Carbohydrate Polymers 127 (2015) 209–214
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Characterization and antioxidant activities of acidic polysaccharides from Gynostemma pentaphyllum (Thunb.) Markino Bo Li a,∗ , Xiaoyu Zhang b , Mingzhu Wang b , Lili Jiao b,∗ a b
School of Pharmeutical Sciences, Changchun University of Chinese Medicine, Changchun 130117, China Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun 130117, China
a r t i c l e
i n f o
Article history: Received 7 February 2015 Received in revised form 6 March 2015 Accepted 14 March 2015 Available online 28 March 2015 Keywords: Polysaccharides Gynostemma pentaphyllum (Thunb.) Markino Antioxidant activity
a b s t r a c t Three acid polysaccharides obtained from Gynostemma pentaphyllum (Thunb.) Markino (named as GPA1, GPA2 and GPA3) using combination of water extraction, ion-exchange and gel permeation chromatography were subjected to composition analysis and valuated for the antioxidant activity. The sugar content of GPA1, GPA2 and GPA3 were 54.55%, 85.70% and 91.34%, respectively. Monosaccharide analysis showed that GPA1, GPA2 and GPA3 were all composed of Man, Rha, GlcA, GalA, Glc, Gal, Ara and Fuc, besides, GPA2 and GPA3 also contained Xyl. The molecular weight of GPA1, GPA2 and GPA3 were 19.6 kDa, 10.6 kDa and 6.7 kDa, respectively. In vitro antioxidant assay, GPA1, GPA2 and GPA3 could scavenge 1, 1-diphenyl2-picrylhydrazyl (DPPH) radical and hydroxyl radical, chelate ferrous ion and reduce ferric ion. The antioxidant activities of GPA3 were stronger than those of GPA1 and GPA2, suggesting that GPA3 has significant potential as a natural antioxidant agent. © 2015 Elsevier Ltd. All rights reserved.
1. Instruction Reactive oxygen species (ROS) involve a series of free radicals such as superoxide anion radicals (1 O2 ), hydroxylradical species (• OH), singletoxygen (O• 2 ) and hydrogen peroxide (H2 O2 ) etc. These active oxygen compounds involving in many cellular metabolic and signaling process, also could damage cellular macromolecules causing lipid peroxidation and nucleic acid and protein alterations. Furthermore, more and more evidence indicated that ROS involved in many disease processes, such as aging, carcinogenesis, atherosclerosis, diabetes and rheumatoid arthritis (Finkel & Holbrook, 2000; Seifried, Anderson, Fisher, & Milner, 2007; Valko et al., 2007). Several lines of evidence from both epidemiological and experimental studies have found that antioxidants, including natural compounds from plants and synthetic compounds, play an important role in scavenging excessive free radicals, to delay or prevent oxidation and maintain the systematic health (Mohammed, 2002; Giles et al., 2003). However, the synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are suspected to be connected with liver damage and
∗ Corresponding authors at: Changchun University of Chinese Medicine, Jingyue Economic Development Zone, 1035, Boshuo Road, Changchun 130117, Jilin Province, China. Tel.: +86 431 86045355; fax: +86431 86045258. E-mail addresses: [email protected] (B. Li), [email protected] (L. Jiao). http://dx.doi.org/10.1016/j.carbpol.2015.03.069 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
carcinogenesis (Grice, 1988; Witschi, 1986). Therefore, more and more interests are aroused to find natural antioxidants without toxicity. Gynostemma pentaphyllum (Thunb.) Markino, a perennial-lianaherb belonging to family Cucurbitaceae and genus Gynostemma Bl., distributed widely in China, particularly in south of the Qinling Moutanis and Yangtze River (Lv et al., 2009). It has been traditionally used as a food and tea in the United States, China and many other Asia countries including Japan. G. pentaphyllum is also a traditional and precious Chinese medicine used for depressing cholesterol levels, promoting the production of body fluid, regulating blood pressure, strengthening the immune system, treating chronic bronchitis and gastritis, and reducing inflammation etc. (Aktan, Henness, Roufogalis, & Ammit, 2003; Circosta, De Pasquale, & Occhiuto, 2005; Huang et al., 2005; Yeo et al., 2008; Lv et al., 2009). G. pentaphyllum contains many active ingredients, such as saponin, amino acid, flavonoids and polysaccharides, which were similarity to those found in ginseng root. Furthermore, G. pentaphyllum is of high value for drug, thus, it was sometimes called “southern ginseng” (Cui, Eneroth, & Bruhn, 1999). Recently, polysaccharides from G. pentaphyllum have been demonstrated to be natural antioxidants. Luo et al. demonstrated that a neutral polysaccharide from G. pentaphyllum exhibited equivalent antioxidant activity in vitro (Wang & Luo, 2007). Jia et al. also isolated a neutral polysaccharide, and demonstrated it had neuroprotective effect via inhibiting oxidative stress. So far, no investigation has been carried out on acidic polysaccharides from G. pentaphyllum that may account
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for antioxidant properties. According to the literature, the acidic polysaccharides from plants had significant antioxidant activities (Sun, Hui, Guo, Pu, & Yan, 2014; He, Yang, Jiao, Tian, & Zhao, 2012; Wu et al., 2013). Hence, we extracted and purified three acidic polysaccharides from G. pentaphyllum, and their antioxidant activity were evaluated by various methods in vitro.
2.5 ml of concentrated H2 SO4 and at 100 ◦ C for 20 min. After cooling to ambient temperature, 40 l m-hydroxydiphenyl was added and incubated for 15 min. The absorbance of reaction mixture was recorded at 525 nm. The total phenols content in GPAs was determined using the Folin–Ciocalteau assay (Sabir & Rocha, 2008). Gallic acid was used in a standard curve and the results were expressed in microgram of gallic acid equivalents per milliliter of dry extract.
