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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effect of different drying methods on chemical composition and bioactivity of finger citron polysaccharides
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Zhen Wu ∗ Chongqing Academy of Chinese Materia Medica, Chongqing 400065, People’s Republic of China
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Article history: Received 7 December 2014 Received in revised form 16 February 2015 Accepted 19 February 2015 Available online xxx
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Keywords: Finger citron Different drying methods Physicochemical properties
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1. Introduction
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Traditionally, people like to take dried finger citron fruits (FC) as adjuvant herbal medicines to treat a diversity of chronic diseases like asthma, hypertension and respiratory tract infections. Many healing properties are attributed to FC polysaccharides (FCPs), one of the main active ingredients of FC. Three drying methods, freeze drying (FDM), hot air drying (HDM) and vacuum drying methods (VDM) were comparatively studied on the physicochemical and antioxidant properties of FCPs. The results showed these FCPs were similar in UV and FT-IR spectrum. However, they showed significant differences (p < 0.05) in yields of crude polysaccharides and contents of protein and ash. Compared with VDM and HDM, FDM resulted in the properties of FCPs with lower molecular weight distribution, higher reducing power and scavenging abilities on DPPH• , OH• , and O2 • − . Available data obtained in vitro models suggested that FDM was an appropriate and effective treatment for obtaining crude polysaccharides from FC fruits. Hence, drying methods used for preparation of FCPs can affect physicochemical and associated functional properties. © 2015 Published by Elsevier B.V.
The finger citron [Citrus medica L. var. sarcodactylis (Noot.) Swingle] fruits (FC), have been long used as traditional Chinese medicinal material and functional vegetables, and preserved as sweetmeats [1]. FC fruits belonging to the family Rutaceae, commonly known as “Five finger orange” in commercial vegetable markets, is an important functional food source in a specific diet [2]. Earlier studies reported that FC fruits possessed antioxidant, insulin secretagogue, anti-inflammatory, anti-microbial and anthelmintic activities [3,4]. Rapid drying process of Chinese herbal pieces is crucial to its quality control and pharmaceutical effect [5,6]. The active ingredients and pharmacological efficacy of Chinese herbal pieces are greatly influenced by different processing technologies. Drying is a very common preservation method used in Chinese herbal pieces and the quality of the final products is strongly dependent on the technique and the process variables used [7]. The World Health Organization estimates that 80% of the world’s population uses medicinal plants medicines and related extracts in some way [8]. According to this growing demand for medicinal extracts, artificial drying has been one of the most important needs of the
∗ Tel.: +86 23 89 02 90 55; fax: +86 23 89 02 90 55. E-mail addresses: [email protected], [email protected]
pharmaceutical industry, which does not have structure to use fresh plants in the quantities required for industrial production. Also, increasing usage of drying procedures within various technological lines in the food industry and biotechnology has made studies of the drying process of important practical interest. In recent years, diets rich in selected natural antioxidants and various extracts from FC fruits are related to reduced risk of incidence of hyperlipidemia, obese and other chronic diseases has lead to the revival of interest in plant-based foods [9,10]. To date, the studies have been mainly focused on the influence of the drying methods on the essential oils or volatile fractions of Citrus herbs in order to optimize the drying [11,12]. Polysaccharides extracted from FC fruits (FCPs) are of particular interest because of their effective bioactivities, such as antioxidant [3]. In general, physicochemical properties and effective bioactivities of crude polysaccharides extracted from fruits, vegetables and medicinal plants was greatly influenced by different drying process including freeze drying (FDM), hot air drying (HDM) and vacuum drying (VDM) [13,14]. Various drying methods have been developed to preserve herb plants and have exhibited their own characteristics [15]. However, there was no information about the effects of drying methods on composition and antioxidant activities of polysaccharides from FC fruits. Additionally, the ability to determine suitable drying methods would increase the grower’s control on both FC fruit yield and its quality. A recommendable drying method should tend to avoid undesirable changes and maintain the good quality
http://dx.doi.org/10.1016/j.ijbiomac.2015.02.043 0141-8130/© 2015 Published by Elsevier B.V.
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of dried product. In spite of our earlier work in this area [3], the situation regarding the influence of different drying methods on the physicochemical properties and antioxidant activities of FCPs is far from clear. The aims of the present study were, therefore, to determine physicochemical properties and antioxidant activities of FCPs as affected by the different drying methods (traditional hot-air, vacuum, and freeze drying).
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2. Materials and methods
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2.1. Materials
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The FC fruits [C. medica L. var. sarcodactylis (Noot.) Swingle], were harvested from different trees in Liangping county (coordinates: lat. 30◦ 40 N, long. 107◦ 48 E), Chongqing, China, at an altitude of 450 m, and authenticated by the Application and Development Institute of Chongqing Academy of Chinese Materia Medica (Chongqing, China). All the collected FC fruits were cut into 5 mm strips before drying at 60 ◦ C for 5 h. All chemicals used in the experiment were of analytical grade.
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2.2. Extract of crude polysaccharides
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The dried FC fruits were refluxed with 95% ethanol at 70 ◦ C in a water bath for 2 h to deactivate the endogenous enzymes and remove some soluble materials, including free sugars, amino acids and some polyphenols. The combined extract was vacuum dried at 60 ◦ C for 12 h, and it was suspended in the water and sonicated at the temperature of 50 ◦ C and actual sonic power of 45 W for 40 min. After rapid cooling to room temperature using ice water, the supernatant was concentrated in a rotary evaporator under reduced pressure (Buchi R-124, Flavil, Sweden). The extract was filtered, and then precipitated using 150 mL of 95% ethanol, 100% ethanol and acetone, respectively. After being left overnight at 4 ◦ C, the precipitates were collected by centrifugation at 3000 rpm for 20 min, redissolved in deionized water, deproteinated by the method of Sevag et al. [16], and dialyzed in a dialysis bag (MWCO 1400 Da, Union Carbide). The precipitates were then hot air dried, vacuum dried or freeze dried, respectively.
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2.3. Drying procedure
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Fig. 1. Scheme of extraction and fractionation of polysaccharides from finger citron fruits. The polysaccharides obtained from finger citron fruits by the hot air drying, vacuum drying and freeze drying methods were named FCPs-H (), FCPs-V (䊉) and FCPs-F (), respectively.
