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Ultrasonic extraction optimization of L. macranthoides polysaccharides and its physicochemical properties
Accepted Manuscript Title: Ultrasonic extraction optimization of L. macranthoides polysaccharides and its physicochemical properties Author: Zhen Wu Hong Li Yong Yang Hongjun Tan PII: DOI: Reference:
S0141-8130(14)00814-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.12.010 BIOMAC 4772
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
29-8-2014 15-11-2014 3-12-2014
Please cite this article as: Z. Wu, H. Li, Y. Yang, H. Tan, Ultrasonic extraction optimization of L. macranthoides polysaccharides and its physicochemical properties, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.12.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights (for review)
Highlights
Ultrasonic-assisted extraction of water-soluble polysaccharides from Lonicera macranthoides. Model was set up to optimization extraction of LMPs.
The best extraction methods were 113.6 W, 71.5 oC, 54.7 min, and W/M ratio 30.7
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mL/g. Structural features of LMPs were investigated.
The LMPs can be a new source of natural antioxidants.
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*Manuscript
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Ultrasonic extraction optimization of L. macranthoides polysaccharides and its
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physicochemical properties
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Zhen Wu a, *, Hong Li b, Yong Yang a, Hongjun Tan a
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a
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Republic of China
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b
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Republic of China
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Chongqing Academy of Chinese Materia Medica, Chongqing 400065, People’s
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Chongqing Institute for Food and Drug Control, Chongqing 401121, People’s
an
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*
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*
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fax +86 23 89 02 90 55; e-mail: [email protected], [email protected] (Z.
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Wu)].
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Author to whom correspondence should be addressed [phone +86 23 89 02 90 55;
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Correspondence author
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Abstract The dried flower buds of L. macranthoides, belong to the item Shan Yin Hua, are
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widely used as raw materials for pharmaceutical, food additive, healthy food and
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cosmetic industry in China. To optimize the effects of the ultrasonic-assisted
27
extraction (UAE) processing parameters on the yield of L. macranthoides
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polysaccharides (LMPs), a response surface methodology with a central composite
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rotatable design was employed. Four independent variables were investigated:
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ultrasonic power (X1), temperature (X2), time (X3), and the ratio of water volume to
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raw material weight (W/M ratio, X4). The experimental data were fitted to a quadratic
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polynomial equation using multiple regression analysis and also examined using
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appropriate statistical methods. The optimum conditions were: X1, 113.6 W; X2, 71.5
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o
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yield of LMPs was (4.81 ± 0.12)%, which is in close agreement with the value
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predicted by the statistical model. Further, LMPs were characterized by FT-IR, XRD,
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TGA/DSC and NMR. In vitro experiments indicated that LMPs had strong
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scavenging capacities towards the DPPH, hydroxyl and superoxide radicals. Overall,
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LMPs may have potential applications in the medical and food industries.
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Keywords: L. macranthoides; Polysaccharides; Physicochemical properties
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C; X3, 54.7 min; and X4, 30.7 mL/g. Under the optimal conditions, the extraction
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1. Introduction Lonicera macranthoides Hand.-Mazz. (L. macranthoides), a plant of the genus
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Lonicera of the Caprifoliaceae family, is commonly used as traditional Chinese
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medicine (TCM) in the southwest of China [1]. Its flower buds have been listed in the
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Chinese Pharmacopoeia since the 2005 edition as a newly added species, which forms
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the item Shan Yin Hua, together with L. hypoglauca Miq. and L. confuse DC. It is
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often used for the treatment of sores, furuncles, carbuncles, swelling and affections
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caused by exopathogenic wind-heat or epidemic febrile diseases [2]. The dried flower
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buds of L. macranthoides harvested during the summer are most commonly consumed
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dissolved in hot water or tea, which is very popular throughout China. Nowadays, a
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number of compounds, such as caffeoylquinic acid derivatives, flavonoids and
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saponins, have been reported from this species [1, 3, 4]. Therefore, L. macranthoides
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have attracted considerable interest due to their biological activities and potential
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applications in the food, pharmaceutical and environmental industries.
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Plant polysaccharides are often identified as immunomodulators or as biological
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response modifiers (BRMs) due to their biological and medicinal properties such as
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anticancer, immunostimulation and potential antioxidant properties [5-8]. However,
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there are few reports on the optimization of L. macranthoides polysaccharides (LMPs)
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production process and its antioxidant activities.
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Hot-water extraction (HWE) is the most frequently used method for extraction of
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plant polysaccharides [7, 9]. However, HWE requires high extraction temperature and
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is quite time consuming [10]. In recent years, ultrasonic-assisted extraction (UAE) has
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been widely used in natural products extraction process and it has been developed as
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an efficient alternative to conventional extraction techniques [10, 11]. This technique
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is fast, consumes less fossil energy and permits the reduction of solvents, thus
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resulting in a purer product and higher yields.
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The objectives of this study were to explore the potential of L. macranthoides in
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producing LMPs and to optimize the extraction conditions of LMPs. Response surface
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methodology (RSM) was applied to fit and to exploit a mathematical model
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representing the relationship between the response (extraction yield) and variables (i.e.
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ultrasonic power, extraction temperature, extraction time, and the ratio of water
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volume to raw material weight (W/M ratio)). Then, the preliminary characterization of
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LMPs was conducted via Fourier transform infrared spectrometry (FT-IR), X-ray
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diffraction (XRD), thermal gravimetric analysis/differential scanning calorimetry
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(TGA/DSC) and nuclear magnetic resonance (NMR). Finally, the antioxidant
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activities in vitro of LMPs against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl,
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and superoxide radicals were investigated.
