Extraction, purification, characterization and antioxidant activities of polysaccharides from Ramaria botrytis (Pers.) Ricken
© The Author(s) 2017
Received: 15 July 2016
Accepted: 9 March 2017
Published: 16 March 2017
Ramaria botrytis (Pers.) Ricken, a member of the family Clavariaceae, has been widely prescribed for anti-aging and improving immunity. To extract and purify the polysaccharides, the main constituent of the fruiting-body, from R. botrytis and explore antioxidant activities was great significant.
Ramaria botrytis polysaccharides (RBP) was extracted with water at 88.47 °C for 1.42 h with a solution to sample ratio of 10.94 mL g−1 employing response surface methodology. Four purified fractions, RBP-1, RBP-2, RBP-3, and RBP-4, were obtained from column chromatography of DEAE-52 and Sephadex G-100. Among these four purified fractions, RBP-1, RBP-2, RBP-4 were mainly composed of glucose, while RBP-3 contained 41.36% mannose and 28.96% glucose. The molecular weights of RBP-1, RBP-2, RBP-3 and RBP-4 were 6.48, 36.12, 96.72 and 8.34 kDa, respectively. These four fractions are also tested for antioxidant activities in vitro, RBP-4 exhibited strong assay of reducing power and high scavenging activity on DPPH radical, while RBP-3 showed the stronger ability of hydroxyl radical scavenging activity.
Response surface methodology was successfully applied to optimize the ultrasonic extraction of polysaccharides from R. botrytis. RBP is an efficient natural antioxidant.
KeywordsRamaria botrytis Polysaccharides Purification Antioxidant activities
Edible mushrooms commonly used as food, flavoring substances or folk traditional medicines, are well-known for their abundant nutrients: carbohydrates, proteins, vitamins, minerals, characteristic flavour components, and other bioactive components . Meanwhile, Products from wild and cultivated edible mushrooms, have acquired considerable attention toward their biological functions, such as improving immunity, antioxidant, anti-cancer and anti-viral activities due to their functional constituents [2–4].
Extensive studies have been done with the structure and bioactivity mechanism of natural polysaccharides and their conjugates, which have been used in food and medicine for a long time [5, 6]. Numerous researches demonstrated that plenty of natural polysaccharides were good at protecting human bodies from oxidative damage in the growth and development of living organism [7–9]. Therefore, natural polysaccharides are considered as a potential resource of novel antioxidants, and the mechanism of polysaccharide are in need of further research [6, 10].
Ramaria botrytis (Pers.) Ricken, one of mushrooms widely consumed as edible food especially prevailing Asian countries including China, mainly due to its special favor and rich nutrients. It is known as cauliflower coral and belongs to Clavariaceae . Polysaccharide, water soluble and water insoluble, is one of the most important bioactive substances in R. botrytis. Recent research revealed that two water insoluble glucans had been isolated from the alkali extract of the fruit bodies of R. botrytis . In this paper, the extraction, purification, characterization and antioxidant activities of polysaccharides isolated from R. botrytis is described. This study aims to purify fractions of water soluble polysaccharides, analyze their preliminary characteristics and investigate their antioxidant activities.
Materials and chemicals
The samples of R. botrytis, collected by the author in Ailao mountains, Yunnan Province, China, in August 2013. Identification of the mushrooms was performed by Prof. Li Yu, the academician of Jilin Agricultural University. Removed impurities and cleaned with water, the samples were air-dried to constant weight at 60 °C. Then the dried sample was ground into fine powder and screened through a 40 mesh sieve. The powder was prepared for the subsequent studies.
Analytical grade of 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) and 1, 10-phenanthroline was purchased from the Sigma-Aldrich Trading Limited Corporation (Shanghai, China) and the Kermel Chemical Corporation (Tianjin, China), respectively. Other reagents used in this study were of analytical grade.
Box–Behnken factorial design (BBD) for the extraction of RBP
Independent variables and their levels for the extraction of RBP
Water to raw material ratio (mL/g)
Extraction temperature (°C)
Extraction time (h)
Analytical method validation
The total content of polysaccharide in R. botrytis was analyzed by phenol–sulfuric acid method using glucose as standard . The regression equation was Y = 0.0124x − 0.0032 with the correlation coefficient as 0.9926, where Y represents absorbance, x represents the concentration of glucose or RBP. A linear relationship between the absorbance and the polysaccharide quantity was observed within the range of 0–40 μg mL−1, detected at 490 nm wavelength.