2. Materials and methods 2.1. Materials
2.5. Analysis of monosaccharide composition
Gynostemma pentaphyllum (Thunb.) Markino was purchased from a local shop (Changchun, Jilin Province, China). The dried material were crushed and passed through 60 mesh sieve. The powders were stored in a freezer for further experiments. Sepharose CL-6B was purchased from Pharmacia Fine Chemical AB, Uppsala, Sweden. 1, 1-diphenyl-2-picrylhydrazyl (DPPH), thiobarbituric acid (TBA), ethylenediaminetetraacetic acid (EDTA), ferrozine, trichloroacetic acid (TCA), deoxyribose (DR), 2,4,6-tris(2-pyridyl)s-triazine (TPTZ), Vitamin C (Vc) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of analytical grade.
The sample (2 mg) was hydrolyzed with 0.5 ml of 2 M trifluoroacetic acid (TFA) in an ampoule (2 ml). The ampoule was sealed under a nitrogen atmosphere and kept at 120 ◦ C for 1 h, and the excess acid was completely removed by co-distillation with ethanol. After removing the acid, the products of hydrolysis were derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) according to the method in the literature (Honda, Akao, Suzuki, Okuda, & Kakehi Kazuaki Nakamura, 1989). The monosaccharide derivatives were analyzed by Aglient RRLC 1200 SL system (Agilent Technologies, Wilmington, USA), equipping with a DIKMA Inertsil ODS-3 column (4.6 i.d. ×150 mm, 5 m, Dikma, Japan), detected by UV–VIS DAD detector and connected to a Chemstation system. The PMP derivative (20 l) was injected, eluted with 82.0% phosphatebuffered saline (PBS, 0.1 M, pH 7.0) and 18.0% acetonitrile (v/v) at a flow rate of 1.0 ml/min at room temperature. The wavelength for UV detection was 245 nm.
2.2. Preparation for the polysaccharides Air-dried G. pentaphyllum were crushed and extracted with 90% ethanol under reflux extraction at 40 ◦ C to remove pigments and small lipophilic molecules. The residue was further extracted thrice (2 h each) with distilled water at 100 ◦ C. All aqueous extracts were combined, centrifuged under reduced pressure and precipitated by adding of 95% ethanol (4 volumes). The crude polysaccharide precipitate was collected by centrifugation, and dried under reduced pressure after washed with dehydrated alcohol and diethyl ether. The crude polysaccharides was dissolved in distilled water and centrifuged, and then the supernatant was applied to a column of DEAE-cellulose (Cl− ) column (7.0 × 30 cm). This column was initially eluted with distilled water (2000 ml), and an unbound fraction was recovered. The bound polysaccharides were eluted by 0.1 M (GPA1), 0.2 M (GPA2) and 0.5 M (GPA3) NaCl solution, respectively. Each eluent was collected and identified as the acid polysaccharides of G. pentaphyllum (GPAs). Subsequently, Sepharose CL-6B column (2.5 × 100 cm) were used to further purify the fractions obtained by anion-exchange step. 2.3. Homogeneity and molecular weight analysis The polysaccharides (10 mg) was dissolved in distilled water (1 ml), and then the homogeneity and the molecular weight distribution of them were determined by high-performance lipid chromatography (HPLC), which was performed on a SHIMADZU HPLC system (Shimadzu, Japan) fitted with one TSK-G3000 PWXL columns (7.8 mm ID × 30.0 cm/l) and a RID-10A detector. The column was calibrated by molecular mass markers (T-150, 80, 40, 20, 10). The mobile phase was distilled water, and the flow rate was 0.5 ml/min at 40 ◦ C, with 1.6 MPa. 2.4. Analysis of chemical composition Total sugar content was determined by phenol-sulfuric acid method using glucose as standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The content of protein was determined by Bradford’s method (Bradford, 1976). The content of uronic acids was measured by the method of m-hydroxydiphenyl method (Blumenkrantz & Asboe-Hansen, 1973). Briefly, 0.4 ml sample (0.1 g/l) was added 40 l of 4 M sulfamic acid-potassium sulfamate (pH 2.5) and mixed thoroughly, then the mixture was add
2.6. FT-IR spectrum analysis The FT-IR spectra were recorded on SPECORD in a range 4000–400 cm−1 . The samples were analyzed as KBr pellets.
2.7. Assay antioxidant activity in vitro 2.7.1. The DPPH scavenging activity The DPPH quenching ability was measured by the method proposed by Konrath et al. (Yamaguchi, Takamura, Matoba, & Terao, 1998) with slight modifications. 1.5 ml freshly prepared DPPH solution (0.1 mM in methanol) was mixed with 4.5 ml of sample (0.1–0.6 mg/ml). The mixture was incubated at 25 ◦ C for 30 min in the dark, and the absorbance of reaction liquid was measured at 517 nm. Vc was used as the positive control. The percentage scavenging radical was calculated using the following equation: Scavenging rate (%) =
(A0 − A1 ) × 100 A0
where A0 is the absorbance of the control (water instead of sample), and A1 is the absorbance of the sample. 2.7.2. Hydroxyl radical scavenging assay The assay was performed as described by the method of Chung, Osawa, and Kawakishi (1997) with minor changes. 0.2 ml solution of various concentrations of sample (0.25–15 mg/ml) was added to 1.2 ml of 10 mM phosphate buffer (PBS) at pH 7.4 containing 2.67 mM 2-deoxyribose and 0.13 mM EDTA. 0.2 ml of iron ammonium sulfate (0.4 mM) was added. Samples were kept in a water bath at 37 ◦ C, the reaction was started by adding 100 l of ascorbic acid (1.0 mM) and 10 l of H2 O2 (0.1 M). Samples were maintained at 37 ◦ C for 15 min, and then 2 ml of 1% TBA and 2 ml of 2% CCl3 COOH was added to the resulting mixture. Finally the mixture was heated in boiling water for 15 min and cooled by ice water. The absorbance was determined against a blank at 532 nm with spectrophotometer.
