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Physicochemical properties and antioxidant activities of polysaccharides from Gynura procumbens leaves by fractional precipitation
International Journal of Biological Macromolecules 95 (2017) 719–724
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Physicochemical properties and antioxidant activities of polysaccharides from Gynura procumbens leaves by fractional precipitation Jing-En Li a,∗ , Wen-Jun Wang a , Guo-Dong Zheng a , Lin-Yan Li b a b
College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang, 330045 Jiangxi Province, China State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 Jiangxi Province, China
a r t i c l e
i n f o
Article history: Received 30 October 2016 Received in revised form 21 November 2016 Accepted 29 November 2016 Available online 2 December 2016 Keywords: Gynura procumbens polysaccharides Physicochemical properties Antioxidant activities
a b s t r a c t Four new polysaccharides (GPP-20, GPP-40, GPP-60 and GPP-80) were fractionated from Gynura procumbens leaves by 20%, 40%, 60% and 80% (v/v) ethanol, successively. Their physicochemical properties including the contents of neutral sugar, uronic acid and protein, as well as the monosaccharide composition were determined. In addition, the antioxidant activities of them were investigated via the reducing power assay and scavenging capacities of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and hydroxyl free radicals, respectively. The results indicated that apart from neutral sugar, they all contained uronic acids and proteins in their structures, which were further proved by the UV–vis and FT-IR spectra. Monosaccharide composition analysis implied that they all belonged to heteropolysaccharides consisted of arabinose, galactose, glucose, xylose and galacturonic acid with different types and ratios. What’s more, GPP-20, GPP-40 and GPP-80 always exhibited better antioxidant activities than GPP-60 among these three antioxidant assays in vitro. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Gynura procumbens is an annual evergreen shrub with a fleshy stem, and widely distributed in Indonesia, Thailand, Malaysia, Vietnam, and China [1]. It belongs to the Asteracea family, and has long been consumed as both vegetables and medicines. As a traditional medicinal plant, the leaves of this plant are usually used for the treatment of many diseases that are caused by oxidative stress, such as inflammation, cancer, diabetes, hypertension, hyperlipidemia and so on [2–8]. And these health benefits have already been supported by the isolation and identification of several possible active chemical constituents from it, including flavonoids, saponins, tannins, and terpenoids [2,9,10]. However, few studies have been carried out about the polysaccharide compounds which might also contribute to the potent biological activities. So far as we know, fractional precipitation with ethanol has been used a lot for the initial purification of aqueous extracts as a simple and rapid method [11]. Importantly, the concentration of ethanol has been proved related to the molecular size, structure feature
and bioactivity of the products [12]. Therefore in our current study, four new polysaccharide fractions from G. procumbens leaves were obtained via this method, and their physicochemical properties and antioxidant activities in vitro were further investigated with the aim of finding new natural antioxidant compounds from G. procumbens leaves. 2. Materials and methods 2.1. Plant materials G. procumbens leaves were collected from the herbal garden of Jiangxi Agricultural university, Jiangxi Province, China. The species was identified by Prof. Guo-dong Zheng, Jiangxi Agricultural university, Nanchang, China. The leaves were dried up and ground into a fine powder using a high speed disintegrator (Model DFY-500, Da De Chinese Traditional Medicine Machine Co., Ltd., Zhejiang, China). The powders were passed through a 20 mesh sieve and stored in a desiccator until analysis. 2.2. Chemicals and reagents
∗ Corresponding author. Present address: Jiangxi Agricultural University, 1101 Zhimin Avenue, Nanchang, 330045 Jiangxi Province, China. E-mail address: [email protected] (J.-E. Li). http://dx.doi.org/10.1016/j.ijbiomac.2016.11.113 0141-8130/© 2016 Elsevier B.V. All rights reserved.
Bovine serum albumin (BSA), sodium tetraborate, mhydroxydiphenyl, 3-phenylphenol, Coomassie brilliant blue
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Fig. 1. Extraction and fractionation procedure of polysaccharide fractions from G. procumbens leaves.