Rotation Viscometer (Jinghai Technology Co. Ltd., Shanghai, China) at a concentration of 10 mg/mL and 25 ◦ C [3,20]. 2.5. Average molecular weight (AMW) determination of FCPs The AMW of FCPs was determined by gel-permeation chromatography (GPC) on a Sephadex G-100 column (1.6 cm × 100 cm) using distilled water as eluent at a flow rate of 24 mL/h [21,22]. The eluate (5 mL/tube) was collected and monitored for carbohydrate content using phenol–sulfuric acid method at 490 nm [17]. The average molecular weight of FCPs was calculated as the following equation [23]:
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2.4. Compositional and relative viscosity analysis
2.6. Ultraviolet–vis and Fourier transform infrared spectroscopy analysis
The percentage FCPs extraction yield (%) was calculated with the formula of y (%) = c/w × 100, where c was the polysaccharides content of extraction, and w represented dried sample weight (1 g). The sugar content was determined by the reaction of sugars with phenol in the presence of sulfuric acid using glucose as a standard [17]. Ash were determined according to AOAC (1990) method [18], while the protein content in the solid polysaccharide was determined using the Kjeldahl method with a conversion factor of 6.25 [19]. Relative viscosity (to deionized water) of LMPs was measured in NDJ-1
The ultraviolet–vis (UV) absorption spectroscopy of the abovementioned FCPs samples was recorded using an UV-2600 spectrophotometer (Shimadzu, Japan) in the wavelength range from 200 and 700 nm. Sample solution was prepared by dissolving 5 mg of sample in distilled water to a concentration of 1.0 mg/mL, and distilled water was taken for the blank. Fourier transform infrared spectrometry (FT-IR) was obtained using a Spectrum 100 FT-IR spectrophotometer (Perkin Elmer, USA). The dried FCPs were grinded with potassium bromide power and pressed into pellet for
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M × Ci i AMW = Ci
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where Mi was the molecular weight of each fraction, obtained from the regression line of the Dextran T-series standard of known molecular weight (T-2000, T-70, T-40, T-20, and T-10) versus elution volume plot. Ci was the total carbohydrate concentration of each fraction, analyzed by phenol–sulfuric method [17].
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The precipitates were hot air dried in an electric thermostatic drying oven (DHG-9240A, Yiheng Scientific Instruments, Shanghai, China) at 50 ◦ C. Vacuum drying was performed at 50 ◦ C, at 95 kPa of vacuum degree for 24 h in a vacuum oven (ZF-6020, Yiheng Scientific Instruments, Shanghai, China). For freeze drying, the precipitates was first frozen for 24 h at −18 ◦ C. Frozen samples were then freeze-dried (Christ ALPHA 1-2 LD plus) at −50 ◦ C. The samples were dried until the moisture contents below the 8%. FCPs drying by hot air drying, vacuum drying and freeze drying methods were named FCPs-H, FCPs-V and FCPs-F, respectively. All experiments were performed at least in duplicate. The flow chart was shown in Fig. 1.
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spectrometric measurement in the range of 4000–450 cm−1 (midinfrared region). 2.7. Antioxidant activity assay 2.7.1. Reducing power assay The reducing power was assessed according to a reported procedure with minor modifications [24]. Various concentrations of FCPs (0.05–1.0 mg/mL) in sodium phosphate buffer (1.5 mL, 0.2 M, pH = 6.6) were mixed with potassium ferricyanide (1.5 mL, 1%, w/v) and the mixture was incubated at 50 ◦ C for 20 min. After that, trichloroacetic acid (TCA, 1.5 mL, 10%, w/v) was added, and the mixture was centrifuged at 12,000 rpm for 15 min. The centrifugate (1.5 mL) was mixed thoroughly with deionized water (1.5 mL) and FeCl3 (0.3 mL, 0.1%, w/v). Finally, the absorbance was measured at 700 nm against a blank. Ascorbic acid (Vc) was used as positive control. A higher absorbance of the reaction mixture indicates a stronger reducing power of the sample. (DPPH• )
2.7.2. 1,1-Diphenyl-2-picrylhydrazyl free radical scavenging assay The scavenging activity of DPPH• was assessed according to a literature procedure with a few modifications [25]. Briefly, 1 mL of DPPH solution (0.10 mM in ethanol) was added into 3 mL FCPs solution at different concentrations ranging from 0.05 to 1.0 mg/mL. The mixture was left in the dark for 30 min and the absorbance was then measured at 517 nm. A control contained all the reaction reagents except the samples (water instead of the sample solution) was prepared and measured as A0 . A1 was the result of samples and A2 contains all the samples but ethanol instead of DPPH• solution. DPPH• scavenging activity was calculated using the formula: [A0 − (A1 − A2 )] × 100 DPPH• scavenging activity (%) = A0
(2)
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2.7.3. Hydroxyl radical (OH• ) scavenging assay The OH• scavenging assay was performed based on a literature procedure with a few modifications [26]. Reaction mixtures in a final volume of 1.0 mL contained deoxyribose (60 mM), phosphate buffer (pH 7.4, 20 mM), ferric trichloride (100 M), EDTA (100 M), H2 O2 (1 mM), ascorbic acid (100 M) and various concentrations of FCPs. The reaction solution was incubated for 1 h at 37 ◦ C, and then 1 mL of 1% thiobarbituric acid (TBA) and 1 mL of 20% (v/v) HCl were added to the mixture. The mixture was boiled for 15 min and cooled on ice. The absorbance of the mixture was measured at 532 nm. A0 is the absorbance of the control (water instead of the sample) and A1 is the absorbance of the sample. The scavenging activity of OH• was calculated according to the following equation:
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[A0 − A1 ] × 100 scavenging activity (%) = A0
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2.7.4. Superoxide anion radical (O2 •− ) scavenging assay O2 •− were generated by pyrogallic acid method with a minor modification [27]. The system contained 2.5 mL of phosphate buffer solution (PBS) (0.1 M, pH 8.2), 4 mL of sample solution, 2.5 mL of pyrogallic acid (6.0 mM), and 0.5 mL of thick hydrochloric acid for termination the reaction. The solution was incubated at 25 ◦ C and determined at 299 nm. The scavenging activity was calculated as follows:
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scavenging activity (%) =
[A0 − (A1 − A2 )] × 100 A0
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where A0 , with the presence of pyrogallic acid but without FCPs; A1 , with the presence of pyrogallic acid and FCPs; and A2 , with the presence of FCPs but without pyrogallic acid.