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Vc was used as the positive control. The scavenging percentage was calculated as following: Scavenging rate (%) =
(A0 − A1 ) × 100 A0
where A0 is the absorbance of the control (water instead of sample), and A1 is the absorbance of the sample. 2.7.3. Ferrous ion-chelating potential assay The ferrous ion-chelating potential of sample was determined as in Dinis, Maderia, and Almeida (1994). The reaction mixture contained 1.0 ml of sample (0.5–10 mg/ml), 0.1 ml of FeCl2 (2 mM) and 0.2 ml of ferrozine (5 mM). After incubating at 25 ◦ C for 10 min, the absorbance was measured at 562 nm using EDTA as positive control. A lower absorbance indicates a higher chelating ability. The chelating ability was calculated using the following equation: Chelating activity (%) =
(A0 − A1 ) × 100 A0
where A0 is the absorbance of the control (water instead of sample), and A1 is the absorbance of the sample. 2.7.4. Assay of ferric reducing power The ferric reducing ability of plasma (FRAP) assay was carried out according to the procedure of Veenashri and Muralikrishna (Veenashri & Muralikrishna, 2011). Briefly, the FRAP reagent was prepared daily from 300 mmol/l acetate buffer (pH 6.3), 10 mmol/l TPTZ solution (in mmol/l HCl) and 20 mmol/l FeCl3 ·6H2 O solution in a proportion of 10:1:1 (v/v). 0.3 ml of sample was added to 2.7 ml of FRAP reagent. The mixture was incubated at 3 ◦ C for 10 min. The absorbance was measured at 593 nm. Fresh working solutions of FeSO4 were used for calibration. The antioxidant capacity based on the ability to reduce ferric ions of sample was calculated from the linear calibration curve and expressed as mmol FeSO4 equivalents per gram of the texted samples. Increased absorbance of FRAP reflected increased reducing power. 2.8. Statistical analysis All values are expressed as the mean ± SD for three independent experiments. Comparison between any two groups was evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons tests. Differences was considered to be statistically significant if P < 0.05. 3. Result and discussion 3.1. Characteristic of GPA1, GPA2 and GPA3 Three acidic polysaccharides, namely, GPA1, GPA2 and GPA3 were purified from G. pentaphyllum by hot-water extraction, ethanol precipitation, ion-exchange and Sepharose CL-6B gel permeation chromatography. The total sugar of GPA3 was higher (91.34%) than GPA2 (87.57%) and GPA1 (54.55%). The protein contents evaluated in GPA1, GPA2 and GPA3 were 3.75%, 4.83% and 5.53%, respectively. Furthermore, uronic acid detected in the three polysaccharides was 23.04%, 32.79% and 27.01%, respectively, suggesting they belonged to acidic polysaccharides. The total phenols contend was relatively lower in GPA3 (256 mg/100 g) than GPA1 (756 mg/100 g) and GPA2 (950 mg/100 g). The HPLC profile showed that each polysaccharide had a single and symmetrically sharp peak revealing that they were homogeneous polysaccharides. According to the retention time, the average molecular weights of GPA1, GPA2 and GPA3 were estimated to be 19.6 kDa, 10.6 kDa and 6.7 kDa, respectively.
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As summarized in Fig. 1 and Table 1, HPLC analysis showed the difference of the three acid polysaccharides, with the presence of Man, Rha, GlcA, GalA, Glc, Gal, Xyl, Ara, Fuc in the molar ratio of 1:0.1:2.2:1.8:2.2:2.6:0.2:1.9:0.2 for GPA2 (Fig. 1b); and Man, Rha, GlcA, GalA, Glc, Gal, Xyl, Ara, Fuc in the molar ratio of 1:0.6:3.9:0.5:5.5:2.6:0.5:1.2:0.2 for GPA3 (Fig. 1c); also Man, Rha, GlcA, GalA, Glc, Gal, Ara, Fuc in the molar ratio of 1:0.04:1.4:0.9:1.3:2.6:2:0.2 for GPA1 (Fig. 1a), and there was no Xyl detected in GPA1. As shown in the FT-IR spectra of GPA1, GPA2 and GPA3 (Fig. 2), the broad and intense band at 3426 cm−1 was the stretch vibration of O H. The signal at 2938 cm−1 was attributed to the stretch vibration of the C H bond. The band at 1630 cm−1 was assigned to the bending vibration of O H, and the signal at 1541 cm−1 was attributed to the vibration of C O. The band at 1416 cm−1 was assigned to C-H bending vibration. the bands in the region of 1741 cm−1 and 1623 cm−1 were ascribed to C = O stretching and asymmetric C O stretching vibration of carboxyl group, respectively. The characteristic absorption band at 833 cm−1 and 877 cm−1 suggested ␣- and -configurations simultaneously existing in GPA1, GPA2 and GPA3. 3.2. Antioxidant activities in vitro of GPA1, GPA2 and GPA3 3.2.1. DPPH radical scavenging activity The antioxidant properties of the acid polysaccharides from G. pentaphyllum were investigated by DPPH assay. DPPH, a stable Ncentered free radial, has been well used to employ the ability of free-radical scavenging properties or hydrogen donation of compounds and medicine materials (Hatano et al., 1989). As shown in Fig. 3, DPPH scavenging activities of the three polysaccharides increased with increasing concentrations. At the concentration of 1.5 mg/ml, the DPPH radical scavenging rate of GPA1, GPA2 and GPA3 was 84.95%, 84.32% and 93.14%, respectively, with IC50 value of 0.08 mg/ml, 0.06 mg/ml and 0.03 mg/ml, respectively. Among the three polysaccharides, GPA3 possessed stronger scavenging activity than that of GPA2 and GPA1. HPLC analysis revealed that GPA2 and GPA3 contained the same monosaccharide composition of Man, Rha, GlcA, GalA, Glc, Gal, Xyl, Ara and Fuc. However, the ratio of the two monosaccharides greatly differed from one another. There was no Xyl detected in GPA1. In addition, there was a significant difference in the average molecular weights among GPA1 (19.6 kDa), GPA2 (10.6 kDa) and GPA3 (6.7 kDa). Of the three, GPA3 exhibited higher antioxidant activity than GPA1 and GPA2, which probably due to the difference in the ratio of monosaccharide, as well as molecular weight between them. In addition, the total phenolic content in them was determined as GPA3 < GPA1 < GPA2, indicating that the DPPH radical scavenging activities of the three polysaccharides were probably owing to their carboxyl group in hexuronic acid, instead of phenolic compounds (Wang, Mao, & Wei, 2012). 