS, nitric oxide synthase; BW, body weight. ∗ Corresponding author. Tel.: +86 20 86361431; fax: +86 20 86361431. E-mail address: [email protected] (X. Lai). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijbiomac.2015.06.024 0141-8130/© 2015 Elsevier B.V. All rights reserved.
the potentially huge medicinal value of this kind of polysaccharide, there is so far limited literature on the purification, characterization and spermatogenesis activities of water-soluble polysaccharides from Ostrea rivularis Gould (ORP). Therefore, we report here the purification, preliminary characterization, anti-oxidative and spermatogenic activities of ORP. In preliminary experiment of our group, we have successfully extracted ORP. On this basis, ORP was separated and purified by DEAE-52 and Sephadex G-100, and then obtained purified ORP (ORPp). After these, the preliminary characterizations of ORPp were analyzed by ultraviolet (UV) spectroscopy, Fourier transforminfrared spectroscopy (FT-IR), gas chromatography (GC) and high performance liquid chromatography (HPLC). Also, the antioxidant activities in vitro and in vivo of ORPp were investigated, followed by the evaluation of spermatogenic activity of ORPp. 2. Materials and methods 2.1. Materials and reagents The oyster was purchased from Huangsha Seafood Market (Guangzhou, PR China). DEAE-52 cellulose and Sephadex G-100 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The
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Wistar rats were provided by the Experiment Animal Center of Academy of Military Medical Sciences (Beijing, China). Assay kits for malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and total antioxidant capacity (TAOC) were the products of Nanjing Jiancheng Biotechnology Co., Ltd (Nanjing, China). Other reagents were of analytical grade.
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120 ◦ C in an oven for 2 h. The hydrolyzed product was acetylated as described [13]. Then the acetylated samples were analyzed by a 7890N GC (Agilent Technologies, Santa Clara, CA, USA) equipped with flame ionization detector and a HP-5 fused silica capillary column (30 m × 0.32 mm × 0.25 mm) using the method reported [14]. UV spectroscopy was analyzed by the UV-2450 UV spectrophotometer (SHIMADZU, Japan) with the wavelength of 200–400 nm.
2.2. Preparation of crude ORP by enzyme assisted extraction The oyster was freeze-dried and then smashed to pass through a screen of 60 meshes. The powder was rinsed twice with 95% ethanol to remove oligosaccharides and small molecule materials. Finally, remove organic solvent to obtain pretreated dry powder. The pretreated dry powder (10.0 g) was soaked in distilled water with the ratio of water to raw material of 45: 1 (v/w, ml/g). The pH value was adjusted to 1.0. ORP was extracted with 2% (m/v) pepsin at 37 ◦ C for 180 min, with a water bath to maintain the given temperature. After the treatment, the mixture with an adjusted pH of 7.0 was centrifuged at 5000 rpm for 20 min. Then the supernatants was collected, and concentrated to a proper volume by using a vacuum rotary evaporator then mix with fourfold amount of absolute ethanol. The mixture was stirred vigorously and then kept overnight at 4 ◦ C. The precipitate was collected through centrifuging the mixture at 5000 rpm for 20 min and air-drying at 50 ◦ C to a constant weight, and the final product of which was the crude ORP. The entire procedure was then repeated with the enzyme being replaced by papain at pH 6.0 to remove the conjugate proteins [4], while dissociative proteins were removed using Sevag method [7]. 2.3. Purification of crude ORP The crude ORP was purified by DEAE-52 and Sephadex G-100 gel filtration chromatography according to the reported method with some modifications [8]. The crude ORP solution (5 ml, 20 mg/ml) was applied to a column (2.6 cm × 40 cm) of DEAE-52 cellulose which was then stepwise eluted with 0, 0.1, 0.3 and 0.5 M NaCl solutions at a flow rate of 1 ml/min. The obtained elute (10 ml/tube) was collected automatically and the polysaccharides were detected by the phenol–sulfuric acid method [9]. As a result, one main fraction of ORP was obtained. The obtained fraction was then collected, concentrated and dialyzed. Dialysis solution was mixed with fourfold amount of absolute ethanol. The mixture was stirred vigorously and then kept overnight at 4 ◦ C. The precipitate was collected through centrifuging the mixture at 5000 rpm for 20 min, and the product was freeze-dried to obtain dry powder. The dry powder was dissolved for further purification through a column (1.6 cm × 60 cm) of Sephadex G-100 at a flow rate of 15 ml/h, to gain refined fraction of ORPp which was then lyophilized for further research. 