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Table 1 Effects of different drying methods on chemical compositions and viscosity of FCPs. SamplesA
FCPs-H
Yield (%, w/w) Total sugar (%, w/w) Protein (%, w/w) Ash (%, w/w) Relative viscosityB
2.74 73.51 4.40 3.46 1.61
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FCPs-F 0.14c 2.62b 0.17a 0.33a 0.09b
3.84 82.31 3.97 3.06 2.56
FCPs-V ± ± ± ± ±
0.21a 2.04a 0.28a 0.31a 0.15a
3.24 78.27 4.45 3.19 1.82
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0.08b 2.11a 0.27a 0.28a 0.11b
A Values are expressed as mean ± SD of three replicated determinations. Means with the different letters in the same line are significantly different (p < 0.05). The polysaccharides obtained from finger citron fruits by the hot air drying, vacuum drying and freeze drying methods were named FCPs-H, FCPs-V and FCPs-F, respectively. B Relative viscosity (to deionized water) of FCPs was measured in NDJ-1 Rotation Viscometer (Jinghai Technology Co. Ltd., Shanghai, China) at a concentration of 10 mg ml−1 and 25 ◦ C.
2.8. Statistical analysis
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Statistical analysis was performed using SPSS (Version 15, SPSS, Chicago, IL) statistical software. Comparison of means was performed by one-way analysis of variance (ANOVA) followed by Duncan’s test.
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3. Results and discussion
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3.1. Yield and physicochemical properties of FCPs
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The yield, content of total sugar, protein, ash and relative viscosity of FCPs were analyzed and summarized in Table 1. The yields of three crude FCPs samples were 2.74% (w/w) for FCPs-H, 3.84% (w/w) for FCPs-F and 3.24% (w/w) for FCPs-V, respectively. The yield of FCPs-F was obviously higher than those obtained by the other two drying methods (p < 0.05). It has been widely acknowledged that antioxidant activities of crude polysaccharides can be affected by many factors including its chemical components, molecular mass, structure and conformation. So it was necessary to analyze the contents of total sugar, protein and ash in FCPs samples. As shown in Table 1, the total sugar content of FCPs-H, FCPs-F and FCPs-V was 73.51 ± 2.62, 82.31 ± 2.04 and 78.27 ± 2.11% (w/w), respectively. The protein content of FCPsH, FCPs-F and FCPs-V was determined to be 4.40, 3.97 and 4.45% (w/w) with an ash content of 3.46, 3.06 and 3.19% (w/w), respectively. The relative viscosity (to deionized water) of FCPs-H, FCPs-V and FCPs-F increased gradually from 1.61 to 2.56. The relative viscosity of FCPs-F was obviously higher than those obtained by the other two drying methods (p < 0.05), which suggested that freeze drying method was a good treatment for obtaining the polysaccharide from finger citron. The chromatographic profiles (Fig. 2) showed that carbohydrate fractions of FCPs ranged in a wide molecular size distribution, which indicated that FCPs were highly dispersive regardless of the drying methods (Table 2). The first peak AMW of sample Table 2 Distribution of average molecular weight (AMW) in all polysaccharides samples extracted from finger citron fruits on Sephdex G-150. Samplesa
FCPs-H FCPs-F FCPs-V
AMW (×104 Da) Peak 1
Peak 2
Peak 3
Peak 4
19.52 ± 1.06 11.87 ± 1.90 16.54 ± 1.22
5.18 ± 0.92 4.39 ± 0.28 7.22 ± 0.41
1.62 ± 0.10 2.26 ± 0.12 3.15 ± 0.19
ndb 0.71 ± 0.11 0.84 ± 0.10
a Values are expressed as mean ± SD of three replicated determinations. The polysaccharides obtained from finger citron fruits by the hot air drying, vacuum drying and freeze drying methods were named FCPs-H, FCPs-V and FCPs-F, respectively. b nd: not detected.
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Fig. 2. Effects of different drying methods on average molecular weight (AMW) distribution of FCPs. The polysaccharides obtained from finger citron fruits by the hot air drying, vacuum drying and freeze drying methods were named FCPs-H (), FCPs-V (䊉) and FCPs-F (), respectively.
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FCPs-F was 11.87 × 104 Da, which was smaller than those obtained by FCPs-H and FCPs-V samples. Bioactivity of polysaccharides mainly depends on several structural parameters including sugar composition, molecular weight, type of glycosidic bond of the main chain, and type and degree of the branched-chain modification [28]. Moreover, Sun et al. [29] reported that high molecular weight polysaccharides from Porphyridium cruentum had no obvious antioxidant activities, but low molecular weight polysaccharides after degradation exhibited an inhibitory effect on oxidative damage. Accordingly, the specific AMW of FCPs-F possibly exerted an important effect on its potent antioxidant activities, as evidenced by the antioxidant capacities in vitro. There were high molecular weight peaks in the FCPs-H and FCPs-V samples, which indicated that the polysaccharide molecules were easy to aggregate at the high temperature condition, especially at the hot air condition. As can be seen from Fig. 3a, three types of FCPs samples obtained by the different drying process are almost consistent in UV scanning spectrum that have stronger absorption at 200–280 nm, showing that the samples may contain unsaturated carbonyl and carboxyl. Fig. 3a also shows the samples had strong absorption peak at 284 nm, which indicates that the samples contain conjugated nucleic acids or proteins. Fig. 3b shows the effect of drying method on the FT-IR spectroscopy of FCPs. No visible differences could be observed between the spectra of the different polysaccharide samples. The band and peak characteristics of the spectra have been reported [3]. The broad band at 3433 cm−1 indicates the presence of hydroxyl ( OH) groups due to moisture but could also arise from the hydroxyl of sugar rings. The small peak at 2926 cm−1 results from stretching modes of the C H bonds of methyl groups ( CH3 ) or the C H bond of methylene and methine groups [14]. An asymmetrical stretching peak at 1635 cm−1 and a weak symmetrical stretching peak near 1401 cm−1 were assigned to the absorbance of the deprotonated carboxylic group (COO ), indicating FCPs be acidic polysaccharides [30]. The wave numbers between 800 and 1200 cm−1 represent the finger print region for carbohydrates [31]. FT-IR characterization of different dried extracts fitted the typical
Fig. 3. (A) UV absorbance spectra of crude FCPs. (B) FT-IR spectra of crude FCPs. The polysaccharides obtained from finger citron fruits by the hot air drying, vacuum drying and freeze drying methods were named FCPs-H, FCPs-V and FCPs-F, respectively.