3.2.2. Hydroxyl radicals scavenging activity The hydroxyl radical is accepted to be the most reactive and poisonous free radical, and induce severe damage to adjacent biomolecules as an active initiator for lipids peroxidation (Chance, Sies, & Boveris, 1979; Ke et al., 2009). An accepted reaction system containing Fe3+ -EDTA-H2 O2 -deoxyribose in the aqueous phase was used to generate •OH and measure inhibitory activity of GPA1, GPA2 and GPA3, and the results were summarized in Fig. 4. The three polysaccharides were able to scavenge hydroxyl radical. At the concentrations ranging from 0.25 to 7.5 mg/ml, the hydroxyl radical scavenging ability of GPA3 increased markedly and dosedependently, and reached 97.42% at 7.5 mg/ml. The IC50 values of GPA1, GPA2 and GPA3 were 0.22, 0.21 and 0.20 mg/ml, respectively. Notably, all the IC50 values of GPAs of hydroxyl radicals were higher
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Fig. 1. HPLC analysis of monosaccharide composition of GPA1 (a), GPA2 (b) and GPA3 (c). The three polysaccharides were hydrolyzed, dried, PMP-labeled and analyzed by HPLC as described in the experimental methods.
Table 1 Content of sugar, uronic acid, protein, total phenols and molecular weight of GPA1, GPA2 and GPA3. Fraction
Sugar contenta
Protein contenta
Uronic acid contenta
Total phenols contentb
Mw (kDa)
GPA1 GPA2 GPA3
54.55 85.70 91.34
3.75 4.38 5.53
23.04 32.79 27.01
756 950 256
19.6 10.6 6.7
a b
Percentage weight in the fraction. mg/100 g of extract.
than that of Vc (0.42 mg/ml). Among the three acid polysaccharides, GPA3 exhibited the strongest scavenging activity. 3.2.3. Fe2+ chelating activity of GPA1, GPA2 and GPA3 Iron (Fe) and copper (Cu) ions are important elements for the human body, whereas these metal ions have potentially dangerous. Fe2+ , with high reactivity, can stimulate lipid peroxidation and accelerate lipid peroxidation, thereby driving the chain reaction of lipid peroxidation (Veenashri & Muralikrishna, 2011). So the Fe2+ chelating activity is considered as an important antioxidant property of materials. In this paper, the Fe2+ chelating ability was determined by the reduction of absorbance at 562 nm, the red color
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Fig. 2. FT-IR spectra of GPA1, GPA2 and GPA3.
Fig. 3. DPPH radical scavenging activity of GPA1, GPA2 and GPA3. Each value represents the mean ± SD (n = 3). FT-IR spectra of GPA1, GPA2 and GPA3.
is quantitatively formed by the reaction of ferrozine with Fe2+ . As shown in Fig. 5, GPA3 exhibited higher chelating activity than GPA2 and GPA1. At the concentrations ranging from 0.05 to 1.5 mg/ml, the Fe2+ chelating activity of WGOS-R was measured as 20.78–79.71%, with IC50 value of 0.34 mg/ml. The Fe2+ chelating activity of GPA1 and GPA2 was 54.75% and 67.67% at 2.5 mg/ml, with the IC50 values of 0.85 and 0.43 mg/ml, respectively. The results indicated that GPA3 possessed a much stronger ferrous chelating activity than GPA1 and GPA2. However, the metal chelating activity of the three polysaccharides was significantly lower than that of EDTA, which had the strongest chelating capacity, and achieved 100% at concentration of 0.5 mg/ml. The bioactivities of polysaccharides are closed related to their molecular weight, monosaccharide composition, configuration of glycosidic linkages, position of glycosidic linkages, as well as the degree of substitution on alcohol or phenolic hydroxyl groups (Bohn & BeMiller, 1995). Thus, the structural features and structure–function relationships involved in Fe2+ chelating activities of Phellinus pini polysaccharides needed to be further studied.
Fig. 4. Hydroxyl radical scavenging activity of GPA1, GPA2 and GPA3. Each value represents the mean ± SD (n = 3).
3.2.4. Ferric reducing powder of GPA1, GPA2 and GPA3 FRAP assay is one of the widely simple and convenient method for determining the antioxidants activity (Li, Ren, & Lin, 2002). The ferrous ions from FRAP reagent was reduced by tested antioxidants in the presence of TPTZ, and formed an intense blue Fe2+ -TPTZ complex with an absorption maximum at 593 nm (Benzie & Strain, 1996). In this work, the FRAP value was used to measure the antioxidant activities of acidic polysaccharides of G. pentaphyllum. The FRAP values was estimated by reference to a linear calibration curve (regression equation: y = 0.085x − 0.016, R2 = 0.9991) made from different concentration of FeSO4 solution. The antioxidant capacity based on the ability to reduce ferric ions of sample was calculated from the linear calibration curve and expressed as mM FeSO4 equivalents per gram of samples. The FRAP values of GPA1, GPA2 and GPA3 (2.5 mg/ml) were obtained as 14.37, 11.01 and 15.07 mM FeSO4 /g, respectively.