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2. Materials and methods
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2.1. Materials
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The samples of L. macranthoides were collected in Xiushan county (coordinates:
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Lat. 28o47′ N. Long. 108o97′ E.), Chongqing, China, at an altitude of 550 m, and
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authenticated by Application and Development Institute of Herbal Medicinal Plants
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(Chongqing, China). All the collected samples were immediately dried at 60 oC for 5
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h. Samples were ground and sieved using a grinder and were passed through a
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40-mesh sieve. DPPH was purchased from Wako Pure Chemical Industries Ltd.
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(Tokyo, Japan). All other reagents and solvents were of analytical purity. All aqueous
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solutions were prepared by using newly double-distilled water.
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2.2. Extraction of crude polysaccharides
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The ultrasonic-assisted extraction of polysaccharides from L. macranthoides
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sample was performed using an ultrasonic clearer (Ningbo Scientz biotechnology Inc.,
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Ningbo, China) with thermostatic temperature control. Twenty grams of dried L.
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macranthoides powders were extracted with distilled water in a 250-mL beaker held
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in the ultrasonic clearer and extracted experimentally at a variety of selected
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ultrasonic powers at different temperatures, and for different lengths of time. The
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extract was left to cool at room temperature, filtered, and then precipitated using 150
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mL of 95% ethanol, 100% ethanol and acetone, respectively. After being left
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overnight at 4 oC, the precipitates were collected by centrifugation at 3, 000 rpm for
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20 min, redissolved in deionized water, deproteinated by the method of Sevag [12],
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dialyzed in a dialysis bag (MWCO 1400 Da, Union Carbide), and then freeze-dried to
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obtain LMPs.
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The polysaccharide extraction yield (Y) is calculated as follows:
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Y (%) = 100 × WLMPs/Wsample
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where WLMPs was defined as weight of LMPs whereas Wsample was defined as weight
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of samples power used (20 g).
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2.3. Experimental design
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(1)
Table 1.
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After determining the preliminary range of the extraction variables though
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preliminary experiments, a central composite design (CCD) with four independent
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variables (X1, ultrasonic power; X2, extraction temperature; X3, extraction time; X4:
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W/M ratio) at five levels was performed [13]. For statistical calculation, the variables
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were coded according to
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χi = (Xi – X0)/∆Xi
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where χi (i = 1, 2, 3 and 4) is a coded value of the variables; Xi the actual value of
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variables; X0 the actual value of the Xi on the center point; and ∆Xi the step change
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value. Thirty experiments (Table 1), which included sixteen factorial points, eight
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axial points and six replicated central points, were randomly performed. Experiments
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at the center point were conducted for evaluation of the experimental error. All trials
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were performed in triplicate. A Design-Expert Software Version 7.0 (STAT-EASE Inc.,
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Minneapolis, USA) was used to generate the experimental designs, statistical analysis,
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and regression model. A second-order polynomial equation was used to express the
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response (Y) as a function of the independent variables:
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Y β0 ∑βi X i ∑βii X i2 ∑∑βij X i X j
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(3)
i 1 j 1
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where Y is the dependent variable (extraction yield), β0 is the constant coefficient, βi,
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βii and βij are the linear, quadratic and interaction coefficients, respectively. The
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statistical significance of the terms in the regression equations was examined.
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2.4. Analysis of polysaccharides characterization
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The obtained LMPs under the optimum condition was stored in a desiccator prior
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to analysis. The sugar content was determined by the reaction of sugars with phenol in 6
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the presence of sulfuric acid using glucose as a standard [14]. Ash were determined
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according to AOAC (1990) method [15], while the protein content in the solid
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polysaccharide was determined using the Kjeldahl method with a conversion factor of
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6.25 [16]. Relative viscosity (to deionized water) of LMPs was measured in NDJ-1
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Rotation Viscometer (Jinghai Technology Co. Ltd., Shanghai, China) at a
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concentration of 10 mg/mL and 25 oC. FT-IR spectrum was obtained using a
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Spectrum 100 FT-IR spectrophotometer (PerkinElmer, USA). The dried LMPs was
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grinded with potassium bromide power and pressed into pellet for spectrometric
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measurement
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analysis/differential scanning calorimetry (Simultaneous TGA/DSC, STA-499 F3,
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NETZSCH) was used to determine the thermodynamic characteristics of crude
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polysaccharides. LMPs was heated from 30 to 600 oC at a heating rate of 10 oC /min
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under an atmosphere of nitrogen. X-ray diffraction pattern for the polysaccharide was
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analyzed using a Siemens D5000 (Japan) diffractometer equipped with a Cu Kα target
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at 40 kV and 30 mA with a scan rate of 4°/min. The diffraction angle ranged from 2θ
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= 5° to 2θ = 70°. 1H and 13C NMR spectra of isolated polysaccharide were recorded in
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an NMR spectrophotometer (Bruker ultrashield 300 NMR); chemical shifts are
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expressed in ppm downfield from tetramethyl silane.
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2.5. Antioxidant activity assay
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2.5.1. DPPH free radical (DPPH•) scavenging assay
range
of
4000–450
cm−1.
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Thermal
gravimetric
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The DPPH• scavenging activity of LMPs was carried out according to the method
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of Liu et al [17] with minor modification. Briefly, 1 mL of DPPH solution (0.1 mM
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DPPH in 95% ethanol) was added with 3 mL LMPs at the concentration of 0.5, 1.0,
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1.5, 2.0 and 2.5 mg/mL and reacted at room temperature. The mixture was shaken and
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the absorbance was measured at 517 nm. Ascorbic acid (Vc) was used as the positive
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control. The DPPH• scavenging activity was calculated using the following formula:
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DPPH• scavenging activity (%) = [A0–(A1–A2)] × 100/A0
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where A0 is the absorbance of the control (water instead of the sample solution), A1 is
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the absorbance of the sample, and A2 is the absorbance of the sample under identical
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condition as A1 with ethanol instead of DPPH• solution.