pattern of polysaccharides well, which suggested that the three drying methods did not destroy the polysaccharide structure. 3.2. Antioxidant activities To assess the possible utilization of the FCPs, we evaluated its antioxidant and radical scavenging activities against DPPH• , OH• , and O2 •− . The methods chosen are most commonly used for the determination of antioxidant activities of plant extracts. It appeared that there was mutual correlation between reducing power and antioxidative activity [32]. The reducing power assay measures the electron-donating ability of antioxidants using the potassium ferricyanide reduction method. The reducing capacity of a compound would possibly serve as a significant indicator of its potential antioxidant activity. Fig. 4a compares the reducing power of FCPs-H, FCPs-V, FCPs-F, and Vc, with a higher absorbance associated with a greater reducing power. In the range of detecting concentration, Vc and all FCPs samples dried by three different methods showed a good linear connection to concentration with R2 > 0.99 and an effective capacity. The FCPs obtained by freeze drying showed greater reducing power than those collected by traditional hot air and vacuum drying (Fig. 4a). The high reducing power of FCPs-F might be existent due to their molecular electron-withdrawing activity, which can eliminate free radicals and terminate radical-mediated oxidative chain reactions.
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Fig. 4. Reducing power (a), scavenging DPPH• (b), OH• (c), and O2 • − (d) capacities of three polysaccharides and ascorbic acid (Vc). Values are means ± SD of three determinations. The polysaccharides obtained from finger citron fruits by the hot air drying, vacuum drying and freeze drying methods were named FCPs-H, FCPs-V and FCPs-F, respectively.
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Applications of antioxidants are increasing due to their multiple roles in minimizing harmful effects of oxidative stress. DPPH• scavenging assay is routinely practiced for the assessment of antiradical properties of different compounds [33]. Further, DPPH• was scavenged by different polysaccharides and Vc through donation of hydrogen to form a stable DPPH molecule. As shown in Fig. 4b, the half-effective concentration (EC50 ) values increased in the order of FCPs-F (0.37 mg/mL) > FCPs-V (0.52 mg/mL) > FCPs-H (0.55 mg/mL), which revealed that FCPs-F showed the best scavenging DPPH• ability and FCPs-H the worst. The maximum value of FCPs-F reached 88.7% that of Vc, suggesting that it may be possible to obtain a novel FCPs with strong radical-scavenging capacity through optimization of the freeze drying parameters. OH• is the most harmful reactive oxygen species (ROS) and is involved in the oxidative injury of biomolecules including carbohydrates, proteins, lipids, and DNA in cells, causing tissue damage or cell death. Removing OH• is important for the protection of biological systems [34]. The OH• scavenging ability of all FCPs and Vc is shown in Fig. 4c. The FCPs-F showed the highest OH• scavenging ability (78.5 ± 1.60%), followed by FCPs-V (75.5 ± 1.50%) and FCPsH (68.8 ± 1.92%). The EC50 values of FCPs-H, FCPs-V, FCPs-F and Vc were 0.45, 0.41, 0.31 and 0.15 mg/mL, respectively. The maximum value of FCPs-F reached 82.8% that of Vc. As shown in Fig. 4d, all three FCPs samples showed O2 •− scavenging activity under the experimental conditions but at a level much lower than that of Vc. Additionally, unlike scavenging of DPPH• , all FCPs exhibited
a lower scavenging activity toward O2 •− (Fig. 4d). However, the EC50 values increased in the order of FCPs-H (0.67 mg/mL) > FCPs-V (0.56 mg/mL) > FCPs-F (0.49 mg/mL). Fan et al. [35] reported that Ganoderma lucidum polysaccharides obtained by vacuum drying and vacuum freeze drying had the higher scavenging effects on OH• , O2 •− , DPPH• , and stronger reducing power than those obtained by hot air drying. In a similar study, Wu et al. [15] had reported that freeze dried ABMP (polysaccharides obtained from Agaricus blazei Murrill) showed some advantages over vacuum drying and air drying ABMP. In the present study, the antioxidant activities of FCPs isolated by ultrasonic extraction were slightly lower compared to those reported previously [3]. However, it showed that the antioxidant activities of FCPs-F isolated by ultrasonic extraction were insignificantly lower compared to the hot water technology [3]. According to the results above, freeze drying treatment may lead to a health beneficial FCPs-F for potential utilization in dietary supplements or functional food products. In conclusion, the results obtained in the present study clearly demonstrated that FCPs-F was a prospective radical scavenging agent. From the four models of antioxidant activities mentioned above, results showed that FCPs-F exhibited the highest antioxidant capacity among the three FCPs samples. This might be due to the different compositions and molecular size distributions of FCPsF, which had higher hydrogen-donating ability [13,36]. Nowadays more and more attention was cast on polysaccharides by biochemical and nutritional researchers due to their various biological
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activities used in health-care food or medicine, especially antioxidant effects. These polysaccharides are often identified as biological response modifiers (BRMs) [37]. Plants, particularly those used as traditional Chinese medicines, contain high amounts of natural polysaccharides that have been identified as free radicals or active oxygen scavengers [28,38]. Dehydration is one of the most important preservation methods employed in storage of finger citron and dehydrated finger citron are valuable ingredients in a variety of traditional Chinese medicinal material and functional vegetables. Further, the selective dehydration of polysaccharides without the protection of cell wall by an appropriate method is very important. Both yield and activity of polysaccharides are strongly dependent on the type of dehydration and drying temperature employed, due to the different potential of compounds with different denaturalization temperature [39]. 4. Conclusion This study showed that the drying method used for preparation of FCPs can significantly affect the physicochemical properties, which in turn affect the associated functionality of polysaccharides. The results of the present work indicated that freeze dried FCPs-F showed some advantages over FCPs-H and FCPs-V owing to its highest polysaccharides yield, neutral sugar content, relative viscosity, and low molecular size distribution, further exhibited best higher reducing power, and scavenging abilities on DPPH• , OH• , and O2 •− . The combined comparison of the physicochemical characterization and bioactivities of polysaccharides from finger citron is helpful to improve their pharmacological activity-based quality control, and beneficial for better understanding the structure–bioactivity relationship of polysaccharides from Chinese herbs. Based on the above studies, further detailed structural characterization and in vivo bioactivity of polysaccharides should be carried out to provide a good opportunity for scientists to elucidate the structure–function relationship and to explore high potential antioxidant agent. Acknowledgement This work was financially supported by Chongqing Municipal Health Bureau Science and Technology Project for Traditional Chinese Medicine (No. zy20132075).