G-250 and ascorbic acid were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Concentrated sulfuric acid, phosphoric acid, salicylic acid, trichloroacetic acid, anhydrous alcohol, acetone, anhydrous ether, 30% hydrogen peroxide (H2 O2 ), phenol, sodium hydroxide, ferrous sulfate (FeSO4 ), ferric chloride (FeCl3 ), sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium ferricyanide (K3 Fe(CN)6 ) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The monosaccharide standards including fucose (Fuc), rhamnose (Rha), arabinose (Ara), mannose (Man), galactose (Gal), glucose (Glc), xylose (Xyl), fructose (Fru), galacturonic acid (GalA), glucuronic acid (GlcA), and 2,2-diphenyl-1-picrylhydrazyl (DPPH), potassium bromide (KBr, spectrum pure grade) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All chemicals and solvents used in this study were of analytical grade. 2.3. Extraction and fractionation procedure Twenty grams of G. procumbens leave dry powders was extracted two times with 500 mL of distilled water at 100 ◦ C for 3 h [13]. The mixture was separated by filtration, and the supernatant was concentrated to one-fifth of the original volume using a rotary evaporator at 55 ◦ C under vacuum. Different volumes of anhydrous ethanol were added to the concentrated supernatant to create a series of concentrations (20%, 40%, 60% and 80%, v/v) of ethanol solution, successively. After the mixture was stored overnight at 4 ◦ C, the precipitate was collected by centrifugation at 4800 rpm for 10 min and then washed with acetone and diethyl ether in turns [14]. The precipitates were then freeze dried, and four different fractions (GPP-20, GPP-40, GPP-60 and GPP-80) were obtained, respectively (Fig. 1). 2.4. Determination of neutral sugar, uronic acid and protein contents The neutral sugar, uronic acid and protein contents of polysaccharides were determined by the phenol-sulfuric acid method
using glucose as standard [15], m-hydroxyphenyl colorimetric method using galacturonic acid as standard [16], and the Bradford assay using bovine serum albumin (BSA) as standard [17], respectively. All samples were analyzed in triplicate. 2.5. Monosaccharide components analysis Monosaccharide composition was determined by a high performance anion exchange chromatography (HPAEC) system equipped with a pulsed amperometric detector (PAD), a Dionex ICS-2500 system and a CarboPacTM PA10 column (2.0 mm × 250 mm) according to the method of Xie et al. [18]. In brief, 10 mg polysaccharide was stirred in 1 mL of 12 M H2 SO4 at room temperature for 30 min. 5 mL of distilled water was added, and then the solution was heated at 100 ◦ C for 2 h. Ten monosaccharide standards (Fuc, Rha, Ara, Man, Gal, Glc, Xyl, Fru, GalA and GlcA) were used to create standard curves, and the monosaccharide composition and molar ratio of samples were calculated. All the samples and standards were filtered through a 0.22 m film before analysis. 2.6. UV-vis spectroscopic analysis The G. procumbens polysaccharides were dissolved in distilled water to a final concentration of 1.0 mg/mL, and then the UV–vis absorption spectra was recorded in the wavelength range of 190–500 nm using a V-5600 UV spectrometer (Shanghai Metash Instruments Co., Ltd., Shanghai, China). 2.7. FT-IR spectroscopic analysis Fourier transform infrared (FT-IR) spectroscopy was recorded on a Nicolet iS5 FT-IR spectrometer (Thermo Electron, Madison, WI, USA) from 4000 to 400 cm−1 at a resolution of 4 cm−1 with 128 co-added scans. The polysaccharide samples were ground with KBr powder, and then pressed into pellets.
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2.8. Determination of antioxidant activities in vitro The polysaccharides (GPP-20, GPP-40, GPP-60, GPP-80) were dissolved with distilled water and prepared into concentrations of 78.1 g/mL, 156.2 g/mL, 312.5 g/mL, 625 g/mL, 1250 g/mL, 2500 g/mL, 5000 g/mL by the two-fold dilution method. The antioxidant activities were investigated by the reducing power, DPPH radical and hydroxyl radical scavenging assays, respectively. 2.8.1. Reducing power assay The reducing power of samples was measured according to the method of Chen et al. [19] with minor modifications. 2 mL of polysaccharide solution was mixed with 2 mL of phosphate buffer (0.2 mol/L, pH 6.6) and 2 mL of K3 Fe(CN)6 (1%, w/v). After through mixing, each solution was incubated at 50 ◦ C for 20 min. After that, the reaction was stopped by adding 2.5 mL of trichloroacetic acid (10%, w/v). The mixture was centrifuged at 3000 rpm for 10 min, and 2 mL of the supernatant was collected and then mixed with 2 mL of distilled water and 1 mL of FeCl3 (0.1%, w/v). After the mixture was kept at room temperature for 10 min, the absorbance was measured at 700 nm. Ascorbic acid was used as positive control.
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2.8.2. DPPH radical scavenging assay DPPH radical scavenging ability was determined according to the method of Zhao et al. [20] with a few modifications. A stock solution of 40 mg/mL of DPPH (in anhydrous ethanol) was freshly prepared. 2 mL of polysaccharide samples were mixed with 2 mL of DPPH stock solution. The mixture was shaken vigorously and incubated at room temperature for 30 min in the dark, and the absorbance was determined at 517 nm. Ascorbic acid and anhydrous ethanol were used as the positive control and blank control. The DPPH radical scavenging rate was calculated according to the following equation:
DPPH radical scavenging rate (%) = [1 − (Ai − Aj )/A0 ] × 100
(1)
where Ai is the absorbance of sample or control, Aj is the background absorbance (anhydrous ethanol instead of DPPH stock solution), and A0 is the absorbance of blank (anhydrous ethanol instead of sample).
Fig. 2. (a) HPAEC profile of monosaccharide standards (1-Fuc, 2-Rha, 3-Ara, 4-Gal, 5-Glc, 6-Xyl, 7-Man, 8-Fru, 9-GalA, 10-GlcA). (b–e) HPAEC profiles of GPP-20, GPP-40, GPP-60 and GPP-80.
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Table 1 The yield and physicochemical properties of polysaccharides from G. procumbens leaves. Samples
GPP-20
GPP-40
GPP-60
GPP-80
Yield (w%)a Neutral sugar content (w%)a Uronic acid content (w%)a Protein content (w%)a
14.00 ± 0.12 16.98 ± 0.00 37.04 ± 1.17 4.91 ± 0.01
32.80 ± 0.31 19.32 ± 2.23 47.28 ± 3.06 3.30 ± 0.02
26.43 ± 0.27 27.48 ± 7.48 64.87 ± 1.69 1.07 ± 0.04
7.96 ± 0.05 14.81 ± 0.54 13.85 ± 0.22 3.13 ± 0.02
Monosaccahride composition (mol%)b Ara Gal Glc Xyl GalA
0.11 ± 0.01 0.37 ± 0.01 5.69 ± 0.23 0.08 ± 0.00 13.50 ± 1.32
0.13 ± 0.00 1.67 ± 0.17 0.49 ± 0.25 4.90 ± 0.73 20.74 ± 2.14
0.12 ± 0.03 1.98 ± 0.05 0.06 ± 0.01 15.97 ± 1.09 29.27 ± 2.68
0.27 ± 0.00 3.69 ± 0.024 0.39 ± 0.03 0.05 ± 0.01 4.48 ± 0.12
a b
Weight percentage. Molar percentage.