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2.5.2. Hydroxyl radical (OH•) scavenging assay
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(4)
Scavenging effects of LMPs on OH• was performed by the method previously
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described by Halliwell et al [18] with a minor modification. Reaction mixtures in a
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final volume of 1.0 mL contained deoxyribose (60 mM), phosphate buffer (pH 7.4, 20
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mM), ferric trichloride (100 μM), EDTA (100 μM), H2O2 (1 mM), ascorbic acid (100
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μM) and different concentrations of LMPs (0.5–2.5 mg/mL). The reaction solution
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was incubated for 1 h at 37 oC, and then 1 mL of 1% thiobarbituric acid and 1 mL of
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20% (v/v) HCl were added to the mixture. The mixture was boiled for 15 min and
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cooled on ice. Vc was used as a reference material. The absorbance of the mixture
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was measured at 532 nm. The OH• scavenging activity was calculated according to
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the following equation:
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OH• scavenging activity (%) = [A0–A1] × 100/A0
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where A0 is the absorbance of the control (water instead of the sample) and A1 is the
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absorbance of the sample.
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(5)
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2.5.3. Superoxide anion radical (O2•–) scavenging assay O2•– were generated by pyrogallic acid method [19] with a minor modification.
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The system contained 2.5 mL of phosphate buffer solution (PBS) (0.1 M, pH 8.2), 4
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mL of LMPs at the concentration of 0.5, 1.0, 1.5, 2.0 and 2.5 mg/mL, 2.5 mL of
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pyrogallic acid (6.0 mM), and 0.5 mL of thick hydrochloric acid for termination the
182
reaction. The solution was incubated at 25 oC and determined at 299 nm. Vc was used
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as the positive control. The O2•– scavenging activity was calculated as follows:
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O2•– scavenging activity (%) = [A0–(A1–A2)] × 100/A0
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where A0, with the presence of pyrogallic acid but without LMPs; A1, with the
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presence of pyrogallic acid and LMPs; and A2, with the presence of LMPs but without
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pyrogallic acid.
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2.6. Statistical analyses
(6)
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The significant terms in the model (Eq. (3)) were found by analysis of variance
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(ANOVA) for each response. The adequacy of the model was checked accounting for
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2 R2 (Coefficient of determination), Radj (the adjusted R2) and PRESS in Eqs. (7)–(9),
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respectively [20]:
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R 1
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2 Radj 1
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PRESS
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SS Residual SS Residual SS Model
(7)
SS Residual / DFResidual ( SS Residual SS Model ) /( DFResidual DFModel )
N
i
(YPred, i YExp,i )2
(8)
(9)
“Adequate precision” compares the range of the predicted values at the design points to the average prediction error. The definition of “Adequate precision” is in Eqs.
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(10) and (11):
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Adequate precision
Max( y ) Min ( y )
(10)
v( y ) v( y )
1 N Nσ 2 v( y ) n i1 n
(11)
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In Eqs. (7)–(11), SS is the sum of squares, DF is the degrees of freedom, YExp, i is
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the experimental responses, YPred, i is the predicted responses, y is the predicted
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value, N is the number of model parameters,
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ANOVA table, and n is the number of experiments.
is the residual mean square from
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Statistical analysis was performed using Design-Expert Software Version 7.0
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(STAT-EASE Inc., Minneapolis, USA) and SPSS (Version 15, SPSS Chicago IL)
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statistical software. Comparison of means was performed by one-way analysis of
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variance (ANOVA) followed by Duncan’s test.
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3. Results and discussion
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3.1. Optimization of LMPs extraction process
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3.1.1. Fitting of second order polynomial equation
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By applying multiple regression analysis on the experimental data, the
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Design-Expert software generated a second-order polynomial equation that can
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express the relationship between process variables and the response. The final
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equation obtained in terms of coded factors is given below:
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Extraction yield = 4.69–0.43X1 +0.26X2 –0.16X3 +0.27X4 –0.038X1 X2 –0.033X1 X3
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+0.36X 1 X 4 –0.25X 2 X 3 –0.34X 2 X 4 –0.30X 3 X 4 –0.60 X 12 –0.47 X 22
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–0.51 X 32 –0.47 X 42
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(12)
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where X1 is ultrasonic power (W), X2 is extraction temperature (oC), X3 is extraction
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time (min), and X4 is W/M ratio (mL/g).
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3.1.2. Model analysis Figure 1.
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Table 2.
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Table 3.
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Table 2 summarized the results of ANOVA, goodness-of-fit and the adequacy of
226
the model. The R2 for Eq. (12) was 0.9925, which was relatively high (close to unity),
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indicating a close agreement between experimental and predicted values of LMPs
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yield. This can be further evidenced by plotting the predicted values against the
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experimental values of LMPs yield as shown in Fig. 1, where the line of the best fit
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with the slope of 0.9929 and R2 = 0.9925 were obtained. This demonstrates that the
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2 established model is very suitable to explain the experimental range studied. The Radj
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is the correlation measure for testing the goodness-of-fit of the regression equation [6].
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The higher it is the better degree of correlation between the actual and predicted
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2 values of the yield of LMPs. The value Radj for Eq. (12) was 0.9854, which indicates
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that 98.54% of the total variation in the yield was attributed to the experimental
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variables studied. Moreover, a low value of coefficient of the variation (C.V.) (4.39%)
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clearly indicated high degree of precision and good deal of reliability for the
238
experimental values [6]. Besides, “Adequate precision” measured the signal to noise
239
ratio. A ratio greater than 4 was desirable [21]. The “Adequate precision” of 42.5691
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indicated that this model could be used to navigate the design space.