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effect of different drying methods on chemical composition and bioactivity of finger citron polysaccharides
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Zhen Wu ∗ Chongqing Academy of Chinese Materia Medica, Chongqing 400065, People’s Republic of China
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Article history: Received 7 December 2014 Received in revised form 16 February 2015 Accepted 19 February 2015 Available online xxx
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Keywords: Finger citron Different drying methods Physicochemical properties
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1. Introduction
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Traditionally, people like to take dried finger citron fruits (FC) as adjuvant herbal medicines to treat a diversity of chronic diseases like asthma, hypertension and respiratory tract infections. Many healing properties are attributed to FC polysaccharides (FCPs), one of the main active ingredients of FC. Three drying methods, freeze drying (FDM), hot air drying (HDM) and vacuum drying methods (VDM) were comparatively studied on the physicochemical and antioxidant properties of FCPs. The results showed these FCPs were similar in UV and FT-IR spectrum. However, they showed significant differences (p < 0.05) in yields of crude polysaccharides and contents of protein and ash. Compared with VDM and HDM, FDM resulted in the properties of FCPs with lower molecular weight distribution, higher reducing power and scavenging abilities on DPPH• , OH• , and O2 • − . Available data obtained in vitro models suggested that FDM was an appropriate and effective treatment for obtaining crude polysaccharides from FC fruits. Hence, drying methods used for preparation of FCPs can affect physicochemical and associated functional properties. © 2015 Published by Elsevier B.V.
The finger citron [Citrus medica L. var. sarcodactylis (Noot.) Swingle] fruits (FC), have been long used as traditional Chinese medicinal material and functional vegetables, and preserved as sweetmeats [1]. FC fruits belonging to the family Rutaceae, commonly known as “Five finger orange” in commercial vegetable markets, is an important functional food source in a specific diet [2]. Earlier studies reported that FC fruits possessed antioxidant, insulin secretagogue, anti-inflammatory, anti-microbial and anthelmintic activities [3,4]. Rapid drying process of Chinese herbal pieces is crucial to its quality control and pharmaceutical effect [5,6]. The active ingredients and pharmacological efficacy of Chinese herbal pieces are greatly influenced by different processing technologies. Drying is a very common preservation method used in Chinese herbal pieces and the quality of the final products is strongly dependent on the technique and the process variables used [7]. The World Health Organization estimates that 80% of the world’s population uses medicinal plants medicines and related extracts in some way [8]. According to this growing demand for medicinal extracts, artificial drying has been one of the most important needs of the
∗ Tel.: +86 23 89 02 90 55; fax: +86 23 89 02 90 55. E-mail addresses: [email protected], [email protected]
pharmaceutical industry, which does not have structure to use fresh plants in the quantities required for industrial production. Also, increasing usage of drying procedures within various technological lines in the food industry and biotechnology has made studies of the drying process of important practical interest. In recent years, diets rich in selected natural antioxidants and various extracts from FC fruits are related to reduced risk of incidence of hyperlipidemia, obese and other chronic diseases has lead to the revival of interest in plant-based foods [9,10]. To date, the studies have been mainly focused on the influence of the drying methods on the essential oils or volatile fractions of Citrus herbs in order to optimize the drying [11,12]. Polysaccharides extracted from FC fruits (FCPs) are of particular interest because of their effective bioactivities, such as antioxidant [3]. In general, physicochemical properties and effective bioactivities of crude polysaccharides extracted from fruits, vegetables and medicinal plants was greatly influenced by different drying process including freeze drying (FDM), hot air drying (HDM) and vacuum drying (VDM) [13,14]. Various drying methods have been developed to preserve herb plants and have exhibited their own characteristics [15]. However, there was no information about the effects of drying methods on composition and antioxidant activities of polysaccharides from FC fruits. Additionally, the ability to determine suitable drying methods would increase the grower’s control on both FC fruit yield and its quality. A recommendable drying method should tend to avoid undesirable changes and maintain the good quality
http://dx.doi.org/10.1016/j.ijbiomac.2015.02.043 0141-8130/© 2015 Published by Elsevier B.V.
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of dried product. In spite of our earlier work in this area [3], the situation regarding the influence of different drying methods on the physicochemical properties and antioxidant activities of FCPs is far from clear. The aims of the present study were, therefore, to determine physicochemical properties and antioxidant activities of FCPs as affected by the different drying methods (traditional hot-air, vacuum, and freeze drying).
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2. Materials and methods
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2.1. Materials
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The FC fruits [C. medica L. var. sarcodactylis (Noot.) Swingle], were harvested from different trees in Liangping county (coordinates: lat. 30◦ 40 N, long. 107◦ 48 E), Chongqing, China, at an altitude of 450 m, and authenticated by the Application and Development Institute of Chongqing Academy of Chinese Materia Medica (Chongqing, China). All the collected FC fruits were cut into 5 mm strips before drying at 60 ◦ C for 5 h. All chemicals used in the experiment were of analytical grade.
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2.2. Extract of crude polysaccharides
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The dried FC fruits were refluxed with 95% ethanol at 70 ◦ C in a water bath for 2 h to deactivate the endogenous enzymes and remove some soluble materials, including free sugars, amino acids and some polyphenols. The combined extract was vacuum dried at 60 ◦ C for 12 h, and it was suspended in the water and sonicated at the temperature of 50 ◦ C and actual sonic power of 45 W for 40 min. After rapid cooling to room temperature using ice water, the supernatant was concentrated in a rotary evaporator under reduced pressure (Buchi R-124, Flavil, Sweden). The extract was filtered, and then precipitated using 150 mL of 95% ethanol, 100% ethanol and acetone, respectively. After being left overnight at 4 ◦ C, the precipitates were collected by centrifugation at 3000 rpm for 20 min, redissolved in deionized water, deproteinated by the method of Sevag et al. [16], and dialyzed in a dialysis bag (MWCO 1400 Da, Union Carbide). The precipitates were then hot air dried, vacuum dried or freeze dried, respectively.
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2.3. Drying procedure
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Fig. 1. Scheme of extraction and fractionation of polysaccharides from finger citron fruits. The polysaccharides obtained from finger citron fruits by the hot air drying, vacuum drying and freeze drying methods were named FCPs-H (), FCPs-V (䊉) and FCPs-F (), respectively.