2.8.3. Hydroxyl radical scavenging assay Hydroxyl radical scavenging assay was performed with the method based on Pu et al. [21]. Briefly, 1 mL of FeSO4 (6 mmol/L) and 1 mL of salicylic acid-ethanol (6 mmol/L) were added to 1 mL of polysaccharide sample, and 1 mL of H2 O2 (6 mmol/L) was added finally to start the reactions. Then the reaction solutions were incubated at 37 ◦ C for 30 min, and the absorbance was measured at 510 nm. The hydroxyl radical scavenging rate was calculated as follows: Hydroxyl radical scavenging rate (%) = [1 − (A1 − A2 )/A0 ] × 100
(2)
where A0 is the absorbance of blank (water instead of sample) and A1 is the absorbance of sample, A2 is the background absorbance (water instead of H2 O2 ). 2.9. Statistical analysis Fig. 3. UV–Vis spectra of polysaccharides from G. procumbens leaves.
All the experiments were carried out in triplicate. The data were analyzed with SPSS software (version 11.0 for Windows, SPSS Inc., Chicago, IL, USA) and recorded as means ± standard deviations (SD). 3. Results and discussion 3.1. Yields and physicochemical properties The yields and physicochemical properties of each polysaccharide fraction from G. procumbens leaves were listed in Table 1. The yields decreased in the following order: GPP-40 > GPP-60 > GPP20 > GPP-80, which suggested that GPP-40 and GPP-60 were the main components of G. procumbens polysaccharides. In addition, both uronic acid and protein were found in all fractions, which indicated that they were all belonged to acid glycoprotein compound. Among them, GPP-60 contained the highest contents of neutral sugar (27.48 ± 7.48%) and uronic acid (64.87 ± 1.69%), as well as the minimum amount of protein (1.07 ± 0.04%). HPAEC-PAD was applied to analyze the monosaccharide composition, and the results indicated that the polysaccharides from G. procumbens leaves were mainly consisted of Ara, Gal, Glc, Xyl and GalA (Fig. 2), and their molar ratios were shown in Table 1. Obviously, more Glc was found in GPP-20, and abundant Xyl was found in GPP-40 and GPP-60. At the same time, GlaA were found rich in all the fractions except GPP-80. 3.2. UV-vis spectra characteristics As shown in Fig. 3, all the samples displayed a maximum absorption peak around 200 nm which belonged to the polysaccharides, as well as some weak absorption peaks between 250 nm and 350 nm
Fig. 4. FT-IR spectra of polysaccharides from G. procumbens leaves.
which indicated the existence of small amount of proteins and nucleic acids [22,23]. 3.3. FT-IR spectra characteristics The FT-IR spectra of polysaccharide samples were shown in Fig. 4. The broad and intense absorption peak around 3439 cm−1 was attributed to the O H stretching vibration, and the weak band near 2936 cm−1 was due to the stretching vibration of C H including CH, CH2 and CH3 in the sugar ring. These two absorption
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Fig. 5. The reducing power of polysaccharides from G. procumbens leaves. Fig. 7. Hydroxyl radical scavenging activity of polysaccharides from G. procumbens leaves.
the DPPH radical scavenging activity and the physicochemical properties of samples. At 5000 g/mL, the scavenging rates of GPP-20, GPP-40, GPP-60 and GPP-80 were 57.1%, 55.1%, 40.8% and 58.2%, respectively.
Fig. 6. DPPH radical scavenging activity of polysaccharides from G. procumbens leaves.
bands were both belonged to the characteristic functional groups of polysaccharides. The absorption peak at approximately 1405 cm−1 could be attributed to the deforming vibration of the C H bond. In addition, the absorption region around 1200 cm−1 –1000 cm−1 were belonged to the stretching vibrations of C O C or C O H which could explain the existence of pyranoid ring conformation in the polysaccharides [24,25]. And the peaks of 1744.4 and 1613.9 cm−1 were represented the esterified and free carboxyl groups, which also proved the existence of uronic acid in the structure [26]. 3.4. Antioxidant activities in vitro 3.4.1. Reducing power The reducing power assay is based on the fact that the polysaccharides could result in the reduction of Fe3+ to Fe2+ by donating an electron. The reducing power was determined at 700 nm and the absorbance values were shown in Fig. 5. Obviously the reducing power of all the samples increased with increasing concentrations. Among the four fractions, GPP-20, GPP-40 and GPP-80 exhibited higher reducing power than GPP-60. At 5000 g/mL, the absorbance of GPP-60 and ascorbic acid were 0.881 and 3.000, which indicated that the reducing power of GPP-60 was only 29.4% of that of ascorbic acid. 3.4.2. DPPH radical scavenging activity The DPPH assay is a very common spectrophotometric method to determine the antioxidant activity of natural products. Fig. 6 indicated that the DPPH radical scavenging activities of GPP-20, GPP-40 and GPP-80 were very close and entangled at each concentration, except GPP-60. And their half inhibition concentration (IC50 ) decreased in the order of GPP-80 (2070.7 g/mL) < GPP-20 (2728.2 g/mL) < GPP-40 (2746.1 g/mL) < GPP-60 (6499.8 g/mL). By comparing with the results in Table 1, no direct correlation could be observed between
3.4.3. Hydroxyl radical scavenging activity Hydroxyl radicals are considered the most harmful free radicals among the reactive oxygen species (ROS), and can damage various macromolecules of human body, such as carbohydrates, nucleic acid, lipids and amino acids [27,28]. Therefore, removing hydroxyl radicals is quite important for the antioxidant defense in living cell systems. As illustrated in Fig. 7, the polysaccharides exhibited strong scavenging activities against hydroxyl radicals in a concentration-dependent manner. At 5000 g/mL, the hydroxyl radical scavenging activities of GPP-20, GPP-40, GPP-60, and GPP80 were 60.7%, 83.8%, 56.6% and 73.6%, with their IC50 values of 3041.8 g/mL, 2568.7 g/mL, 4665.3 g/mL and 2503.8 g/mL, respectively. The results were quite similar to the hydroxyl radical scavenging activity of polysaccharides from peony seed dreg reported by Shi et al. [29].