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The adequacy of the fitted quadratic polynomial model of the LMPs yield was
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further justified through ANOVA (Table 2). The “Model F-Value” of 141.0251
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implied the model was significant. There was only a 0.01% (p < 0.0001) chance that a
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“Model F-Value” this large could occur due to noise. At the same time, the “Lack of
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Fit F-Value” of 2.1785 implied the Lack of Fit was not significant relative to the pure
246
error, meaning that all models accurately predicted the related responses. The p-values
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were used as a tool to check the significance of each coefficient and indicated the
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pattern of interactions between variables [22]. As shown in Table 3, the independent
249
variables (X1, X2, X3, and X4), the interaction terms (X1X4, X2X3, X2X4, and X3X4), and
250
all two quadratic terms ( X 12 , X 22 , X 32 , and X 42 ) significantly affected the yield of
251
LMPs (p < 0.05).
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3.1.3. Analysis of contour and response surface plots
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Figure 2.
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Figure 3.
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In order to gain a better understanding of the results, the predicted models are
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presented in Figs. 2 and 3 as the 3-D response surface plot and contour plot. From
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Figs. 2a and 3a, it can be seen that the maximum extraction yield of LMPs could be
258
achieved when X1 and X2 were 112.8 W and 71.5 oC, respectively.
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Figs. 2b and 3b showed the effects of X1 and X3 on the yield of LMPs. With X2
260
set at 70 oC and X4 at 30.0 mL/g, it indicated that the maximum extraction yield of
261
LMPs can be achieved when X1 and X3 were at the threshold level of 115.6 W and
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57.7 min, respectively.
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Figs. 2c and 3c showed the 3-D response surface plot and the contour plot at
264
varying X1 and X4 at fixed X2 70 oC and X3 60 min. It can be seen that the maximum
265
extraction yield of LMPs could be achieved when X1 and X4 were 112.4 W and 30.9
266
mL/g, respectively.
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In Figs. 2d and 3d, when the 3-D response surface plot and the contour plot were
268
developed for the extraction yield of LMPs with varying X2 and X3 at fixed X1 120 W
269
and X4 30 mL/g. It indicated that the maximum extraction yield of LMPs can be
270
achieved when X2 and X3 at the threshold level of 71.3 oC and 57.0 min, respectively.
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In Figs. 2e and 3e, when the 3-D response surface plot and the contour plot were
272
developed for the extraction yield of LMPs with varying X2 and X4 at fixed X1 120 W
273
and X3 60 min. It indicated that the maximum extraction yield of LMPs can be
274
achieved when X2 and X4 at the threshold level of 70.9 oC and 31.2 mL/g,
275
respectively.
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When X1 and X2 was fixed at 120 W and 70 oC, the 3-D response surface plot and
277
the contour plot based on independent variables X3 and X4 were shown in Figs. 2f and
278
3f. It can be seen that the yield of LMPs increased with the increase of X3 from 40 to
279
56.1 min, then dropped slightly from 56.1 to 80 min, and the yield of LMPs increased
280
rapidly with the increase of X4 from 25 to 32.3 mL/g, but when beyond 32.3 mL/g, the
281
yield of LMPs did not further increase.
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In general, efficiency of UAE is influenced by multiple parameters such as
283
ultrasonic power, extraction temperature, extraction time, and W/M ratio, among
284
others, and their effects may be either independent or interactive [10, 23]. Ultrasonic
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enhancement of extraction was attributed to disruption of cell walls, particle-size
286
reduction, and enhanced mass transfer of the cell contents as a result of cavitation
287
bubble collapse [24, 25]. Our studies have shown that UAE with water is an
288
alternative means of increasing the speed of polysaccharide extraction.
289
3.1.4. Model adequacy checking
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Figure 4.
291
Generally, it is necessary to check that the fitted quadratic polynomial model
292
gives a sufficient approximation to the actual values. Unless the model shows an
293
adequate fit, proceeding with an investigation and optimization of the fitted response
294
surface likely gives poor or misleading results [26]. In addition to determination
295
coefficient, the adequacy of the models was also evaluated by the residuals [23]. As
296
shown in Fig. 4(a), the normal probability plot is a suitable graphical method for
297
judging residuals normality. The normality assumption was satisfied as the residual
298
plot approximated along a straight line. Fig. 4(b) shows that the residuals scatter
299
randomly on the display, suggesting that the variance of the original observation is
300
constant for all values of Y. Hence, trends observed in Fig. 4 revealed that, no obvious
301
patterns were found and residuals appeared to be randomly scattered.
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3.1.5. Experimental validation of the optimized condition
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Table 4.
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Optimization of the extraction procedure was based upon higher extraction yield
305
[7]. The optimal values of the selected variables were obtained by solving the
306
regression equation using the Design-Expert software. The suitability of the model
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equation for predicting optimum response value was investigated under the following
308
optimal conditions: ultrasonic power 113.6 W, extraction temperature 71.5 oC,
309
extraction time 54.7 min, and W/M ratio 30.7 mL/g (Table 4). The conditions were
310
determined to be optimum by RSM optimization process and were also used to
311
predict the values of the response. Under these conditions, the experimental extraction
312
yield of LMPs was (4.81 ± 0.12)%, which was agreed with predicted value 4.84%. No
313
significant difference (p > 0.05) was found between the experimental and the
314
predicted value. Therefore, the results indicated the suitability of the model employed
315
and the success of RSM in optimizing the extraction conditions.
316
3.2. Preliminary characterization of LMPs
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In the present study, LMPs was prepared through a series procedure of UAE
318
based on the optimal extraction conditions, centrifugation, ethanol precipitation and
319
drying. Then, LMPs was preliminary characterized by physicochemical analysis. The
320
contents of total sugar, protein, and ash in LMPs were 80.61 ± 2.03, 2.31 ± 0.22 and
321
4.33 ± 0.14%, respectively. Notably, the relative viscosity (to deionized water) was
322
2.74 ± 0.21.
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Figure 5.