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Fig. 5. Fe2+ chelating activity of GPA1, GPA2 and GPA3. Each value represents the mean ± SD (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Characterization and antioxidant activities of acidic polysaccharides from Gynostemma pentaphyllum (Thunb.) Markino Bo Li a,∗ , Xiaoyu Zhang b , Mingzhu Wang b , Lili Jiao b,∗ a b
School of Pharmeutical Sciences, Changchun University of Chinese Medicine, Changchun 130117, China Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun 130117, China
a r t i c l e
i n f o
Article history: Received 7 February 2015 Received in revised form 6 March 2015 Accepted 14 March 2015 Available online 28 March 2015 Keywords: Polysaccharides Gynostemma pentaphyllum (Thunb.) Markino Antioxidant activity
a b s t r a c t Three acid polysaccharides obtained from Gynostemma pentaphyllum (Thunb.) Markino (named as GPA1, GPA2 and GPA3) using combination of water extraction, ion-exchange and gel permeation chromatography were subjected to composition analysis and valuated for the antioxidant activity. The sugar content of GPA1, GPA2 and GPA3 were 54.55%, 85.70% and 91.34%, respectively. Monosaccharide analysis showed that GPA1, GPA2 and GPA3 were all composed of Man, Rha, GlcA, GalA, Glc, Gal, Ara and Fuc, besides, GPA2 and GPA3 also contained Xyl. The molecular weight of GPA1, GPA2 and GPA3 were 19.6 kDa, 10.6 kDa and 6.7 kDa, respectively. In vitro antioxidant assay, GPA1, GPA2 and GPA3 could scavenge 1, 1-diphenyl2-picrylhydrazyl (DPPH) radical and hydroxyl radical, chelate ferrous ion and reduce ferric ion. The antioxidant activities of GPA3 were stronger than those of GPA1 and GPA2, suggesting that GPA3 has significant potential as a natural antioxidant agent. © 2015 Elsevier Ltd. All rights reserved.
1. Instruction Reactive oxygen species (ROS) involve a series of free radicals such as superoxide anion radicals (1 O2 ), hydroxylradical species (• OH), singletoxygen (O• 2 ) and hydrogen peroxide (H2 O2 ) etc. These active oxygen compounds involving in many cellular metabolic and signaling process, also could damage cellular macromolecules causing lipid peroxidation and nucleic acid and protein alterations. Furthermore, more and more evidence indicated that ROS involved in many disease processes, such as aging, carcinogenesis, atherosclerosis, diabetes and rheumatoid arthritis (Finkel & Holbrook, 2000; Seifried, Anderson, Fisher, & Milner, 2007; Valko et al., 2007). Several lines of evidence from both epidemiological and experimental studies have found that antioxidants, including natural compounds from plants and synthetic compounds, play an important role in scavenging excessive free radicals, to delay or prevent oxidation and maintain the systematic health (Mohammed, 2002; Giles et al., 2003). However, the synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are suspected to be connected with liver damage and
∗ Corresponding authors at: Changchun University of Chinese Medicine, Jingyue Economic Development Zone, 1035, Boshuo Road, Changchun 130117, Jilin Province, China. Tel.: +86 431 86045355; fax: +86431 86045258. E-mail addresses: [email protected] (B. Li), [email protected] (L. Jiao). http://dx.doi.org/10.1016/j.carbpol.2015.03.069 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
carcinogenesis (Grice, 1988; Witschi, 1986). Therefore, more and more interests are aroused to find natural antioxidants without toxicity. Gynostemma pentaphyllum (Thunb.) Markino, a perennial-lianaherb belonging to family Cucurbitaceae and genus Gynostemma Bl., distributed widely in China, particularly in south of the Qinling Moutanis and Yangtze River (Lv et al., 2009). It has been traditionally used as a food and tea in the United States, China and many other Asia countries including Japan. G. pentaphyllum is also a traditional and precious Chinese medicine used for depressing cholesterol levels, promoting the production of body fluid, regulating blood pressure, strengthening the immune system, treating chronic bronchitis and gastritis, and reducing inflammation etc. (Aktan, Henness, Roufogalis, & Ammit, 2003; Circosta, De Pasquale, & Occhiuto, 2005; Huang et al., 2005; Yeo et al., 2008; Lv et al., 2009). G. pentaphyllum contains many active ingredients, such as saponin, amino acid, flavonoids and polysaccharides, which were similarity to those found in ginseng root. Furthermore, G. pentaphyllum is of high value for drug, thus, it was sometimes called “southern ginseng” (Cui, Eneroth, & Bruhn, 1999). Recently, polysaccharides from G. pentaphyllum have been demonstrated to be natural antioxidants. Luo et al. demonstrated that a neutral polysaccharide from G. pentaphyllum exhibited equivalent antioxidant activity in vitro (Wang & Luo, 2007). Jia et al. also isolated a neutral polysaccharide, and demonstrated it had neuroprotective effect via inhibiting oxidative stress. So far, no investigation has been carried out on acidic polysaccharides from G. pentaphyllum that may account
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for antioxidant properties. According to the literature, the acidic polysaccharides from plants had significant antioxidant activities (Sun, Hui, Guo, Pu, & Yan, 2014; He, Yang, Jiao, Tian, & Zhao, 2012; Wu et al., 2013). Hence, we extracted and purified three acidic polysaccharides from G. pentaphyllum, and their antioxidant activity were evaluated by various methods in vitro.
2.5 ml of concentrated H2 SO4 and at 100 ◦ C for 20 min. After cooling to ambient temperature, 40 l m-hydroxydiphenyl was added and incubated for 15 min. The absorbance of reaction mixture was recorded at 525 nm. The total phenols content in GPAs was determined using the Folin–Ciocalteau assay (Sabir & Rocha, 2008). Gallic acid was used in a standard curve and the results were expressed in microgram of gallic acid equivalents per milliliter of dry extract.