4. Conclusions In summary, four polysaccharides (GPP-20, GPP-40, GPP-60 and GPP-80) were isolated from G. procumbens leaves via ethanol fractional precipitation method. Their physiochemical properties and antioxidant activities were preliminary investigated. The results showed that the four polysaccharides were all belonged to heteropolysaccharides that consisted of Ara, Gal, Glc, Xyl and GalA in different molar ratios. The UV–vis spectra showed a strong characteristic absorption peak of carbohydrates around 200 nm and some weak absorption peaks of proteins and nucleic acids between 250 and 350 nm. FTIR spectra also implied the typical vibrations of carbohydrates at 3439, 2936, 1405 cm−1 , and the typical vibrations of uronic acids around 1744 cm−1 and 1614 cm−1 . Antioxidant activity assays indicated that the four polysaccharide fractions all exhibited strong reducing power and scavenging capacities on DPPH and hydroxyl radicals in a concentrationdependent manner. Among them, GPP-60 always exhibited the weakest antioxidant activity, while GPP-20, GPP-40 and GPP-80 showed very close scavenging activities which were better than that of GPP-60. However, no direct correlation could be found yet between the antioxidant activity and their physicochemical properties. Therefore, further study will be performed to purify the four polysaccharide fractions and reveal the relationship between their structure characteristics and antioxidant activities.
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Conflicts of interest The authors declare no conflict of interest. Acknowledgments This research was supported financially by Science and Technology Research Projects of the Education Department of Jiangxi Province (Grant No. GJJ150433) and Open Project Program of State Key Laboratory of Food Science and Technology, Nanchang University (Grant No. SKLF-KF-201601). The authors would also like to thank Dr. Hui Zhang from the College of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, for his helpful suggestions and assistance. References [1] J. Kim, C.W. Lee, E.K. Kim, S.J. Lee, N.H. Park, H.S. Kim, H.K. Kim, K. Char, Y.P. Jang, J.W. Kim, Inhibition effect of Gynura procumbens extract on UV-B-induced matrix-metalloproteinase expression in human dermal fibroblasts, J. Ethnopharmacol. 137 (2011) 427–433. [2] G.A. Akowuah, A. Sadikun, M. Ahmad, Flavonoid identification and hypoglycaemic studies of the butanol fraction from Gynura procumbens, Pharm. Biol. 40 (2008) 405–410. [3] M.N. Iskander, Y. Song, I.M. Coupar, W. Jiratchariyakul, Antiinflammatory screening of the medicinal plant Gynura procumbens, Plant Food Hum. Nutr. 57 (2002) 233–244. [4] M.J. Kim, H.J. Lee, S. Wiryowidagdo, H.K. Kim, Antihypertensive effects of Gynura procumbens extract in spontaneously hypertensive rats, J. Med. Food. 9 (2006) 587–590. [5] S.K. Lam, A. Idris, Z.A.A. Bakar, R. Ismail, Gynura procumbens and blood pressure in the rats: preliminary study, Asia Pac. J. Pharmacol. 13 (1998) 14–15. [6] H.W. Lee, P. Hakim, A. Rabu, H.A. Sani, Antidiabetic effect of Gynura procumbens leaves extracts involve modulation of hepatic carbohydrate metabolism in streptozotocin-induced diabetic rats, J. Med. Plant Res. 6 (2012) 796–812. [7] L.M. Perry, Medicinal Plants of East and Southeast Asia: Attributed Properties and Uses, Massachusetts Institute of Technology Press, Cambridge, 1980. [8] X.F. Zhang, B.K.H. Tan, Effects of an ethanolic extract of Gynura procumbens on serum glucose, cholesterol and triglyceride levels in normal and streptozotocin-induced diabetic rats, Singapore Med. J. 41 (2000) 9–13. [9] G.F. Deng, X. Lin, X.R. Xu, L.L. Gao, J.F. Xie, H.B. Li, Antioxidant capacities and total phenolic contents of 56 vegetables, J. Funct. Foods 5 (2013) 260–266. [10] U.K.S. Khanam, S. Oba, E. Yanase, Y. Murakami, Phenolic acids, flavonoids and total antioxidant capacity of selected leafy vegetables, J. Funct. Foods 4 (2012) 979–987. [11] G.Y. Koh, G.X. Chou, Z.J. Liu, Purification of a water extract of Chinese sweet tea plant (Rubus suavissimus S. Lee) by alcohol precipitation, J. Agr. Food Chem. 57 (2009) 5000–5006.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Physicochemical properties and antioxidant activities of polysaccharides from Gynura procumbens leaves by fractional precipitation Jing-En Li a,∗ , Wen-Jun Wang a , Guo-Dong Zheng a , Lin-Yan Li b a b