2.4. Preliminary characterization of ORPp 2.4.1. Determination of contents of carbohydrate, sulfuric radical, protein and uronic acid The contents of carbohydrate in ORPp were determined by phenol–sulfuric acid method using glucose as a standard [9]. The content of sulfate radical was determined according to the reported method [10]. The content of protein was determined by the method of Bradford using bovine serum albumin as the standard [11]. The content of uronic acid was determined according to the method of Blumenkrantz and Asboe-Hansen by using d-glucuronic acid as the standard [12]. 2.4.2. Analysis of monosaccharide composition of ORPp Gas chromatography was used for identification and quantification of monosaccharides. Firstly, the polysaccharide sample (5.0 mg) was hydrolyzed with 4 ml trifluoroacetic acid (TFA, 2 M) at
2.4.3. Molecular weight determination of ORPp The molecular weight of ORPp was characterized by highperformance liquid chromatography (HPLC) according to the reported method with slight modifications [15]. Briefly, the ORPp sample (3.6 mg) were dissolved in distilled water (2 ml), passed through a 0.45 m filter, and applied to a gel-filtration chromatographic column of TSK-GEL G3000SWxl column (7.5 mm × 300 mm, Tosoh Corp., Japan). The column was maintained at a temperature of 25 ◦ C, eluted with 0.1 M Na2 SO4 solution in PBS buffer (0.01 M, pH 6.8) at a flow rate of 0.8 ml/min. Preliminary calibration of the column was conducted by using the dextrans with various molecular weights. 2.4.4. Infrared spectroscopy analysis of ORP A Nicolet 5700 spectrometer (Thermo Electron, Madison, WI, US) was applied to record FT-IR spectrum of ORPp by KBr disks method [16]. 2.5. Determination of antioxidant activities in vitro of ORP 2.5.1. Assay of superoxide radical scavenging activity The superoxide radical scavenging activity of ORPp was determined by the reported method [17] with a little modifications. Briefly, each 1.0 ml of NBT solution (145 mol/L), PMS solution (62 mol/L) and NADH solution (435 mol/L) were mixed with 1 ml sample solution with different concentrations (0.5, 1.0, 1.5, 3.0 and 5.0 mg/ml). The absorbance at 560 nm was measured against the blank (water and 0.1 M phosphate buffer instead of ORPp sample and NBT solution, respectively). The scavenging activity on superoxide radical was calculated according to the following formula: Superoxide radical scavenging activity (%) =
A0 − A1 + A2 × 100 A0 (1)
where A0 is the absorbance of control sample, A1 is the absorbance of the tested sample, and A2 is the absorbance of test sample solution without NBT solution. Ascorbic acid was used as positive control. 2.5.2. Assay of reductive potential The reductive potential of ORPp was determined according to the method of Li [18] with some modification. ORPp samples of 0.5 ml (0.5, 1.0, 1.5, 3.0 and 5.0 mg/ml), 1.0 ml pH 6.6 phosphate buffer (0.2 M) and 1.0 ml 1% (w/v) K3 Fe(CN)6 solution were incubated at 50 ◦ C for 20 min, then left to cool down. 1.0 ml trichloroacetic acid (10%, w/v) and 0.25 ml fresh 0.1% (w/v) FeCl3 were added to the mixture in sequence. The mixture was shaken and its absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicates an increase of reduction capability. 2.5.3. Ferrous metal ions chelating activity The chelating of ferrous ions by ORPp was estimated by the method of Liu [19] with some modification. Briefly, five concentrations (0.5, 1.0, 1.5, 3.0 and 5.0 mg/ml) of ORPp samples were each prepared in deionized water, 0.4 ml samples were added to a solution of 2.0 mM FeCl2 (0.05 ml). The reaction was initiated by the