Rotation Viscometer (Jinghai Technology Co. Ltd., Shanghai, China) at a concentration of 10 mg/mL and 25 ◦ C [3,20]. 2.5. Average molecular weight (AMW) determination of FCPs The AMW of FCPs was determined by gel-permeation chromatography (GPC) on a Sephadex G-100 column (1.6 cm × 100 cm) using distilled water as eluent at a flow rate of 24 mL/h [21,22]. The eluate (5 mL/tube) was collected and monitored for carbohydrate content using phenol–sulfuric acid method at 490 nm [17]. The average molecular weight of FCPs was calculated as the following equation [23]:
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2.4. Compositional and relative viscosity analysis
2.6. Ultraviolet–vis and Fourier transform infrared spectroscopy analysis
The percentage FCPs extraction yield (%) was calculated with the formula of y (%) = c/w × 100, where c was the polysaccharides content of extraction, and w represented dried sample weight (1 g). The sugar content was determined by the reaction of sugars with phenol in the presence of sulfuric acid using glucose as a standard [17]. Ash were determined according to AOAC (1990) method [18], while the protein content in the solid polysaccharide was determined using the Kjeldahl method with a conversion factor of 6.25 [19]. Relative viscosity (to deionized water) of LMPs was measured in NDJ-1
The ultraviolet–vis (UV) absorption spectroscopy of the abovementioned FCPs samples was recorded using an UV-2600 spectrophotometer (Shimadzu, Japan) in the wavelength range from 200 and 700 nm. Sample solution was prepared by dissolving 5 mg of sample in distilled water to a concentration of 1.0 mg/mL, and distilled water was taken for the blank. Fourier transform infrared spectrometry (FT-IR) was obtained using a Spectrum 100 FT-IR spectrophotometer (Perkin Elmer, USA). The dried FCPs were grinded with potassium bromide power and pressed into pellet for
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M × Ci i AMW = Ci
(1)
where Mi was the molecular weight of each fraction, obtained from the regression line of the Dextran T-series standard of known molecular weight (T-2000, T-70, T-40, T-20, and T-10) versus elution volume plot. Ci was the total carbohydrate concentration of each fraction, analyzed by phenol–sulfuric method [17].
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The precipitates were hot air dried in an electric thermostatic drying oven (DHG-9240A, Yiheng Scientific Instruments, Shanghai, China) at 50 ◦ C. Vacuum drying was performed at 50 ◦ C, at 95 kPa of vacuum degree for 24 h in a vacuum oven (ZF-6020, Yiheng Scientific Instruments, Shanghai, China). For freeze drying, the precipitates was first frozen for 24 h at −18 ◦ C. Frozen samples were then freeze-dried (Christ ALPHA 1-2 LD plus) at −50 ◦ C. The samples were dried until the moisture contents below the 8%. FCPs drying by hot air drying, vacuum drying and freeze drying methods were named FCPs-H, FCPs-V and FCPs-F, respectively. All experiments were performed at least in duplicate. The flow chart was shown in Fig. 1.
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spectrometric measurement in the range of 4000–450 cm−1 (midinfrared region). 2.7. Antioxidant activity assay 2.7.1. Reducing power assay The reducing power was assessed according to a reported procedure with minor modifications [24]. Various concentrations of FCPs (0.05–1.0 mg/mL) in sodium phosphate buffer (1.5 mL, 0.2 M, pH = 6.6) were mixed with potassium ferricyanide (1.5 mL, 1%, w/v) and the mixture was incubated at 50 ◦ C for 20 min. After that, trichloroacetic acid (TCA, 1.5 mL, 10%, w/v) was added, and the mixture was centrifuged at 12,000 rpm for 15 min. The centrifugate (1.5 mL) was mixed thoroughly with deionized water (1.5 mL) and FeCl3 (0.3 mL, 0.1%, w/v). Finally, the absorbance was measured at 700 nm against a blank. Ascorbic acid (Vc) was used as positive control. A higher absorbance of the reaction mixture indicates a stronger reducing power of the sample. (DPPH• )
2.7.2. 1,1-Diphenyl-2-picrylhydrazyl free radical scavenging assay The scavenging activity of DPPH• was assessed according to a literature procedure with a few modifications [25]. Briefly, 1 mL of DPPH solution (0.10 mM in ethanol) was added into 3 mL FCPs solution at different concentrations ranging from 0.05 to 1.0 mg/mL. The mixture was left in the dark for 30 min and the absorbance was then measured at 517 nm. A control contained all the reaction reagents except the samples (water instead of the sample solution) was prepared and measured as A0 . A1 was the result of samples and A2 contains all the samples but ethanol instead of DPPH• solution. DPPH• scavenging activity was calculated using the formula: [A0 − (A1 − A2 )] × 100 DPPH• scavenging activity (%) = A0
(2)
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2.7.3. Hydroxyl radical (OH• ) scavenging assay The OH• scavenging assay was performed based on a literature procedure with a few modifications [26]. Reaction mixtures in a final volume of 1.0 mL contained deoxyribose (60 mM), phosphate buffer (pH 7.4, 20 mM), ferric trichloride (100 M), EDTA (100 M), H2 O2 (1 mM), ascorbic acid (100 M) and various concentrations of FCPs. The reaction solution was incubated for 1 h at 37 ◦ C, and then 1 mL of 1% thiobarbituric acid (TBA) and 1 mL of 20% (v/v) HCl were added to the mixture. The mixture was boiled for 15 min and cooled on ice. The absorbance of the mixture was measured at 532 nm. A0 is the absorbance of the control (water instead of the sample) and A1 is the absorbance of the sample. The scavenging activity of OH• was calculated according to the following equation:
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[A0 − A1 ] × 100 scavenging activity (%) = A0
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2.7.4. Superoxide anion radical (O2 •− ) scavenging assay O2 •− were generated by pyrogallic acid method with a minor modification [27]. The system contained 2.5 mL of phosphate buffer solution (PBS) (0.1 M, pH 8.2), 4 mL of sample solution, 2.5 mL of pyrogallic acid (6.0 mM), and 0.5 mL of thick hydrochloric acid for termination the reaction. The solution was incubated at 25 ◦ C and determined at 299 nm. The scavenging activity was calculated as follows:
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•−
scavenging activity (%) =
[A0 − (A1 − A2 )] × 100 A0
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where A0 , with the presence of pyrogallic acid but without FCPs; A1 , with the presence of pyrogallic acid and FCPs; and A2 , with the presence of FCPs but without pyrogallic acid.