College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang, 330045 Jiangxi Province, China State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 Jiangxi Province, China
a r t i c l e
i n f o
Article history: Received 30 October 2016 Received in revised form 21 November 2016 Accepted 29 November 2016 Available online 2 December 2016 Keywords: Gynura procumbens polysaccharides Physicochemical properties Antioxidant activities
a b s t r a c t Four new polysaccharides (GPP-20, GPP-40, GPP-60 and GPP-80) were fractionated from Gynura procumbens leaves by 20%, 40%, 60% and 80% (v/v) ethanol, successively. Their physicochemical properties including the contents of neutral sugar, uronic acid and protein, as well as the monosaccharide composition were determined. In addition, the antioxidant activities of them were investigated via the reducing power assay and scavenging capacities of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and hydroxyl free radicals, respectively. The results indicated that apart from neutral sugar, they all contained uronic acids and proteins in their structures, which were further proved by the UV–vis and FT-IR spectra. Monosaccharide composition analysis implied that they all belonged to heteropolysaccharides consisted of arabinose, galactose, glucose, xylose and galacturonic acid with different types and ratios. What’s more, GPP-20, GPP-40 and GPP-80 always exhibited better antioxidant activities than GPP-60 among these three antioxidant assays in vitro. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Gynura procumbens is an annual evergreen shrub with a fleshy stem, and widely distributed in Indonesia, Thailand, Malaysia, Vietnam, and China [1]. It belongs to the Asteracea family, and has long been consumed as both vegetables and medicines. As a traditional medicinal plant, the leaves of this plant are usually used for the treatment of many diseases that are caused by oxidative stress, such as inflammation, cancer, diabetes, hypertension, hyperlipidemia and so on [2–8]. And these health benefits have already been supported by the isolation and identification of several possible active chemical constituents from it, including flavonoids, saponins, tannins, and terpenoids [2,9,10]. However, few studies have been carried out about the polysaccharide compounds which might also contribute to the potent biological activities. So far as we know, fractional precipitation with ethanol has been used a lot for the initial purification of aqueous extracts as a simple and rapid method [11]. Importantly, the concentration of ethanol has been proved related to the molecular size, structure feature
and bioactivity of the products [12]. Therefore in our current study, four new polysaccharide fractions from G. procumbens leaves were obtained via this method, and their physicochemical properties and antioxidant activities in vitro were further investigated with the aim of finding new natural antioxidant compounds from G. procumbens leaves. 2. Materials and methods 2.1. Plant materials G. procumbens leaves were collected from the herbal garden of Jiangxi Agricultural university, Jiangxi Province, China. The species was identified by Prof. Guo-dong Zheng, Jiangxi Agricultural university, Nanchang, China. The leaves were dried up and ground into a fine powder using a high speed disintegrator (Model DFY-500, Da De Chinese Traditional Medicine Machine Co., Ltd., Zhejiang, China). The powders were passed through a 20 mesh sieve and stored in a desiccator until analysis. 2.2. Chemicals and reagents
∗ Corresponding author. Present address: Jiangxi Agricultural University, 1101 Zhimin Avenue, Nanchang, 330045 Jiangxi Province, China. E-mail address: [email protected] (J.-E. Li). http://dx.doi.org/10.1016/j.ijbiomac.2016.11.113 0141-8130/© 2016 Elsevier B.V. All rights reserved.
Bovine serum albumin (BSA), sodium tetraborate, mhydroxydiphenyl, 3-phenylphenol, Coomassie brilliant blue
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Fig. 1. Extraction and fractionation procedure of polysaccharide fractions from G. procumbens leaves.