2. Materials and methods 2.1. Materials
2.5. Analysis of monosaccharide composition
Gynostemma pentaphyllum (Thunb.) Markino was purchased from a local shop (Changchun, Jilin Province, China). The dried material were crushed and passed through 60 mesh sieve. The powders were stored in a freezer for further experiments. Sepharose CL-6B was purchased from Pharmacia Fine Chemical AB, Uppsala, Sweden. 1, 1-diphenyl-2-picrylhydrazyl (DPPH), thiobarbituric acid (TBA), ethylenediaminetetraacetic acid (EDTA), ferrozine, trichloroacetic acid (TCA), deoxyribose (DR), 2,4,6-tris(2-pyridyl)s-triazine (TPTZ), Vitamin C (Vc) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of analytical grade.
The sample (2 mg) was hydrolyzed with 0.5 ml of 2 M trifluoroacetic acid (TFA) in an ampoule (2 ml). The ampoule was sealed under a nitrogen atmosphere and kept at 120 ◦ C for 1 h, and the excess acid was completely removed by co-distillation with ethanol. After removing the acid, the products of hydrolysis were derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) according to the method in the literature (Honda, Akao, Suzuki, Okuda, & Kakehi Kazuaki Nakamura, 1989). The monosaccharide derivatives were analyzed by Aglient RRLC 1200 SL system (Agilent Technologies, Wilmington, USA), equipping with a DIKMA Inertsil ODS-3 column (4.6 i.d. ×150 mm, 5 m, Dikma, Japan), detected by UV–VIS DAD detector and connected to a Chemstation system. The PMP derivative (20 l) was injected, eluted with 82.0% phosphatebuffered saline (PBS, 0.1 M, pH 7.0) and 18.0% acetonitrile (v/v) at a flow rate of 1.0 ml/min at room temperature. The wavelength for UV detection was 245 nm.
2.2. Preparation for the polysaccharides Air-dried G. pentaphyllum were crushed and extracted with 90% ethanol under reflux extraction at 40 ◦ C to remove pigments and small lipophilic molecules. The residue was further extracted thrice (2 h each) with distilled water at 100 ◦ C. All aqueous extracts were combined, centrifuged under reduced pressure and precipitated by adding of 95% ethanol (4 volumes). The crude polysaccharide precipitate was collected by centrifugation, and dried under reduced pressure after washed with dehydrated alcohol and diethyl ether. The crude polysaccharides was dissolved in distilled water and centrifuged, and then the supernatant was applied to a column of DEAE-cellulose (Cl− ) column (7.0 × 30 cm). This column was initially eluted with distilled water (2000 ml), and an unbound fraction was recovered. The bound polysaccharides were eluted by 0.1 M (GPA1), 0.2 M (GPA2) and 0.5 M (GPA3) NaCl solution, respectively. Each eluent was collected and identified as the acid polysaccharides of G. pentaphyllum (GPAs). Subsequently, Sepharose CL-6B column (2.5 × 100 cm) were used to further purify the fractions obtained by anion-exchange step. 2.3. Homogeneity and molecular weight analysis The polysaccharides (10 mg) was dissolved in distilled water (1 ml), and then the homogeneity and the molecular weight distribution of them were determined by high-performance lipid chromatography (HPLC), which was performed on a SHIMADZU HPLC system (Shimadzu, Japan) fitted with one TSK-G3000 PWXL columns (7.8 mm ID × 30.0 cm/l) and a RID-10A detector. The column was calibrated by molecular mass markers (T-150, 80, 40, 20, 10). The mobile phase was distilled water, and the flow rate was 0.5 ml/min at 40 ◦ C, with 1.6 MPa. 2.4. Analysis of chemical composition Total sugar content was determined by phenol-sulfuric acid method using glucose as standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The content of protein was determined by Bradford’s method (Bradford, 1976). The content of uronic acids was measured by the method of m-hydroxydiphenyl method (Blumenkrantz & Asboe-Hansen, 1973). Briefly, 0.4 ml sample (0.1 g/l) was added 40 l of 4 M sulfamic acid-potassium sulfamate (pH 2.5) and mixed thoroughly, then the mixture was add
2.6. FT-IR spectrum analysis The FT-IR spectra were recorded on SPECORD in a range 4000–400 cm−1 . The samples were analyzed as KBr pellets.
2.7. Assay antioxidant activity in vitro 2.7.1. The DPPH scavenging activity The DPPH quenching ability was measured by the method proposed by Konrath et al. (Yamaguchi, Takamura, Matoba, & Terao, 1998) with slight modifications. 1.5 ml freshly prepared DPPH solution (0.1 mM in methanol) was mixed with 4.5 ml of sample (0.1–0.6 mg/ml). The mixture was incubated at 25 ◦ C for 30 min in the dark, and the absorbance of reaction liquid was measured at 517 nm. Vc was used as the positive control. The percentage scavenging radical was calculated using the following equation: Scavenging rate (%) =
(A0 − A1 ) × 100 A0
where A0 is the absorbance of the control (water instead of sample), and A1 is the absorbance of the sample. 2.7.2. Hydroxyl radical scavenging assay The assay was performed as described by the method of Chung, Osawa, and Kawakishi (1997) with minor changes. 0.2 ml solution of various concentrations of sample (0.25–15 mg/ml) was added to 1.2 ml of 10 mM phosphate buffer (PBS) at pH 7.4 containing 2.67 mM 2-deoxyribose and 0.13 mM EDTA. 0.2 ml of iron ammonium sulfate (0.4 mM) was added. Samples were kept in a water bath at 37 ◦ C, the reaction was started by adding 100 l of ascorbic acid (1.0 mM) and 10 l of H2 O2 (0.1 M). Samples were maintained at 37 ◦ C for 15 min, and then 2 ml of 1% TBA and 2 ml of 2% CCl3 COOH was added to the resulting mixture. Finally the mixture was heated in boiling water for 15 min and cooled by ice water. The absorbance was determined against a blank at 532 nm with spectrophotometer.