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addition of 5.0 mM ferrozine (0.2 ml) and total volume was adjusted to 4 ml with water. Then, the mixture was shaken vigorously and left at room temperature for 10 min. Absorbance of the solution was then measured spectrophotometrically at 562 nm against the blank (water instead of ORPp sample and FeCl2 solution, respectively). The percentage of inhibition of ferrozine–Fe2+ complex formation was calculated according to the following formula: A0 − A1 + A2 Metal chelating effect (%) = × 100 A0
slide and then covered with a cover slip. The slide was then examined under the microscope at 400×, and the motility was scored in different fields of view. Spermatozoa showing any degree of movement were considered to be motile. All spermatozoa (motile as well as immotile) were counted with the help of a blood cell counter. Sperm motility was calculated using the following formula: Sperm motility (%) =
(2)
where A0 is the absorbance of control sample, A1 is the absorbance of the tested sample, and A2 is the absorbance of test sample solution without FeCl2 solution. 2.6. Determination of antioxidant and spermatogenic activities in vivo of ORPp 2.6.1. Animal grouping and experimental design The assay of antioxidant activities in vivo of ORPp was carried out according to the reported method [20]. Wistar male rats (8-weeksold), grade of specific pathogen free, were used in this study. The mice were maintained in separated cages, under the conditions of 21 ± 1 ◦ C and 50–60% relative humidity. They were free to access to food and water and kept on a 12 h light/dark cycles during the experiments. After adapting to their environment for 1 week, these male rats were randomly divided into 6 groups (eight for each) for experiment. Rats in Group I (normal control group) were treated with 0.9% sodium chloride (NaCl, 25 ml/kg BW) per day by hypodermic injection and gastric gavage. Rats in Group II (model control group) were treated with 9% d-galactose (d-Gal, 25 ml/kg BW) by hypodermic injection and 0.9% NaCl (25 ml/kg BW) by gastric gavage per day. Rats in Group III (positive control group) were treated with 9% d-Gal (25 ml/kg BW) by hypodermic injection and vitamin E (VE , 50 mg/kg BW) by gastric gavage each day. Mice in Group IV (low dose of ORPp), Group V (medium dose of ORPp) and Group VI (high dose of ORPp) were treated with 9% d-Gal (25 ml/kg BW) by hypodermic injection, and crude ORPp of 50, 100 and 200 mg/kg BW, respectively, by gastric gavage, was administrated once a day for 21 consecutive days. After overnight fasting following the last drug administration, the rats were weighed and killed by decapitation. 2.6.2. Assay of antioxidant activity in serums Blood samples were harvested immediately in centrifuge tube. After 1 h, the blood samples were centrifuged at 4000 g for 10 min to afford the serums required. All above treatments were done at 4 ◦ C. Activities of SOD, CAT and GSH-Px, level of MDA and TAOC were measured by using commercial reagent kits guided by the instruction manuals. 2.6.3. Effect on the weight of sexual organs The testis, seminal vesicles, epididymis, and prostate glands were carefully removed and weighed. Then calculate the proportion of above organs. 2.6.4. Sperm analysis The motility, viability, and number of spermatozoa in the cauda epididymis were assessed. The cauda epididymis was transected at the point of origin of the vas deferens at the distal end and placed in a watch glass containing 0.5 ml of normal saline (0.9% NaCl) maintained at 37 ◦ C. The tissue was minced carefully with the help of fine forceps and scissors to ensure the extrusion of spermatozoa. The tissue fractions were removed using the fine forceps and a needle, and the suspension was used for sperm analysis. 2.6.5. Assay of sperm motility Sperm motility was tested by observing the movement under microscope. A drop of sperm suspension was placed on a clear glass
n1 × 100 n2
(3)