G-250 and ascorbic acid were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Concentrated sulfuric acid, phosphoric acid, salicylic acid, trichloroacetic acid, anhydrous alcohol, acetone, anhydrous ether, 30% hydrogen peroxide (H2 O2 ), phenol, sodium hydroxide, ferrous sulfate (FeSO4 ), ferric chloride (FeCl3 ), sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium ferricyanide (K3 Fe(CN)6 ) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The monosaccharide standards including fucose (Fuc), rhamnose (Rha), arabinose (Ara), mannose (Man), galactose (Gal), glucose (Glc), xylose (Xyl), fructose (Fru), galacturonic acid (GalA), glucuronic acid (GlcA), and 2,2-diphenyl-1-picrylhydrazyl (DPPH), potassium bromide (KBr, spectrum pure grade) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All chemicals and solvents used in this study were of analytical grade. 2.3. Extraction and fractionation procedure Twenty grams of G. procumbens leave dry powders was extracted two times with 500 mL of distilled water at 100 ◦ C for 3 h [13]. The mixture was separated by filtration, and the supernatant was concentrated to one-fifth of the original volume using a rotary evaporator at 55 ◦ C under vacuum. Different volumes of anhydrous ethanol were added to the concentrated supernatant to create a series of concentrations (20%, 40%, 60% and 80%, v/v) of ethanol solution, successively. After the mixture was stored overnight at 4 ◦ C, the precipitate was collected by centrifugation at 4800 rpm for 10 min and then washed with acetone and diethyl ether in turns [14]. The precipitates were then freeze dried, and four different fractions (GPP-20, GPP-40, GPP-60 and GPP-80) were obtained, respectively (Fig. 1). 2.4. Determination of neutral sugar, uronic acid and protein contents The neutral sugar, uronic acid and protein contents of polysaccharides were determined by the phenol-sulfuric acid method
using glucose as standard [15], m-hydroxyphenyl colorimetric method using galacturonic acid as standard [16], and the Bradford assay using bovine serum albumin (BSA) as standard [17], respectively. All samples were analyzed in triplicate. 2.5. Monosaccharide components analysis Monosaccharide composition was determined by a high performance anion exchange chromatography (HPAEC) system equipped with a pulsed amperometric detector (PAD), a Dionex ICS-2500 system and a CarboPacTM PA10 column (2.0 mm × 250 mm) according to the method of Xie et al. [18]. In brief, 10 mg polysaccharide was stirred in 1 mL of 12 M H2 SO4 at room temperature for 30 min. 5 mL of distilled water was added, and then the solution was heated at 100 ◦ C for 2 h. Ten monosaccharide standards (Fuc, Rha, Ara, Man, Gal, Glc, Xyl, Fru, GalA and GlcA) were used to create standard curves, and the monosaccharide composition and molar ratio of samples were calculated. All the samples and standards were filtered through a 0.22 m film before analysis. 2.6. UV-vis spectroscopic analysis The G. procumbens polysaccharides were dissolved in distilled water to a final concentration of 1.0 mg/mL, and then the UV–vis absorption spectra was recorded in the wavelength range of 190–500 nm using a V-5600 UV spectrometer (Shanghai Metash Instruments Co., Ltd., Shanghai, China). 2.7. FT-IR spectroscopic analysis Fourier transform infrared (FT-IR) spectroscopy was recorded on a Nicolet iS5 FT-IR spectrometer (Thermo Electron, Madison, WI, USA) from 4000 to 400 cm−1 at a resolution of 4 cm−1 with 128 co-added scans. The polysaccharide samples were ground with KBr powder, and then pressed into pellets.
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2.8. Determination of antioxidant activities in vitro The polysaccharides (GPP-20, GPP-40, GPP-60, GPP-80) were dissolved with distilled water and prepared into concentrations of 78.1 g/mL, 156.2 g/mL, 312.5 g/mL, 625 g/mL, 1250 g/mL, 2500 g/mL, 5000 g/mL by the two-fold dilution method. The antioxidant activities were investigated by the reducing power, DPPH radical and hydroxyl radical scavenging assays, respectively. 2.8.1. Reducing power assay The reducing power of samples was measured according to the method of Chen et al. [19] with minor modifications. 2 mL of polysaccharide solution was mixed with 2 mL of phosphate buffer (0.2 mol/L, pH 6.6) and 2 mL of K3 Fe(CN)6 (1%, w/v). After through mixing, each solution was incubated at 50 ◦ C for 20 min. After that, the reaction was stopped by adding 2.5 mL of trichloroacetic acid (10%, w/v). The mixture was centrifuged at 3000 rpm for 10 min, and 2 mL of the supernatant was collected and then mixed with 2 mL of distilled water and 1 mL of FeCl3 (0.1%, w/v). After the mixture was kept at room temperature for 10 min, the absorbance was measured at 700 nm. Ascorbic acid was used as positive control.
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2.8.2. DPPH radical scavenging assay DPPH radical scavenging ability was determined according to the method of Zhao et al. [20] with a few modifications. A stock solution of 40 mg/mL of DPPH (in anhydrous ethanol) was freshly prepared. 2 mL of polysaccharide samples were mixed with 2 mL of DPPH stock solution. The mixture was shaken vigorously and incubated at room temperature for 30 min in the dark, and the absorbance was determined at 517 nm. Ascorbic acid and anhydrous ethanol were used as the positive control and blank control. The DPPH radical scavenging rate was calculated according to the following equation:
DPPH radical scavenging rate (%) = [1 − (Ai − Aj )/A0 ] × 100
(1)
where Ai is the absorbance of sample or control, Aj is the background absorbance (anhydrous ethanol instead of DPPH stock solution), and A0 is the absorbance of blank (anhydrous ethanol instead of sample).
Fig. 2. (a) HPAEC profile of monosaccharide standards (1-Fuc, 2-Rha, 3-Ara, 4-Gal, 5-Glc, 6-Xyl, 7-Man, 8-Fru, 9-GalA, 10-GlcA). (b–e) HPAEC profiles of GPP-20, GPP-40, GPP-60 and GPP-80.
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Table 1 The yield and physicochemical properties of polysaccharides from G. procumbens leaves. Samples
GPP-20
GPP-40
GPP-60
GPP-80
Yield (w%)a Neutral sugar content (w%)a Uronic acid content (w%)a Protein content (w%)a
14.00 ± 0.12 16.98 ± 0.00 37.04 ± 1.17 4.91 ± 0.01
32.80 ± 0.31 19.32 ± 2.23 47.28 ± 3.06 3.30 ± 0.02
26.43 ± 0.27 27.48 ± 7.48 64.87 ± 1.69 1.07 ± 0.04
7.96 ± 0.05 14.81 ± 0.54 13.85 ± 0.22 3.13 ± 0.02
Monosaccahride composition (mol%)b Ara Gal Glc Xyl GalA
0.11 ± 0.01 0.37 ± 0.01 5.69 ± 0.23 0.08 ± 0.00 13.50 ± 1.32
0.13 ± 0.00 1.67 ± 0.17 0.49 ± 0.25 4.90 ± 0.73 20.74 ± 2.14
0.12 ± 0.03 1.98 ± 0.05 0.06 ± 0.01 15.97 ± 1.09 29.27 ± 2.68
0.27 ± 0.00 3.69 ± 0.024 0.39 ± 0.03 0.05 ± 0.01 4.48 ± 0.12
a b
Weight percentage. Molar percentage.