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Vc was used as the positive control. The scavenging percentage was calculated as following: Scavenging rate (%) =
(A0 − A1 ) × 100 A0
where A0 is the absorbance of the control (water instead of sample), and A1 is the absorbance of the sample. 2.7.3. Ferrous ion-chelating potential assay The ferrous ion-chelating potential of sample was determined as in Dinis, Maderia, and Almeida (1994). The reaction mixture contained 1.0 ml of sample (0.5–10 mg/ml), 0.1 ml of FeCl2 (2 mM) and 0.2 ml of ferrozine (5 mM). After incubating at 25 ◦ C for 10 min, the absorbance was measured at 562 nm using EDTA as positive control. A lower absorbance indicates a higher chelating ability. The chelating ability was calculated using the following equation: Chelating activity (%) =
(A0 − A1 ) × 100 A0
where A0 is the absorbance of the control (water instead of sample), and A1 is the absorbance of the sample. 2.7.4. Assay of ferric reducing power The ferric reducing ability of plasma (FRAP) assay was carried out according to the procedure of Veenashri and Muralikrishna (Veenashri & Muralikrishna, 2011). Briefly, the FRAP reagent was prepared daily from 300 mmol/l acetate buffer (pH 6.3), 10 mmol/l TPTZ solution (in mmol/l HCl) and 20 mmol/l FeCl3 ·6H2 O solution in a proportion of 10:1:1 (v/v). 0.3 ml of sample was added to 2.7 ml of FRAP reagent. The mixture was incubated at 3 ◦ C for 10 min. The absorbance was measured at 593 nm. Fresh working solutions of FeSO4 were used for calibration. The antioxidant capacity based on the ability to reduce ferric ions of sample was calculated from the linear calibration curve and expressed as mmol FeSO4 equivalents per gram of the texted samples. Increased absorbance of FRAP reflected increased reducing power. 2.8. Statistical analysis All values are expressed as the mean ± SD for three independent experiments. Comparison between any two groups was evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons tests. Differences was considered to be statistically significant if P < 0.05. 3. Result and discussion 3.1. Characteristic of GPA1, GPA2 and GPA3 Three acidic polysaccharides, namely, GPA1, GPA2 and GPA3 were purified from G. pentaphyllum by hot-water extraction, ethanol precipitation, ion-exchange and Sepharose CL-6B gel permeation chromatography. The total sugar of GPA3 was higher (91.34%) than GPA2 (87.57%) and GPA1 (54.55%). The protein contents evaluated in GPA1, GPA2 and GPA3 were 3.75%, 4.83% and 5.53%, respectively. Furthermore, uronic acid detected in the three polysaccharides was 23.04%, 32.79% and 27.01%, respectively, suggesting they belonged to acidic polysaccharides. The total phenols contend was relatively lower in GPA3 (256 mg/100 g) than GPA1 (756 mg/100 g) and GPA2 (950 mg/100 g). The HPLC profile showed that each polysaccharide had a single and symmetrically sharp peak revealing that they were homogeneous polysaccharides. According to the retention time, the average molecular weights of GPA1, GPA2 and GPA3 were estimated to be 19.6 kDa, 10.6 kDa and 6.7 kDa, respectively.
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As summarized in Fig. 1 and Table 1, HPLC analysis showed the difference of the three acid polysaccharides, with the presence of Man, Rha, GlcA, GalA, Glc, Gal, Xyl, Ara, Fuc in the molar ratio of 1:0.1:2.2:1.8:2.2:2.6:0.2:1.9:0.2 for GPA2 (Fig. 1b); and Man, Rha, GlcA, GalA, Glc, Gal, Xyl, Ara, Fuc in the molar ratio of 1:0.6:3.9:0.5:5.5:2.6:0.5:1.2:0.2 for GPA3 (Fig. 1c); also Man, Rha, GlcA, GalA, Glc, Gal, Ara, Fuc in the molar ratio of 1:0.04:1.4:0.9:1.3:2.6:2:0.2 for GPA1 (Fig. 1a), and there was no Xyl detected in GPA1. As shown in the FT-IR spectra of GPA1, GPA2 and GPA3 (Fig. 2), the broad and intense band at 3426 cm−1 was the stretch vibration of O H. The signal at 2938 cm−1 was attributed to the stretch vibration of the C H bond. The band at 1630 cm−1 was assigned to the bending vibration of O H, and the signal at 1541 cm−1 was attributed to the vibration of C O. The band at 1416 cm−1 was assigned to C-H bending vibration. the bands in the region of 1741 cm−1 and 1623 cm−1 were ascribed to C = O stretching and asymmetric C O stretching vibration of carboxyl group, respectively. The characteristic absorption band at 833 cm−1 and 877 cm−1 suggested ␣- and -configurations simultaneously existing in GPA1, GPA2 and GPA3. 3.2. Antioxidant activities in vitro of GPA1, GPA2 and GPA3 3.2.1. DPPH radical scavenging activity The antioxidant properties of the acid polysaccharides from G. pentaphyllum were investigated by DPPH assay. DPPH, a stable Ncentered free radial, has been well used to employ the ability of free-radical scavenging properties or hydrogen donation of compounds and medicine materials (Hatano et al., 1989). As shown in Fig. 3, DPPH scavenging activities of the three polysaccharides increased with increasing concentrations. At the concentration of 1.5 mg/ml, the DPPH radical scavenging rate of GPA1, GPA2 and GPA3 was 84.95%, 84.32% and 93.14%, respectively, with IC50 value of 0.08 mg/ml, 0.06 mg/ml and 0.03 mg/ml, respectively. Among the three polysaccharides, GPA3 possessed stronger scavenging activity than that of GPA2 and GPA1. HPLC analysis revealed that GPA2 and GPA3 contained the same monosaccharide composition of Man, Rha, GlcA, GalA, Glc, Gal, Xyl, Ara and Fuc. However, the ratio of the two monosaccharides greatly differed from one another. There was no Xyl detected in GPA1. In addition, there was a significant difference in the average molecular weights among GPA1 (19.6 kDa), GPA2 (10.6 kDa) and GPA3 (6.7 kDa). Of the three, GPA3 exhibited higher antioxidant activity than GPA1 and GPA2, which probably due to the difference in the ratio of monosaccharide, as well as molecular weight between them. In addition, the total phenolic content in them was determined as GPA3 < GPA1 < GPA2, indicating that the DPPH radical scavenging activities of the three polysaccharides were probably owing to their carboxyl group in hexuronic acid, instead of phenolic compounds (Wang, Mao, & Wei, 2012). 