where n1 was number of motile spermatozoa, n2 was total number of spermatozoa. 2.6.6. Assay of sperm viability Sperm viability was assessed by a supravital staining technique based on the principle that cells with damaged plasma membrane take up the stain, whereas viable ones do not. A drop of sperm suspension and a drop of 1% Congo red were placed on a clear glass slide and mixed thoroughly. A portion of the mixture was transferred to a second slide, and a thin film was prepared. The slide was then examined under the microscope (400×), and about 100 spermatozoa (viable and dead) were counted from different fields of the slide. Spermatozoa appearing pinkish (stained) were considered to be dead, whereas those appearing colorless (unstained) were counted as being viable. Sperm viability was calculated in percent by using the following formula: Sperm viability (%) =
n1 × 100 n2
(4)
where n1 was the number of viable spermatozoa and n2 was the total number of spermatozoa. 2.6.7. Assay of sperm count Sperm count was investigated according to the reported method [21]. A hemocytometer with improved Neubauer ruling was employed for counting the spermatozoa. The sperm suspension was diluted to 20 times with normal saline. The preparation was then thoroughly mixed, and one drop was added to both sides of the hemocytometer. The number of spermatozoa was counted in the four corner squares of the hemocytometer under a microscope at 400×. Spermatozoa on both sides of the hemocytometer were counted, and the average number was recorded. The number of spermatozoa per cauda epididymis was expressed as follows: Sperm number = N × 0.2 × 106
(5)
where N was averaged number of spermatozoa counted. 2.7. Statistical analysis Data were statistically analyzed, using the SPSS 18.0 software package (SPSS, Chicago, IL, USA), by one-way ANOVA. Significant differences between two means were determined by Tukey’s test. P-values < 0.05 were regarded as significant. 3. Results and discussion 3.1. Separation and purification of ORP The crude ORP was obtained by enzyme-assisted extraction and it was then separated through DEAE-52 cellulose. As the result, one main independent elution peak (F1 ) was obtained as shown in Fig. 1A. This fraction was collected, concentrated, dialyzed, freezedried and loaded into a column of Sephadex G-100. The column was eluted with deioned water, and the resulting elute was collected. As showed in Fig. 1B, the fraction generated one single elution peak, representing of ORPp. The fraction was collected, concentrated,
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Fig. 1. Elution profiles of crude ORP on DEAE-52 cellulose anion-exchange chromatography column with 0–0.5 M NaCl elute (A) and elution curve of polysaccharide fraction from DEAE-52 cellulose on size-exclusion chromatography column of Sephadex G-100 (B).
freeze-dried and the purified products were obtained. The yield of ORPp was 32.8%. 3.2. Preliminary characterization of ORPp 3.2.1. Contents of carbohydrate, sulfate, protein and uronic acid in ORPp As shown in Fig. 2A, there was a weak optical absorption of ORPp at 280 nm manifesting the existence of protein or nucleic acid in ORPp. The contents of carbohydrate, sulfate, protein and uronic acid in ORPp were 68.2%, 7.64%, 4.33% and 9.48%, respectively. The existence of protein was consistent with the UV spectrum analysis, there was a weak absorption at 260–280 nm in the UV spectrum. ORPp was characterized by FT-IR spectroscopy as showed in Fig. 2B. There was a strong and broad peak around the 3415 cm−1 of ORPp infrared spectroscopy, which was belonged to the hydroxyl groups stretching vibration [22]. The weak peaks around 2930 and 1320 cm−1 were assigned to the C-H asymmetric stretching vibration [23]. The bands at 1633.7 and 1337.2 cm−1 were characteristic signals for the deprotonated carboxylic group (COO− ), indicating ORPp being acidic polysaccharides. The weak peak around 1240 cm−1 was assigned to the O SO3 − . A strong extensive absorption in the region of 1000–1200 cm−1 for stretching vibrations of C OH side groups and the C O C glycosidic band vibrations were observed in the spectra [24]. Furthermore, absorption around 1071.9 cm−1 was the furan glycosides characteristic absorption peaks [25]. All these were characteristic absorptions of polysaccharides. GC analysis showed that ORPp was composed of galactose [26] and glucose (Glu) in a molar percent of 23.7% and 76.3%, respectively (Fig. 2C). The average molecular weight of ORPp was
Fig. 2. UV (A), FT-IR spectra (B), and GC chromatogram of monosaccharide (C) of ORPp.