FT-IR spectroscopy of LMPs is shown in Fig. 5a. A strong and broad absorption
325
peak at 3380 cm−1 for O−H stretching vibrations, a peak at 2930 cm−1 for C−H
326
stretching vibrations, and a strong extensive absorption in the region of 900–1200
327
cm−1 for coupled C−O and C−C stretching and C–OH bending vibrations were
328
observed in LMPs, indicating the characteristic absorptions of polysaccharides [27].
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Furthermore, an asymmetrical stretching peak at 1610 cm−1 and a weak symmetrical
330
stretching peak near 1400 cm−1 were assigned to the absorbance of the deprotonated
331
carboxylic group (COO−), indicating LMPs be acidic polysaccharides [28]. In
332
addition, the absorption at 1265 cm−1 was related to S＝O stretching vibration of the
333
sulfate group.
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TGA and DSC curves of LMPs are shown in Fig. 5b. Three different stages were
335
well defined during TGA and DSC analysis. The first one was basically associated
336
with the weight loss (moisture) due to dehydration, which covered a temperature
337
range between 25 oC and 120 oC. Subsequently, pyrolysis reactions of LMPs started at
338
120 oC. The second stage started at 185 oC and consisted in the devolatilization of the
339
sample, with evolution of the volatile matter mainly occurring between 220 oC and
340
540 oC. Finally, the third stage began close to 540 oC and was maintained up to 600
341
o
ed
M
an
us
cr
334
C.
The XRD pattern of LMPs is shown in Fig. 5c. The sample shows peaks at
343
approximately 28o, 31o, 33o and 42o 2θ. However, other peaks are very weak and
344
unresolved or are shoulders on more intense peaks. The result of the XRD confirms
345
that of the DSC, which shows that LMPs exhibits both crystalline and amorphous
346
portions [29].
Ac
ce pt
342
347
The signals of 1H NMR were 4.91 ppm (α-C-1), 4.76 ppm (β-C-1), 3.70 ppm
348
(C-5), 3.52 ppm (C-4), 3.65 ppm (C-3), and 3.37 ppm (C-2) are shown in Fig. 5d. The
349
anomeric protons have been assigned to β-sugar and α-sugar residues due to presence
350
of signals between 4.47–4.91 ppm and 5.08–5.09 ppm, respectively [30]. The signals
16
Page 17 of 36
351
at 60.74 ppm can be attributed to an O–methyl group attached to the 4-position of the
352
D-glucuronic
353
β-D-galactopyranose 103.84 ppm (C-1), 30.42 ppm (C-3), 69.71 ppm (C-4), 75.28
354
ppm (C-5), and 62.59 ppm (C-6) [31].
355
3.3. Antioxidant activity analysis
ip t
acid [29, 30]. Additionally, signals in Fig. 5e were also observed for
Table 5.
357
The result of antioxidant activities of LMPs are shown in Table 5 and compared
358
with Vc as control standards. The DPPH• scavenging ability increased from 10.94 to
359
75.69%, when the concentration of the polysaccharides increased from 0.50 to 2.5
360
mg/mL. The scavenging ability was lower than that of Vc. Similar results have been
361
reported in other plant polysaccharides [7, 21].
M
an
us
cr
356
The OH• scavenging ability increased from 13.06 to 69.39%, when the
363
concentration of LMPs increased from 0.50 to 2.50 mg/mL (Table 5). This shows
364
LMPs exhibited scavenging activity towards OH• in a concentration-dependent
365
manner and the scavenging effect increased based on the concentration of LMPs.
366
However, the antioxidant activity of LMPs was detected to be lower than that of Vc at
367
each concentration point. Further, O2•– scavenging activity of LMPs followed a
368
dose-dependent manner at all tested concentrations (Table 5). O2•– scavenging effects
369
of LMPs and Vc were 61.18% and 80.63%, respectively, at the concentration of 2.50
370
mg/mL.
Ac
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ed
362
371
As known to all, free radicals, chemical reactions and several redox reactions of
372
various compounds may cause protein oxidation, DNA damage, and lipid
17
Page 18 of 36
peroxidation in living cells [32]. In order to reduce damage to the human body and
374
prolong the storage stability of foods, synthetic antioxidants are used widely at
375
present. However, recent research suggested that synthetic antioxidants were
376
responsible for liver damage and carcinogenesis [33-35]. Our data indicate that
377
polysaccharides isolated from L. macranthoides have high antioxidant activities and
378
can be explored as a novel and potential natural antioxidant and anticancer agent for
379
use in functional or medicinal foods.
380
4. Conclusion
us
cr
ip t
373
RSM was used to determine the optimal process parameters that gave a high
382
extraction yield. ANOVA showed that the effects of ultrasonic power, temperature,
383
time, and W/M ratio were significant and quadratic models were obtained for
384
predicting the response. The optimal conditions were: ultrasonic power 113.6 W,
385
temperature 71.5 oC, time 54.7 min, and W/M ratio 30.7 mL/g. The extraction
386
information on L. macranthoides obtained in this work should also be helpful in other
387
species. The polysaccharides were characterized by FT-IR, DSC, TGA/XRD, 1H and
388
13
389
antioxidant activities assays demonstrated that LMPs had strong scavenging activities
390
in vitro on DPPH•, OH• and O2•–. LMPs should be explored as a novel potential
391
antioxidant, and further studies are essential to evaluate antioxidant activities in vivo
392
and elucidate the antioxidant mechanism.
393
Acknowledgements
394
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an
381
Ac
C NMR, which showed typical chemical structure of polysaccharides. The results of
This work was financially supported by the Chongqing Health Bureau for
18
Page 19 of 36
395
Traditional Chinese Medicine (No. zy20132075).
396 397
ip t
398 399
cr
400
us
401 402
an
403
M
404 405
ed
406
409 410 411 412
Ac
408
ce pt
407
413 414 415 416
19
Page 20 of 36
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418
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[31] G.L. de Pinto, M. Martinez, A.L. de Corredor, C. Rivas, E. Ocando,
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Phytochemistry 37 (1994) 1311-1315.