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Table 1 Effects of different drying methods on chemical compositions and viscosity of FCPs. SamplesA
FCPs-H
Yield (%, w/w) Total sugar (%, w/w) Protein (%, w/w) Ash (%, w/w) Relative viscosityB
2.74 73.51 4.40 3.46 1.61
± ± ± ± ±
FCPs-F 0.14c 2.62b 0.17a 0.33a 0.09b
3.84 82.31 3.97 3.06 2.56
FCPs-V ± ± ± ± ±
0.21a 2.04a 0.28a 0.31a 0.15a
3.24 78.27 4.45 3.19 1.82
± ± ± ± ±
0.08b 2.11a 0.27a 0.28a 0.11b
A Values are expressed as mean ± SD of three replicated determinations. Means with the different letters in the same line are significantly different (p < 0.05). The polysaccharides obtained from finger citron fruits by the hot air drying, vacuum drying and freeze drying methods were named FCPs-H, FCPs-V and FCPs-F, respectively. B Relative viscosity (to deionized water) of FCPs was measured in NDJ-1 Rotation Viscometer (Jinghai Technology Co. Ltd., Shanghai, China) at a concentration of 10 mg ml−1 and 25 ◦ C.
2.8. Statistical analysis
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Statistical analysis was performed using SPSS (Version 15, SPSS, Chicago, IL) statistical software. Comparison of means was performed by one-way analysis of variance (ANOVA) followed by Duncan’s test.
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3. Results and discussion
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3.1. Yield and physicochemical properties of FCPs
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The yield, content of total sugar, protein, ash and relative viscosity of FCPs were analyzed and summarized in Table 1. The yields of three crude FCPs samples were 2.74% (w/w) for FCPs-H, 3.84% (w/w) for FCPs-F and 3.24% (w/w) for FCPs-V, respectively. The yield of FCPs-F was obviously higher than those obtained by the other two drying methods (p < 0.05). It has been widely acknowledged that antioxidant activities of crude polysaccharides can be affected by many factors including its chemical components, molecular mass, structure and conformation. So it was necessary to analyze the contents of total sugar, protein and ash in FCPs samples. As shown in Table 1, the total sugar content of FCPs-H, FCPs-F and FCPs-V was 73.51 ± 2.62, 82.31 ± 2.04 and 78.27 ± 2.11% (w/w), respectively. The protein content of FCPsH, FCPs-F and FCPs-V was determined to be 4.40, 3.97 and 4.45% (w/w) with an ash content of 3.46, 3.06 and 3.19% (w/w), respectively. The relative viscosity (to deionized water) of FCPs-H, FCPs-V and FCPs-F increased gradually from 1.61 to 2.56. The relative viscosity of FCPs-F was obviously higher than those obtained by the other two drying methods (p < 0.05), which suggested that freeze drying method was a good treatment for obtaining the polysaccharide from finger citron. The chromatographic profiles (Fig. 2) showed that carbohydrate fractions of FCPs ranged in a wide molecular size distribution, which indicated that FCPs were highly dispersive regardless of the drying methods (Table 2). The first peak AMW of sample Table 2 Distribution of average molecular weight (AMW) in all polysaccharides samples extracted from finger citron fruits on Sephdex G-150. Samplesa
FCPs-H FCPs-F FCPs-V
AMW (×104 Da) Peak 1
Peak 2
Peak 3
Peak 4
19.52 ± 1.06 11.87 ± 1.90 16.54 ± 1.22
5.18 ± 0.92 4.39 ± 0.28 7.22 ± 0.41
1.62 ± 0.10 2.26 ± 0.12 3.15 ± 0.19
ndb 0.71 ± 0.11 0.84 ± 0.10
a Values are expressed as mean ± SD of three replicated determinations. The polysaccharides obtained from finger citron fruits by the hot air drying, vacuum drying and freeze drying methods were named FCPs-H, FCPs-V and FCPs-F, respectively. b nd: not detected.
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Fig. 2. Effects of different drying methods on average molecular weight (AMW) distribution of FCPs. The polysaccharides obtained from finger citron fruits by the hot air drying, vacuum drying and freeze drying methods were named FCPs-H (), FCPs-V (䊉) and FCPs-F (), respectively.
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FCPs-F was 11.87 × 104 Da, which was smaller than those obtained by FCPs-H and FCPs-V samples. Bioactivity of polysaccharides mainly depends on several structural parameters including sugar composition, molecular weight, type of glycosidic bond of the main chain, and type and degree of the branched-chain modification [28]. Moreover, Sun et al. [29] reported that high molecular weight polysaccharides from Porphyridium cruentum had no obvious antioxidant activities, but low molecular weight polysaccharides after degradation exhibited an inhibitory effect on oxidative damage. Accordingly, the specific AMW of FCPs-F possibly exerted an important effect on its potent antioxidant activities, as evidenced by the antioxidant capacities in vitro. There were high molecular weight peaks in the FCPs-H and FCPs-V samples, which indicated that the polysaccharide molecules were easy to aggregate at the high temperature condition, especially at the hot air condition. As can be seen from Fig. 3a, three types of FCPs samples obtained by the different drying process are almost consistent in UV scanning spectrum that have stronger absorption at 200–280 nm, showing that the samples may contain unsaturated carbonyl and carboxyl. Fig. 3a also shows the samples had strong absorption peak at 284 nm, which indicates that the samples contain conjugated nucleic acids or proteins. Fig. 3b shows the effect of drying method on the FT-IR spectroscopy of FCPs. No visible differences could be observed between the spectra of the different polysaccharide samples. The band and peak characteristics of the spectra have been reported [3]. The broad band at 3433 cm−1 indicates the presence of hydroxyl ( OH) groups due to moisture but could also arise from the hydroxyl of sugar rings. The small peak at 2926 cm−1 results from stretching modes of the C H bonds of methyl groups ( CH3 ) or the C H bond of methylene and methine groups [14]. An asymmetrical stretching peak at 1635 cm−1 and a weak symmetrical stretching peak near 1401 cm−1 were assigned to the absorbance of the deprotonated carboxylic group (COO ), indicating FCPs be acidic polysaccharides [30]. The wave numbers between 800 and 1200 cm−1 represent the finger print region for carbohydrates [31]. FT-IR characterization of different dried extracts fitted the typical
Fig. 3. (A) UV absorbance spectra of crude FCPs. (B) FT-IR spectra of crude FCPs. The polysaccharides obtained from finger citron fruits by the hot air drying, vacuum drying and freeze drying methods were named FCPs-H, FCPs-V and FCPs-F, respectively.