The extraction method was validated in terms of precision and accuracy. The precision was estimated by analyzing the intra-day (repeatability) and inter-day (intermediate) precision variations. The repeatability was evaluated by testing standard solution at three different concentrations (0.05, 0.10 and 0.20 mg mL−1) with five replicates during one day, and the intermediate precision was evaluated by testing standard solution at three different concentrations (0.05, 0.10 and 0.20 mg mL−1) for three days. The accuracy was evaluated with the spiked recovery test. Three different standards (0.05, 0.10 and 0.20 mg mL−1) were added to blank sample separately for further extraction and analysis.
Preparation of crude RBP
The Sevage solution was adopted to remove the proteins in the crude RBP after extracted under the optimal condition. The deproteinized RBP was extracted with the reaction mixture (chloroform: butyl alcohol, 5:1) for three times. After centrifugation (15 min, 4000 rpm, 20 °C), ethanol was added into the supernatant until the final concentration of ethanol was 50%. The mixture was standing at 4 °C for 18 h, then centrifugal separated at 4000 rpm for 15 min. The supernatant was collected and repeated the same procedure until the final concentration of ethanol was 60, 75, 85 and 95%. The precipitate was collected, freeze-dried and accurately weighed respectively, for further study.
Purification of RBPs
Crude RBP was purified sequentially by DEAE-52 cellulose and Sephadex G-100 filtration chromatography according to a previous study with little modifications . In detail, the RBP solution (3 mL, 10 mg mL−1) was applied tardily to a column (2.6 × 40 cm) of DEAE-52 cellulose. The column was stepwise eluted with 0, 0.1, 0.3 and 0.5 mol L−1 NaCl solutions at a flow rate of 1.0 mL min−1. Then the obtained elutes (5 mL per tube) were collected by the automatic collector. According to the phenol–sulfuric acid method, each fraction of polysaccharides of RBP was collected. Repeat the process and collect the same fractions together. Each fraction was concentrated, dialyzed and freeze-dried. The solution (2 mL, 30 mg mL−1) of each fraction was further purified through the Sephadex G-100 column (2.6 × 60 cm). The elutes were collected automatically eluted with deionized water, then concentrated and freeze-dried for further research.
Characterization of RBP
The monosaccharide composition of RBP-1, RBP-2, RBP-3 and RBP-4 were analyzed by high performance anion exchange chromatography (Dionex ICS-3000, Sunnyvale, CA, USA) in combination with a carbopac PA-1 ion exchange column (4 × 250 mm).
The average molecular weights of polysaccharide fractions were determined by gel permeation chromatography (GPC). Each sample (2.0 mg) was dissolved in distilled water (2 mL), passed through a 0.45 μm filter, and then applied to a column of gel-permeation chromatographic at a flow rate of 0.5 mL min−1 . The calibration curve was conducted by reference of the dextrans with various molecular weight (P-400, P-100, P-50, P-10, and P-5).
Determination of antioxidant activities
DPPH radical-scavenging activity
Hydroxyl radical-scavenging activity
The reducing power was determined by the method  with some modifications. The four RBPs were dissolved in distilled water to form various sample solutions (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg mL−1). A volume of 2 mL sample solution was added into 2.5 mL phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1%, w/v). Incubated at 50 °C for 20 min, 2.5 mL of trichloroacetic acid (TCA) was added to the mixture and centrifuged at 3000 rpm for 10 min. The final mixture solution was formed by adding 2.5 mL distilled water and 0.5 mL ferric chloride (0.1%, w/v) to 2.5 mL of the supernatant. The absorbance of the reaction mixture was measured at 700 nm. Ascorbic acid was used as the positive control. A higher absorbance indicates a stronger reducing power of the sample.
Results and discussion
Optimization for the extraction parameters of RBP
Model fitting preliminary
The Box–Behnken design and the yield of Ramaria botrytis polysaccharide
X 1/water to raw material ratio (mL g−1)
X 2/extraction temperature (°C)
X 3/extraction time (h)
Extraction yield (%)
Predicted yield (%)
where Y represents the yield of RBP (%), X 1, X 2 and X 3 represent ratio of water to solid, extraction temperature and extraction time, respectively.