calculated as 118 kDa by HPLC according to the calibration curve with standard dextran–glucose (D-Glc). 3.3. Antioxidant activity in vitro of ORPp 3.3.1. Superoxide radical scavenging activity of ORPp In reactive oxygen species (ROS), superoxide radical is usually formed as a precursor, which could form stronger ROS, such as singlet oxygen and hydroxyl. Although superoxide radical is a relatively weak oxidant, it can cause lipid peroxidation triggered. Beside these, superoxide can also form the hydrogen peroxide, precursor of hydroxyl and so on, then indirectly lead to lipid peroxidation triggered [27]. Taking these reasons into account, it is important to remove superoxide radicals in the process of organism antioxidant. As it is shown in Fig. 3A, the scavenging effects of ORPp were the most evident at all of the tested concentrations and even stronger than ascorbic acid. Also, the superoxide radical scavenging effects of ORPp and ascorbic acid were positively correlated well with
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3.3.3. Chelating ability of ferrous ion Chelation is an important parameter in the sense that iron is essential for oxygen transport, respiration and activity of many enzymes, which are required for life. However, iron is an extremely reactive metal and will catalyze oxidative changes in lipids, proteins and other cellular components. Among the transition metals, iron is known as the most important lipid oxidation pro-oxidant due to its high reactivity. The ferrous state of iron accelerates lipid oxidation by breaking down hydrogen and lipid peroxides to reactive free radicals via the Fenton reaction. Fe3+ ion also produces radicals from peroxides although the rate is ten times slower than that of Fe2+ ion. Fe2+ ion is the most powerful pro-oxidant among the various species of metal ions. Ferrozine can quantitatively form complexes with Fe2+ . In the presence of chelating agents, the complex formation can be disrupted, resulting in a decreasingly intense red color of the complex. Therefore, measuring the level of color reduction allows the estimation of metal chelating activity in terms of the co-existing chelator [29,30]. Fig. 3C showed chelating activities of ORPp for Fe2+ . At a concentration of 0.5–5 mg/ml, ferrous metalions chelating activity of ORPp was significantly stronger than Vc. The results indicated that ORPp has strong chelating ability of ferrous ion. 3.4. Antioxidant activities of ORPp in vivo
Fig. 3. Scavenging affects on superoxide radical (A), reductive potential (B) and metal chelating activity (C) of ORPp.
the increase of concentration from 0.5 to 5.0 mg/ml. The results indicated that ORPp has strong scavenging activity on superoxide radical.
3.3.2. Reductive potential of ORPp Reducing capacity of compounds is a symbol of potential antioxidant capacity. Various mechanisms, including reducing capacity, decomposition of peroxides, prevention of chain initiation, prevention of continued hydrogen abstraction binding of transition metal ion catalysts, and radical scavenging have been claimed to explain the antioxidant activities [28]. Fig. 3B showed the reductive activity of ORPp using the potassium ferricyanide reduction method. The reducing power of ORPp increased significantly with increasing concentration of samples. When the concentration exceeded 5 mg/ml, reductive power of ORPp was as high as 49%. This indicated that the reduction potential of ORPp was strong.