pattern of polysaccharides well, which suggested that the three drying methods did not destroy the polysaccharide structure. 3.2. Antioxidant activities To assess the possible utilization of the FCPs, we evaluated its antioxidant and radical scavenging activities against DPPH• , OH• , and O2 •− . The methods chosen are most commonly used for the determination of antioxidant activities of plant extracts. It appeared that there was mutual correlation between reducing power and antioxidative activity [32]. The reducing power assay measures the electron-donating ability of antioxidants using the potassium ferricyanide reduction method. The reducing capacity of a compound would possibly serve as a significant indicator of its potential antioxidant activity. Fig. 4a compares the reducing power of FCPs-H, FCPs-V, FCPs-F, and Vc, with a higher absorbance associated with a greater reducing power. In the range of detecting concentration, Vc and all FCPs samples dried by three different methods showed a good linear connection to concentration with R2 > 0.99 and an effective capacity. The FCPs obtained by freeze drying showed greater reducing power than those collected by traditional hot air and vacuum drying (Fig. 4a). The high reducing power of FCPs-F might be existent due to their molecular electron-withdrawing activity, which can eliminate free radicals and terminate radical-mediated oxidative chain reactions.
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Fig. 4. Reducing power (a), scavenging DPPH• (b), OH• (c), and O2 • − (d) capacities of three polysaccharides and ascorbic acid (Vc). Values are means ± SD of three determinations. The polysaccharides obtained from finger citron fruits by the hot air drying, vacuum drying and freeze drying methods were named FCPs-H, FCPs-V and FCPs-F, respectively.
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Applications of antioxidants are increasing due to their multiple roles in minimizing harmful effects of oxidative stress. DPPH• scavenging assay is routinely practiced for the assessment of antiradical properties of different compounds [33]. Further, DPPH• was scavenged by different polysaccharides and Vc through donation of hydrogen to form a stable DPPH molecule. As shown in Fig. 4b, the half-effective concentration (EC50 ) values increased in the order of FCPs-F (0.37 mg/mL) > FCPs-V (0.52 mg/mL) > FCPs-H (0.55 mg/mL), which revealed that FCPs-F showed the best scavenging DPPH• ability and FCPs-H the worst. The maximum value of FCPs-F reached 88.7% that of Vc, suggesting that it may be possible to obtain a novel FCPs with strong radical-scavenging capacity through optimization of the freeze drying parameters. OH• is the most harmful reactive oxygen species (ROS) and is involved in the oxidative injury of biomolecules including carbohydrates, proteins, lipids, and DNA in cells, causing tissue damage or cell death. Removing OH• is important for the protection of biological systems [34]. The OH• scavenging ability of all FCPs and Vc is shown in Fig. 4c. The FCPs-F showed the highest OH• scavenging ability (78.5 ± 1.60%), followed by FCPs-V (75.5 ± 1.50%) and FCPsH (68.8 ± 1.92%). The EC50 values of FCPs-H, FCPs-V, FCPs-F and Vc were 0.45, 0.41, 0.31 and 0.15 mg/mL, respectively. The maximum value of FCPs-F reached 82.8% that of Vc. As shown in Fig. 4d, all three FCPs samples showed O2 •− scavenging activity under the experimental conditions but at a level much lower than that of Vc. Additionally, unlike scavenging of DPPH• , all FCPs exhibited
a lower scavenging activity toward O2 •− (Fig. 4d). However, the EC50 values increased in the order of FCPs-H (0.67 mg/mL) > FCPs-V (0.56 mg/mL) > FCPs-F (0.49 mg/mL). Fan et al. [35] reported that Ganoderma lucidum polysaccharides obtained by vacuum drying and vacuum freeze drying had the higher scavenging effects on OH• , O2 •− , DPPH• , and stronger reducing power than those obtained by hot air drying. In a similar study, Wu et al. [15] had reported that freeze dried ABMP (polysaccharides obtained from Agaricus blazei Murrill) showed some advantages over vacuum drying and air drying ABMP. In the present study, the antioxidant activities of FCPs isolated by ultrasonic extraction were slightly lower compared to those reported previously [3]. However, it showed that the antioxidant activities of FCPs-F isolated by ultrasonic extraction were insignificantly lower compared to the hot water technology [3]. According to the results above, freeze drying treatment may lead to a health beneficial FCPs-F for potential utilization in dietary supplements or functional food products. In conclusion, the results obtained in the present study clearly demonstrated that FCPs-F was a prospective radical scavenging agent. From the four models of antioxidant activities mentioned above, results showed that FCPs-F exhibited the highest antioxidant capacity among the three FCPs samples. This might be due to the different compositions and molecular size distributions of FCPsF, which had higher hydrogen-donating ability [13,36]. Nowadays more and more attention was cast on polysaccharides by biochemical and nutritional researchers due to their various biological
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activities used in health-care food or medicine, especially antioxidant effects. These polysaccharides are often identified as biological response modifiers (BRMs) [37]. Plants, particularly those used as traditional Chinese medicines, contain high amounts of natural polysaccharides that have been identified as free radicals or active oxygen scavengers [28,38]. Dehydration is one of the most important preservation methods employed in storage of finger citron and dehydrated finger citron are valuable ingredients in a variety of traditional Chinese medicinal material and functional vegetables. Further, the selective dehydration of polysaccharides without the protection of cell wall by an appropriate method is very important. Both yield and activity of polysaccharides are strongly dependent on the type of dehydration and drying temperature employed, due to the different potential of compounds with different denaturalization temperature [39]. 4. Conclusion This study showed that the drying method used for preparation of FCPs can significantly affect the physicochemical properties, which in turn affect the associated functionality of polysaccharides. The results of the present work indicated that freeze dried FCPs-F showed some advantages over FCPs-H and FCPs-V owing to its highest polysaccharides yield, neutral sugar content, relative viscosity, and low molecular size distribution, further exhibited best higher reducing power, and scavenging abilities on DPPH• , OH• , and O2 •− . The combined comparison of the physicochemical characterization and bioactivities of polysaccharides from finger citron is helpful to improve their pharmacological activity-based quality control, and beneficial for better understanding the structure–bioactivity relationship of polysaccharides from Chinese herbs. Based on the above studies, further detailed structural characterization and in vivo bioactivity of polysaccharides should be carried out to provide a good opportunity for scientists to elucidate the structure–function relationship and to explore high potential antioxidant agent. Acknowledgement This work was financially supported by Chongqing Municipal Health Bureau Science and Technology Project for Traditional Chinese Medicine (No. zy20132075).
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