2.8.3. Hydroxyl radical scavenging assay Hydroxyl radical scavenging assay was performed with the method based on Pu et al. [21]. Briefly, 1 mL of FeSO4 (6 mmol/L) and 1 mL of salicylic acid-ethanol (6 mmol/L) were added to 1 mL of polysaccharide sample, and 1 mL of H2 O2 (6 mmol/L) was added finally to start the reactions. Then the reaction solutions were incubated at 37 ◦ C for 30 min, and the absorbance was measured at 510 nm. The hydroxyl radical scavenging rate was calculated as follows: Hydroxyl radical scavenging rate (%) = [1 − (A1 − A2 )/A0 ] × 100
(2)
where A0 is the absorbance of blank (water instead of sample) and A1 is the absorbance of sample, A2 is the background absorbance (water instead of H2 O2 ). 2.9. Statistical analysis Fig. 3. UV–Vis spectra of polysaccharides from G. procumbens leaves.
All the experiments were carried out in triplicate. The data were analyzed with SPSS software (version 11.0 for Windows, SPSS Inc., Chicago, IL, USA) and recorded as means ± standard deviations (SD). 3. Results and discussion 3.1. Yields and physicochemical properties The yields and physicochemical properties of each polysaccharide fraction from G. procumbens leaves were listed in Table 1. The yields decreased in the following order: GPP-40 > GPP-60 > GPP20 > GPP-80, which suggested that GPP-40 and GPP-60 were the main components of G. procumbens polysaccharides. In addition, both uronic acid and protein were found in all fractions, which indicated that they were all belonged to acid glycoprotein compound. Among them, GPP-60 contained the highest contents of neutral sugar (27.48 ± 7.48%) and uronic acid (64.87 ± 1.69%), as well as the minimum amount of protein (1.07 ± 0.04%). HPAEC-PAD was applied to analyze the monosaccharide composition, and the results indicated that the polysaccharides from G. procumbens leaves were mainly consisted of Ara, Gal, Glc, Xyl and GalA (Fig. 2), and their molar ratios were shown in Table 1. Obviously, more Glc was found in GPP-20, and abundant Xyl was found in GPP-40 and GPP-60. At the same time, GlaA were found rich in all the fractions except GPP-80. 3.2. UV-vis spectra characteristics As shown in Fig. 3, all the samples displayed a maximum absorption peak around 200 nm which belonged to the polysaccharides, as well as some weak absorption peaks between 250 nm and 350 nm
Fig. 4. FT-IR spectra of polysaccharides from G. procumbens leaves.
which indicated the existence of small amount of proteins and nucleic acids [22,23]. 3.3. FT-IR spectra characteristics The FT-IR spectra of polysaccharide samples were shown in Fig. 4. The broad and intense absorption peak around 3439 cm−1 was attributed to the O H stretching vibration, and the weak band near 2936 cm−1 was due to the stretching vibration of C H including CH, CH2 and CH3 in the sugar ring. These two absorption
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Fig. 5. The reducing power of polysaccharides from G. procumbens leaves. Fig. 7. Hydroxyl radical scavenging activity of polysaccharides from G. procumbens leaves.
the DPPH radical scavenging activity and the physicochemical properties of samples. At 5000 g/mL, the scavenging rates of GPP-20, GPP-40, GPP-60 and GPP-80 were 57.1%, 55.1%, 40.8% and 58.2%, respectively.
Fig. 6. DPPH radical scavenging activity of polysaccharides from G. procumbens leaves.
bands were both belonged to the characteristic functional groups of polysaccharides. The absorption peak at approximately 1405 cm−1 could be attributed to the deforming vibration of the C H bond. In addition, the absorption region around 1200 cm−1 –1000 cm−1 were belonged to the stretching vibrations of C O C or C O H which could explain the existence of pyranoid ring conformation in the polysaccharides [24,25]. And the peaks of 1744.4 and 1613.9 cm−1 were represented the esterified and free carboxyl groups, which also proved the existence of uronic acid in the structure [26]. 3.4. Antioxidant activities in vitro 3.4.1. Reducing power The reducing power assay is based on the fact that the polysaccharides could result in the reduction of Fe3+ to Fe2+ by donating an electron. The reducing power was determined at 700 nm and the absorbance values were shown in Fig. 5. Obviously the reducing power of all the samples increased with increasing concentrations. Among the four fractions, GPP-20, GPP-40 and GPP-80 exhibited higher reducing power than GPP-60. At 5000 g/mL, the absorbance of GPP-60 and ascorbic acid were 0.881 and 3.000, which indicated that the reducing power of GPP-60 was only 29.4% of that of ascorbic acid. 3.4.2. DPPH radical scavenging activity The DPPH assay is a very common spectrophotometric method to determine the antioxidant activity of natural products. Fig. 6 indicated that the DPPH radical scavenging activities of GPP-20, GPP-40 and GPP-80 were very close and entangled at each concentration, except GPP-60. And their half inhibition concentration (IC50 ) decreased in the order of GPP-80 (2070.7 g/mL) < GPP-20 (2728.2 g/mL) < GPP-40 (2746.1 g/mL) < GPP-60 (6499.8 g/mL). By comparing with the results in Table 1, no direct correlation could be observed between
3.4.3. Hydroxyl radical scavenging activity Hydroxyl radicals are considered the most harmful free radicals among the reactive oxygen species (ROS), and can damage various macromolecules of human body, such as carbohydrates, nucleic acid, lipids and amino acids [27,28]. Therefore, removing hydroxyl radicals is quite important for the antioxidant defense in living cell systems. As illustrated in Fig. 7, the polysaccharides exhibited strong scavenging activities against hydroxyl radicals in a concentration-dependent manner. At 5000 g/mL, the hydroxyl radical scavenging activities of GPP-20, GPP-40, GPP-60, and GPP80 were 60.7%, 83.8%, 56.6% and 73.6%, with their IC50 values of 3041.8 g/mL, 2568.7 g/mL, 4665.3 g/mL and 2503.8 g/mL, respectively. The results were quite similar to the hydroxyl radical scavenging activity of polysaccharides from peony seed dreg reported by Shi et al. [29].