Rats injected with d-Gal have been used as an aging animal model in some previous studies. d-Gal can cause accumulation of ROS, or stimulate free radical production indirectly by forming advanced glycation end-product (AGE) in vivo. On the other hand, VE has been applied in early studies as a positive control agent because of its highly efficient antioxidation and prevention of lipid peroxidation [31]. Thus, in this study, mice treated with d-Gal were used as an aging animal model while VE was used as positive control medicine. Effects of ORPp on the activities of SOD, CAT, GSH-Px and the levels of MDA and TAOC in serums in aging mice are presented in Table 1. Apparently, a marked increase in MDA and significant decreases (P < 0.05) of antioxidant enzymes activities (SOD, GSH-Px, and CAT) and TAOC were observed in serums between the treatments of Group I (normal control group) and Group II (model control group). ORPp and VE treatments inhibited significantly (P < 0.05) the formation of MDA in mice serums and raised the activities of antioxidant enzymes and the level of TAOC in a dose-dependent manner (Groups IV–VI). The results shown that, injection of d-Gal can cause oxidative injury of the body, and VE and ORPp could overcome this damage. The mechanism of this damage-preventing function may be a result of the enhanced activity of endogenous antioxidant enzyme (SOD, GSH-Px, and CAT), promoted TOAC and decreased the content of MDA. 3.5. Effect on sexual organ weight The structure and functional degradation of gonad is closely related to aging process. Gonad atrophy is the most obvious degradation of endocrine system during aging, resulting in an impaired immune function and inducing aging [32]. Therefore, the research of aging of gonad helps to reveal the aging mechanism. As showed in Table 2, the sexual organs of aging rats with the treatment of d-Gal were markedly atrophic. Administration of the ORPp at the doses of 50, 100 and 200 mg/kg BW resulted in a significantly dose-dependent increase in weights of all sexual organs. Then weight increase caused by the injection of middle and high dose of ORPp was even higher than that of VE . These indicated that ORPp can prevent the gonad atrophy resulted from aging, the mechanism of which may be (or partially) related to the antioxidant activity of ORPp.
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Table 1 Effects of ORPp on the activities of SOD, CAT, GSH-Px and levels of MDA and TAOC in serums. Group
SOD (U/ml)
I II III IV V VI
134 122 158 151 162 168
± ± ± ± ± ±
CAT (U/ml)
3 5* 4# 6# 4# 5#
71 63 75 72 85 87
± ± ± ± ± ±
3 4* 2# 3# 5# 4#
GSH-Px (U/ml) 7389 7046 7834 7537 7763 7712
± ± ± ± ± ±
128 158* 101# 116# 142# 135#
MDA (nmol/ml) 13.8 16.4 10.4 11.4 10.7 9.6
± ± ± ± ± ±
TAOC (U/ml)
0.52 0.26* 0.37# 0.53# 0.59# 0.75#
19.3 16.3 24.4 23.0 26.4 29.6
± ± ± ± ± ±
1.28 0.69* 0.89# 1.39# 0.95# 1.17#
The data were presented as mean ± SD (n = 8). * Compared with normal control group (Group I), P < 0.05. # Compared with model control group (Group II), P < 0.05. Table 2 Effect of ORPp on body and sexual organ weights. Group
Weight of testis (mg/100 g BW)
I II III IV V VI
870 790 845 837 856 867
± ± ± ± ± ±
17.3 14.8* 21.9# 19.7# 15.9# 22.4#
Weight of prostate (mg/100 g BW) 261 228 257 244 266 271
± ± ± ± ± ±
10.4 13.2* 13.4# 16.4# 11.2# 15.6#
Weight of seminal (mg/100 g BW) 315 274 329 307 325 322
± ± ± ± ± ±
19.4 11.7* 16.4# 14.8# 15.5# 18.3#
Weight of epididymis (mg/100 g BW) 635 551 619 612 644 651
± ± ± ± ± ±
22.7 24.3* 27.4# 23.3# 28.9# 28.2#
The data were presented as mean ± SD (n = 8). * Compared with normal control group (Group I), P < 0.05. # Compared with model control group (Group II), P < 0.05. Table 3 Effect of ORPp on male rats’ sperm motility and number of spermatozoa in cauda epididymis. Group
Sperm motility (%)
I II III IV V VI
80.4 69.3 82.4 77.2 81.9 83.3
± ± ± ± ± ±
2.5 1.9* 2.9# 3.8# 4.2# 3.2#
Sperm viability (%) 76.4 63.9 75.9 73.5 79.3 78.7
± ± ± ± ± ±
1.88 2.16* 1.64# 3.24# 2.93# 2.37#
Sperm number (106 /mL) 6.3 5.1 6.2 5.5 5.8 6.2
± ± ± ± ± ±
0.13 0.08* 0.09# 0.15# 0.11# 0.16#
The data were presented as mean ± SD (n = 8). * Compared with normal control group (Group I), P < 0.05. # Compared with model control group (Group II), P < 0.05.