3.2.2. Hydroxyl radicals scavenging activity The hydroxyl radical is accepted to be the most reactive and poisonous free radical, and induce severe damage to adjacent biomolecules as an active initiator for lipids peroxidation (Chance, Sies, & Boveris, 1979; Ke et al., 2009). An accepted reaction system containing Fe3+ -EDTA-H2 O2 -deoxyribose in the aqueous phase was used to generate •OH and measure inhibitory activity of GPA1, GPA2 and GPA3, and the results were summarized in Fig. 4. The three polysaccharides were able to scavenge hydroxyl radical. At the concentrations ranging from 0.25 to 7.5 mg/ml, the hydroxyl radical scavenging ability of GPA3 increased markedly and dosedependently, and reached 97.42% at 7.5 mg/ml. The IC50 values of GPA1, GPA2 and GPA3 were 0.22, 0.21 and 0.20 mg/ml, respectively. Notably, all the IC50 values of GPAs of hydroxyl radicals were higher
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Fig. 1. HPLC analysis of monosaccharide composition of GPA1 (a), GPA2 (b) and GPA3 (c). The three polysaccharides were hydrolyzed, dried, PMP-labeled and analyzed by HPLC as described in the experimental methods.
Table 1 Content of sugar, uronic acid, protein, total phenols and molecular weight of GPA1, GPA2 and GPA3. Fraction
Sugar contenta
Protein contenta
Uronic acid contenta
Total phenols contentb
Mw (kDa)
GPA1 GPA2 GPA3
54.55 85.70 91.34
3.75 4.38 5.53
23.04 32.79 27.01
756 950 256
19.6 10.6 6.7
a b
Percentage weight in the fraction. mg/100 g of extract.
than that of Vc (0.42 mg/ml). Among the three acid polysaccharides, GPA3 exhibited the strongest scavenging activity. 3.2.3. Fe2+ chelating activity of GPA1, GPA2 and GPA3 Iron (Fe) and copper (Cu) ions are important elements for the human body, whereas these metal ions have potentially dangerous. Fe2+ , with high reactivity, can stimulate lipid peroxidation and accelerate lipid peroxidation, thereby driving the chain reaction of lipid peroxidation (Veenashri & Muralikrishna, 2011). So the Fe2+ chelating activity is considered as an important antioxidant property of materials. In this paper, the Fe2+ chelating ability was determined by the reduction of absorbance at 562 nm, the red color
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Fig. 2. FT-IR spectra of GPA1, GPA2 and GPA3.
Fig. 3. DPPH radical scavenging activity of GPA1, GPA2 and GPA3. Each value represents the mean ± SD (n = 3). FT-IR spectra of GPA1, GPA2 and GPA3.
is quantitatively formed by the reaction of ferrozine with Fe2+ . As shown in Fig. 5, GPA3 exhibited higher chelating activity than GPA2 and GPA1. At the concentrations ranging from 0.05 to 1.5 mg/ml, the Fe2+ chelating activity of WGOS-R was measured as 20.78–79.71%, with IC50 value of 0.34 mg/ml. The Fe2+ chelating activity of GPA1 and GPA2 was 54.75% and 67.67% at 2.5 mg/ml, with the IC50 values of 0.85 and 0.43 mg/ml, respectively. The results indicated that GPA3 possessed a much stronger ferrous chelating activity than GPA1 and GPA2. However, the metal chelating activity of the three polysaccharides was significantly lower than that of EDTA, which had the strongest chelating capacity, and achieved 100% at concentration of 0.5 mg/ml. The bioactivities of polysaccharides are closed related to their molecular weight, monosaccharide composition, configuration of glycosidic linkages, position of glycosidic linkages, as well as the degree of substitution on alcohol or phenolic hydroxyl groups (Bohn & BeMiller, 1995). Thus, the structural features and structure–function relationships involved in Fe2+ chelating activities of Phellinus pini polysaccharides needed to be further studied.
Fig. 4. Hydroxyl radical scavenging activity of GPA1, GPA2 and GPA3. Each value represents the mean ± SD (n = 3).
3.2.4. Ferric reducing powder of GPA1, GPA2 and GPA3 FRAP assay is one of the widely simple and convenient method for determining the antioxidants activity (Li, Ren, & Lin, 2002). The ferrous ions from FRAP reagent was reduced by tested antioxidants in the presence of TPTZ, and formed an intense blue Fe2+ -TPTZ complex with an absorption maximum at 593 nm (Benzie & Strain, 1996). In this work, the FRAP value was used to measure the antioxidant activities of acidic polysaccharides of G. pentaphyllum. The FRAP values was estimated by reference to a linear calibration curve (regression equation: y = 0.085x − 0.016, R2 = 0.9991) made from different concentration of FeSO4 solution. The antioxidant capacity based on the ability to reduce ferric ions of sample was calculated from the linear calibration curve and expressed as mM FeSO4 equivalents per gram of samples. The FRAP values of GPA1, GPA2 and GPA3 (2.5 mg/ml) were obtained as 14.37, 11.01 and 15.07 mM FeSO4 /g, respectively.
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Fig. 5. Fe2+ chelating activity of GPA1, GPA2 and GPA3. Each value represents the mean ± SD (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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