ANOVA for the quadratic regression model in BBD
Sum of squares
X 1 X 2
X 1 X 3
X 2 X 3
X 1 2
X 2 2
X 3 2
Lack of fit
Optimization for the extraction of RBP
Verification of the model
The relative standard deviation (RSD) value of repeatability was 3.25%, and the RSD value of intermediate precision was 2.68%, which showed the precision of instruments was good. The spiked recoveries of glucose were 91.20–104.30%. In summary, the method was effective and reliable. The polysaccharide yield was 9.08% according to the optimal extraction condition, in which the extraction temperature 90 °C, extraction time 1.5 h and ratio of water to solid 11.00 mL g−1.
Fractional precipitation of polysaccharides
The yield of the precipitation was 58.06, 12.08, 18.78 and 11.08%, as the concentration of ethanol 50, 75, 85 and 95%. No precipitate appeared when the concentration of ethanol was up to 95%. From the yield, the polysaccharide collected with the concentration of ethanol 50% was the main component and was acted as crude polysaccharide to purify further.
Purification of crude RBP
Characterization of RBP
Monosaccharide composition of RBP
Monosaccharide composition for RBP-1, RBP-2, RBP-3, RBP-4
Molecular weight determination of RBPs
The molecular weight of RBP-1, RBP-2, RBP-3, and RBP-4 was determined by GPC method. According to the different molecular weight of dextran standards, the average molecular weights of RBP-1, RBP-2, RBP-3 and RBP-4 were 6.48, 36.12, 96.72 and 8.34 kDa, respectively.
Antioxidant activity in vitro of RBP
Scavenging activity on DPPH radical of RBP
Assay of hydroxyl radical scavenging activity
The hydroxyl radical, which has high reactivity and a very short half-life of approximately 10−9 s in vivo, is the most reactive and dangerous compound generated through the Fenton reaction to organisms . The hydroxyl radical-scavenging activity of RBP-1, RBP-2, RBP-3, RBP-4 and ascorbic acid determined at 510 nm were depicted in Fig. 4b. The results showed the scavenging activity of RBP-3 was higher than RBP-4, RBP-2, RBP-1, but lower than ascorbic acid. The hydroxyl radical-scavenging activity of ascorbic acid and all the polysaccharides increased gradually as their concentrations increased. With the increase of amount in the range of 0–1.2 mg mL−1, hydroxyl radical-scavenging activityof each compound increased, whereas the activity of RBP-3 (90%) was approximatelythe same as ascorbic acid (95.33%) at the concentration of 1.2 mg mL−1.
Assay of reducing power
Served as a significant indicator of its potential antioxidant activity, the reducing power of a compound may directly reflect the production condition of electron donor [20, 21]. The reducing power of RBP-1, RBP-2, RBP-3, RBP-4 and ascorbic acid determined at 700 nm is depicted in Fig. 4c. Ascorbic acid is a well-recognized reducing agent. As shown in the figure, the reducing power of ascorbic acid increased quickly as the concentration increased from 0.2 to 1.2 mg mL−1. All four samples showed higher reducing power with the increasing of their concentrations, but much lower than ascorbic acid. RBP-4 had the strongest reducing power among the four fractions.
It can be concluded that the water-soluble and purified polysaccharides from the sporocarp of R. botrytis could be obtained with the optimized method. Firstly, The BBD method provided the optimal extraction condition of the crude polysaccharide. And the crude polysaccharide was eluted and purified by two column chromatographies of DEAE-52 and Sephadex G-100 successively. Four purified fractions of polysaccharides, RBP-1, RBP-2, RBP-3 and RBP-4 were obtained in this study, which average molecular weights were 6.48, 36.12, 96.72 and 8.34 kDa, respectively. Moreover, RBP-1, RBP-2, RBP-4 were mainly composed of glucose, with a percentage of 88.24, 95.42 and 65.62%, respectively; while RBP-3 contained 41.36% mannose, 28.96% glucose, 15.01% xylose and 14.37% galactose. Furthermore, the antioxidant activity tests showed that RBP-4 had strong assay of reducing power and high scavenging activity on DPPH radical, while RBP-3 exhibited the strongest ability of hydroxyl radical scavenging activity. All the results implied that RBP could be a promising new natural antioxidant in food industry or drug therapies.
This work was supported by the National Natural Science Foundation of China (31401548), Special Fund for Agro-scientific Research in the Public Interest (No. 201303070) and the fundamental research funds for special projects of Henan University of Technology (2014YWQQ04).
The author declares that she has no competing interests.
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