4. Conclusions In summary, four polysaccharides (GPP-20, GPP-40, GPP-60 and GPP-80) were isolated from G. procumbens leaves via ethanol fractional precipitation method. Their physiochemical properties and antioxidant activities were preliminary investigated. The results showed that the four polysaccharides were all belonged to heteropolysaccharides that consisted of Ara, Gal, Glc, Xyl and GalA in different molar ratios. The UV–vis spectra showed a strong characteristic absorption peak of carbohydrates around 200 nm and some weak absorption peaks of proteins and nucleic acids between 250 and 350 nm. FTIR spectra also implied the typical vibrations of carbohydrates at 3439, 2936, 1405 cm−1 , and the typical vibrations of uronic acids around 1744 cm−1 and 1614 cm−1 . Antioxidant activity assays indicated that the four polysaccharide fractions all exhibited strong reducing power and scavenging capacities on DPPH and hydroxyl radicals in a concentrationdependent manner. Among them, GPP-60 always exhibited the weakest antioxidant activity, while GPP-20, GPP-40 and GPP-80 showed very close scavenging activities which were better than that of GPP-60. However, no direct correlation could be found yet between the antioxidant activity and their physicochemical properties. Therefore, further study will be performed to purify the four polysaccharide fractions and reveal the relationship between their structure characteristics and antioxidant activities.
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Conflicts of interest The authors declare no conflict of interest. Acknowledgments This research was supported financially by Science and Technology Research Projects of the Education Department of Jiangxi Province (Grant No. GJJ150433) and Open Project Program of State Key Laboratory of Food Science and Technology, Nanchang University (Grant No. SKLF-KF-201601). The authors would also like to thank Dr. Hui Zhang from the College of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, for his helpful suggestions and assistance. References [1] J. Kim, C.W. Lee, E.K. Kim, S.J. Lee, N.H. Park, H.S. Kim, H.K. Kim, K. Char, Y.P. Jang, J.W. Kim, Inhibition effect of Gynura procumbens extract on UV-B-induced matrix-metalloproteinase expression in human dermal fibroblasts, J. Ethnopharmacol. 137 (2011) 427–433. [2] G.A. Akowuah, A. Sadikun, M. Ahmad, Flavonoid identification and hypoglycaemic studies of the butanol fraction from Gynura procumbens, Pharm. Biol. 40 (2008) 405–410. [3] M.N. Iskander, Y. Song, I.M. Coupar, W. Jiratchariyakul, Antiinflammatory screening of the medicinal plant Gynura procumbens, Plant Food Hum. Nutr. 57 (2002) 233–244. [4] M.J. Kim, H.J. Lee, S. Wiryowidagdo, H.K. Kim, Antihypertensive effects of Gynura procumbens extract in spontaneously hypertensive rats, J. Med. Food. 9 (2006) 587–590. [5] S.K. Lam, A. Idris, Z.A.A. Bakar, R. Ismail, Gynura procumbens and blood pressure in the rats: preliminary study, Asia Pac. J. Pharmacol. 13 (1998) 14–15. [6] H.W. Lee, P. Hakim, A. Rabu, H.A. Sani, Antidiabetic effect of Gynura procumbens leaves extracts involve modulation of hepatic carbohydrate metabolism in streptozotocin-induced diabetic rats, J. Med. Plant Res. 6 (2012) 796–812. [7] L.M. Perry, Medicinal Plants of East and Southeast Asia: Attributed Properties and Uses, Massachusetts Institute of Technology Press, Cambridge, 1980. [8] X.F. Zhang, B.K.H. Tan, Effects of an ethanolic extract of Gynura procumbens on serum glucose, cholesterol and triglyceride levels in normal and streptozotocin-induced diabetic rats, Singapore Med. J. 41 (2000) 9–13. [9] G.F. Deng, X. Lin, X.R. Xu, L.L. Gao, J.F. Xie, H.B. Li, Antioxidant capacities and total phenolic contents of 56 vegetables, J. Funct. Foods 5 (2013) 260–266. [10] U.K.S. Khanam, S. Oba, E. Yanase, Y. Murakami, Phenolic acids, flavonoids and total antioxidant capacity of selected leafy vegetables, J. Funct. Foods 4 (2012) 979–987. [11] G.Y. Koh, G.X. Chou, Z.J. Liu, Purification of a water extract of Chinese sweet tea plant (Rubus suavissimus S. Lee) by alcohol precipitation, J. Agr. Food Chem. 57 (2009) 5000–5006.
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