3.6. Sperm analysis With the development of aging, different degrees of degradations can appear in terms of the structure and functions of testis, such as the decrease of spermatogenesis function and sperm activity, which finally lead to senescence of testis. Increased oxidative stress is closely related to aging, whereas anti-aging medicine can function against the oxidative damage of oxidative stress on the body, through which it can effectively prolong the life span of the body in normal range, so that the aging process can be delayed [33]. Therefore, the research of spermatogenesis activity is highly useful for the investigation of anti-aging effects. The motility of caudal epididymal spermatozoa was decreased significantly (P < 0.05) in model control group compared with the normal control group. Sperm motility in the Group IV-VI was 77.2%, 81.9%, and 83.3%, respectively with no significant difference compared with 80.4% in the Group I. The motility of spermatozoa in Group V and VI were higher than Group III. Sperm viability of model control group was significantly lower than normal control group, indicating that sperm viability was related to the aging process. ORPp and VE potentially have a favorable effect on sperm viability. The amounts of epididymal sperm counts were significantly increased in the treated groups: 5.5, 5.8 and 6.2 × 106 /ml in Group IV, V and VI, respectively, compared with 5.1 × 106 /ml in the Group II (Table 3).
All these showed that ORPp has a novel spermatogenesis activity, which might be related to the antioxidant activity of ORPp. Generally, the excess free radicals is a factor that leads to damaged organ, declined immunity, the onset of various diseases and the process of aging [34]. However, the strong antioxidant activity of ORPp could scavenge excess free radicals so that the normal functions and operations of organs can be maintained and thus the aging process can be delayed. 4. Conclusion Polysaccharide was extracted from Ostrea rivularis Gould, and one purified fraction ORPp was obtained from the crude ORP through sequential purification by chromatography of DEAE-52 and Sephadex G-100. Then, the ORPp was characterized by chemical analysis, GC, HPLC and FT-IR. Experimental results showed that ORPp was mainly composed of glucose (76.3%) and galactose (23.7%). The average molecular weight of ORPp was determined to be 118 kDa. And there was infrared characteristic absorption peak of polysaccharides in the FT-IR spectroscopy of ORPp. Beside these, the antioxidant and spermatogenic activities of ORPp were evaluated. The results demonstrated that ORPp possessed strong scavenging activities on superoxide radical, reductive potential and metal chelating activities. For antioxidant activity in vivo, ORPp significantly raised the level of TAOC, reduced the formation of MDA and enhanced the activities of SOD and GSH-Px. Furthermore, ORPp could significant increased the weights of all sexual organs of rats, promoted sperm motility and raised epididymal sperm counts. Further work on detailed characterization of ORPp is in progress. Acknowledgement This research is supported by the Special Science and Technology Cooperation Fund of Hongkong, Macao and Taiwan in China (2014DFH30010). References [1] H. Que, X. Liu, H. Wang, S. Zhang, G. Zhang, F. Zhang, Chin. J. Zool. 4 (2003) 110–113. [2] Z. Duan, X. Liu, Meat Res. 7 (2011) 29–31.
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