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[32] V. Sindhi, V. Gupta, K. Sharma, S. Bhatnagar, R. Kumari, N. Dhaka, J. Pharm.
471
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[33] N. Singh, P.S. Rajini, Food Chem. 85 (2004) 611-616.
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[34] A. Takagi, K. Sekita, M. Saitoh, J. Kanno, J. Toxicol. Sci. 30 (2005) 275-285.
474
[35] A.L. Branen, J. Am. Oil Chem. Soc. 52 (1975) 59-63.
477 478
cr
us
an
M
ed
ce pt
476
Ac
475
ip t
461
479 480 481 482
22
Page 23 of 36
Figure captions
484
Fig. 1. Correlation between the predicted and experimental yield of L. macranthoides
485
polysaccharides (LMPs).
486
Fig. 2. Response surface plots (a–f) showing the interactive effects of ultrasonic
487
power (X1), extraction temperature (X2), extraction time (X3), and W/M ratio (X4) on
488
the extraction yield of L. macranthoides polysaccharides (LMPs). Experimental data
489
and conditions are shown in Table 1.
490
Fig. 3. Contour plots (a–f) showing the interactive effects of ultrasonic power (X1),
491
extraction temperature (X2), extraction time (X3), and W/M ratio (X4) on the extraction
492
yield of L. macranthoides polysaccharides (LMPs). Experimental data and conditions
493
are shown in Table 1.
494
Fig. 4. (a) Normal probability of internally studentized residuals. (b) Plot of internally
495
studentized residuals vs. predicted response.
496
Fig. 5. Preliminary characterization of L. macranthoides polysaccharides (LMPs)
497
obtained under the optimum UAE conditions. (a) Fourier transform infrared spectrum
498
(FT-IR). (b) Thermal gravimetric analysis and differential scanning calorimetry (TGA
499
and DSC) thermograms. (c) X-ray diffraction (XRD) pattern. (d) 1 H NMR spectra
500
and (e) 13 C NMR spectra.
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483
23
Page 24 of 36
Table 1
Table 1 Factors and levels for response surface methodology, central composite design matrix, experimental data and predicted values for five-level-four-factor response surface analysis. Variable levelsb
LMPs yield (%)c
ip t
Numbera
X2
X3
X4
Observed
Predicted
1
-1(100)
-1(65)
-1(40)
-1(25)
1.98±0.09
2.08
2
1(140)
-1
-1
-1
0.78±0.12
0.66
3
-1
1(75)
-1
4
1
1
-1
5
-1
-1
1(80)
6
1
-1
7
-1
1
8
1
9
-1
us
3.87
-1
2.28±0.08
2.29
-1
3.08±0.16
2.93
1
-1
1.46±0.17
1.37
1
-1
3.77±0.08
3.71
ed
M
an
3.89±0.11
1
1
-1
1.94±0.12
2.00
-1
-1
1(35)
3.27±0.07
3.21
1
-1
-1
1
3.15±0.11
3.21
-1
1
-1
1
3.53±0.21
3.62
Ac
11
-1
ce pt
10
cr
X1
12
1
1
-1
1
3.31±0.18
3.46
13
-1
-1
1
1
2.86±0.09
2.85
14
1
-1
1
1
2.69±0.15
2.72
15
-1
1
1
1
2.13±0.11
2.25
16
1
1
1
1
2.07±0.07
1.97
17
-2(80)
0
0
0
3.17±0.21
3.16
Page 25 of 36
0
0
0
1.45±0.15
1.45
19
0(120)
-2(60)
0
0
2.17±0.17
2.29
20
0
2(80)
0
0
3.45±0.16
3.32
21
0
0(70)
-2(20)
0
3.07±0.11
2.96
22
0
0
2(100)
0
2.21±0.07
2.31
23
0
0
0(60)
-2(20)
2.12±0.12
2.25
24
0
0
0
2(40)
3.49±0.21
3.35
25
0
0
0
0(30)
4.71±0.21
26
0
0
0
us
4.69
0
4.75±0.18
4.69
27
0
0
0
0
4.78±0.17
4.69
28
0
0
0
0
4.55±0.16
4.69
29
0
0
0
0
4.58±0.17
4.69
30
0
ed
0
4.77±0.20
4.69
0
M 0
cr
ip t
2(160)
an
18
Experiments were conducted in a random order.
b
X1: ultrasonic power (W), X2: extraction temperature (oC), X3: extraction time (min),
ce pt
a
c
Ac
X4: W/M ratio (mL/g).
LMPs: L. macranthoides polysaccharides. Each value represented the mean ± SD (n
= 3).
Page 26 of 36
Table 2
Table 2 Analysis of variance (ANOVA) testing the fitness of the regression equation. Sum of squares df
Mean squares F-value
Model
35.3968
14 2.5283
Residual
0.2689
15 0.0179
Lack of fit 0.2187
10 0.0219
Pure error
0.0502
5
Cor total
35.6657
29
Probability prob > F
141.0251 < 0.0001
2.1785
us
cr
0.0100
0.2018
ip t
Source
2 2 R2=0.9925, Radj =0.9854, Rpred =0.9626, Adequate precision=42.5691, C.V.=4.39%,
Ac
ce pt
ed
M
an
PRESS=1.33.
Page 27 of 36
Table 3
Table 3 Testing of the significance of the regression coefficients associated with different experimental factors.
Factor
Standard
95% CI
95% CI
df
F-value low
high
prob > F b
0.0547
4.5735
4.8065
–
X1
-0.4279
1
0.0273
-0.4862
-0.3697
245.1262 < 0.0001
X2
0.2588
1
0.0273
0.2005
0.3170
89.6256
< 0.0001
X3
-0.1629
1
0.0273
-0.2212
-0.1047
35.5306
< 0.0001
X4
0.2738
1
0.0273
0.2155
0.3320
100.3182 < 0.0001
X1X2
-0.0381
1
0.0335
-0.1095
0.0332
1.2972
0.2726 (ns)
X1X3
-0.0331
1
0.0335
-0.1045
0.0382
0.9792
0.3381 (ns)
X1X4
0.3556
1
0.0335
0.2843
0.4270
112.8664 < 0.0001
X2X3
-0.2506
1
X2X4
-0.3444
1
X3X4
-0.3019
X 12
-0.5957
ed 0.0335
-0.3220
-0.1793
56.0569
0.0335
-0.4157
-0.2730
105.8384 < 0.0001
1
0.0335
-0.3732
-0.2305
81.3269
1
0.0256
-0.6502
-0.5412
542.9515 < 0.0001
ce pt
Ac
–
cr
1
an
Intercept 4.6900
us
error
M
estimate
Probability
ip t
Coefficient a
< 0.0001
< 0.0001
X 22
-0.4707
1
0.0256
-0.5252
-0.4162
339.0045 < 0.0001
X 32
-0.5132
1
0.0256
-0.5677
-0.4587
402.9823 < 0.0001
X 42
-0.4720
1
0.0256
-0.5265
-0.4175
340.8073 < 0.0001
a
X1: ultrasonic power (W), X2: extraction temperature (oC), X3: extraction time (min),
X4: W/M ratio (mL/g). b
p < 0.05 indicates statistical significance. ns = not significant at p ≤ 0.05.
Page 28 of 36
Table 4
Table 4 Predicted and experimental values of the responses at the optimum and modified conditions. Conditions a
X1 (W) X2 (oC) X3 (min) X4 (mL/g) LMPs yield (%) 54.72
30.72
4.84 (predicted)
Modified conditions
54.7
30.7
4.81 ± 0.12 (actual)
71.5
X1: ultrasonic power (W), X2: extraction temperature (oC), X3: extraction time (min),
cr
a
113.6
ip t
Optimum conditions 113.64 71.53
Ac
ce pt
ed
M
an
us
X4: W/M ratio (mL/g), LMPs: L. macranthoides polysaccharides.
Page 29 of 36
Accepted Manuscript Title: Ultrasonic extraction optimization of L. macranthoides polysaccharides and its physicochemical properties Author: Zhen Wu Hong Li Yong Yang Hongjun Tan PII: DOI: Reference:
S0141-8130(14)00814-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.12.010 BIOMAC 4772
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
29-8-2014 15-11-2014 3-12-2014
Please cite this article as: Z. Wu, H. Li, Y. Yang, H. Tan, Ultrasonic extraction optimization of L. macranthoides polysaccharides and its physicochemical properties, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.12.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Figure 2
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Figure 3
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Figure 4(a)
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Figure 4(b)
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Figure 5
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S0141-8130(14)00814-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.12.010 BIOMAC 4772
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
29-8-2014 15-11-2014 3-12-2014
Please cite this article as: Z. Wu, H. Li, Y. Yang, H. Tan, Ultrasonic extraction optimization of L. macranthoides polysaccharides and its physicochemical properties, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.12.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights (for review)
Highlights
Ultrasonic-assisted extraction of water-soluble polysaccharides from Lonicera macranthoides. Model was set up to optimization extraction of LMPs.
The best extraction methods were 113.6 W, 71.5 oC, 54.7 min, and W/M ratio 30.7
ip t
cr
mL/g. Structural features of LMPs were investigated.
The LMPs can be a new source of natural antioxidants.
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*Manuscript
1
Ultrasonic extraction optimization of L. macranthoides polysaccharides and its
2
physicochemical properties
3
Zhen Wu a, *, Hong Li b, Yong Yang a, Hongjun Tan a
5
a
6
Republic of China
7
b
8
Republic of China
ip t
4
cr
Chongqing Academy of Chinese Materia Medica, Chongqing 400065, People’s
us
Chongqing Institute for Food and Drug Control, Chongqing 401121, People’s
an
9 10
*
11
*
12
fax +86 23 89 02 90 55; e-mail: [email protected], [email protected] (Z.
13
Wu)].
16 17 18
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15
Author to whom correspondence should be addressed [phone +86 23 89 02 90 55;
Ac
14
Correspondence author
19 20 21 22
1
Page 2 of 36
23
Abstract The dried flower buds of L. macranthoides, belong to the item Shan Yin Hua, are
25
widely used as raw materials for pharmaceutical, food additive, healthy food and
26
cosmetic industry in China. To optimize the effects of the ultrasonic-assisted
27
extraction (UAE) processing parameters on the yield of L. macranthoides
28
polysaccharides (LMPs), a response surface methodology with a central composite
29
rotatable design was employed. Four independent variables were investigated:
30
ultrasonic power (X1), temperature (X2), time (X3), and the ratio of water volume to
31
raw material weight (W/M ratio, X4). The experimental data were fitted to a quadratic
32
polynomial equation using multiple regression analysis and also examined using
33
appropriate statistical methods. The optimum conditions were: X1, 113.6 W; X2, 71.5
34
o
35
yield of LMPs was (4.81 ± 0.12)%, which is in close agreement with the value
36
predicted by the statistical model. Further, LMPs were characterized by FT-IR, XRD,
37
TGA/DSC and NMR. In vitro experiments indicated that LMPs had strong
38
scavenging capacities towards the DPPH, hydroxyl and superoxide radicals. Overall,
39
LMPs may have potential applications in the medical and food industries.
40
Keywords: L. macranthoides; Polysaccharides; Physicochemical properties
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24
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C; X3, 54.7 min; and X4, 30.7 mL/g. Under the optimal conditions, the extraction
41 42 43 44
2
Page 3 of 36
45
1. Introduction Lonicera macranthoides Hand.-Mazz. (L. macranthoides), a plant of the genus
47
Lonicera of the Caprifoliaceae family, is commonly used as traditional Chinese
48
medicine (TCM) in the southwest of China [1]. Its flower buds have been listed in the
49
Chinese Pharmacopoeia since the 2005 edition as a newly added species, which forms
50
the item Shan Yin Hua, together with L. hypoglauca Miq. and L. confuse DC. It is
51
often used for the treatment of sores, furuncles, carbuncles, swelling and affections
52
caused by exopathogenic wind-heat or epidemic febrile diseases [2]. The dried flower
53
buds of L. macranthoides harvested during the summer are most commonly consumed
54
dissolved in hot water or tea, which is very popular throughout China. Nowadays, a
55
number of compounds, such as caffeoylquinic acid derivatives, flavonoids and
56
saponins, have been reported from this species [1, 3, 4]. Therefore, L. macranthoides
57
have attracted considerable interest due to their biological activities and potential
58
applications in the food, pharmaceutical and environmental industries.
ce pt
ed
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an
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cr
ip t
46
Plant polysaccharides are often identified as immunomodulators or as biological
60
response modifiers (BRMs) due to their biological and medicinal properties such as
61
anticancer, immunostimulation and potential antioxidant properties [5-8]. However,
62
there are few reports on the optimization of L. macranthoides polysaccharides (LMPs)
63
production process and its antioxidant activities.
Ac
59
64
Hot-water extraction (HWE) is the most frequently used method for extraction of
65
plant polysaccharides [7, 9]. However, HWE requires high extraction temperature and
66
is quite time consuming [10]. In recent years, ultrasonic-assisted extraction (UAE) has
3
Page 4 of 36
been widely used in natural products extraction process and it has been developed as
68
an efficient alternative to conventional extraction techniques [10, 11]. This technique
69
is fast, consumes less fossil energy and permits the reduction of solvents, thus
70
resulting in a purer product and higher yields.
ip t
67
The objectives of this study were to explore the potential of L. macranthoides in
72
producing LMPs and to optimize the extraction conditions of LMPs. Response surface
73
methodology (RSM) was applied to fit and to exploit a mathematical model
74
representing the relationship between the response (extraction yield) and variables (i.e.
75
ultrasonic power, extraction temperature, extraction time, and the ratio of water
76
volume to raw material weight (W/M ratio)). Then, the preliminary characterization of
77
LMPs was conducted via Fourier transform infrared spectrometry (FT-IR), X-ray
78
diffraction (XRD), thermal gravimetric analysis/differential scanning calorimetry
79
(TGA/DSC) and nuclear magnetic resonance (NMR). Finally, the antioxidant
80
activities in vitro of LMPs against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl,
81
and superoxide radicals were investigated.
82
2. Materials and methods
83
2.1. Materials
us
an
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ed
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84
cr
71
The samples of L. macranthoides were collected in Xiushan county (coordinates:
85
Lat. 28o47′ N. Long. 108o97′ E.), Chongqing, China, at an altitude of 550 m, and
86
authenticated by Application and Development Institute of Herbal Medicinal Plants
87
(Chongqing, China). All the collected samples were immediately dried at 60 oC for 5
88
h. Samples were ground and sieved using a grinder and were passed through a
4
Page 5 of 36
40-mesh sieve. DPPH was purchased from Wako Pure Chemical Industries Ltd.
90
(Tokyo, Japan). All other reagents and solvents were of analytical purity. All aqueous
91
solutions were prepared by using newly double-distilled water.
92
2.2. Extraction of crude polysaccharides
ip t
89
The ultrasonic-assisted extraction of polysaccharides from L. macranthoides
94
sample was performed using an ultrasonic clearer (Ningbo Scientz biotechnology Inc.,
95
Ningbo, China) with thermostatic temperature control. Twenty grams of dried L.
96
macranthoides powders were extracted with distilled water in a 250-mL beaker held
97
in the ultrasonic clearer and extracted experimentally at a variety of selected
98
ultrasonic powers at different temperatures, and for different lengths of time. The
99
extract was left to cool at room temperature, filtered, and then precipitated using 150
100
mL of 95% ethanol, 100% ethanol and acetone, respectively. After being left
101
overnight at 4 oC, the precipitates were collected by centrifugation at 3, 000 rpm for
102
20 min, redissolved in deionized water, deproteinated by the method of Sevag [12],
103
dialyzed in a dialysis bag (MWCO 1400 Da, Union Carbide), and then freeze-dried to
104
obtain LMPs.
us
an
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ed
ce pt
Ac
105
cr
93
The polysaccharide extraction yield (Y) is calculated as follows:
106
Y (%) = 100 × WLMPs/Wsample
107
where WLMPs was defined as weight of LMPs whereas Wsample was defined as weight
108
of samples power used (20 g).
109
2.3. Experimental design
110
(1)
Table 1.
5
Page 6 of 36
After determining the preliminary range of the extraction variables though
112
preliminary experiments, a central composite design (CCD) with four independent
113
variables (X1, ultrasonic power; X2, extraction temperature; X3, extraction time; X4:
114
W/M ratio) at five levels was performed [13]. For statistical calculation, the variables
115
were coded according to
116
χi = (Xi – X0)/∆Xi
117
where χi (i = 1, 2, 3 and 4) is a coded value of the variables; Xi the actual value of
118
variables; X0 the actual value of the Xi on the center point; and ∆Xi the step change
119
value. Thirty experiments (Table 1), which included sixteen factorial points, eight
120
axial points and six replicated central points, were randomly performed. Experiments
121
at the center point were conducted for evaluation of the experimental error. All trials
122
were performed in triplicate. A Design-Expert Software Version 7.0 (STAT-EASE Inc.,
123
Minneapolis, USA) was used to generate the experimental designs, statistical analysis,
124
and regression model. A second-order polynomial equation was used to express the
125
response (Y) as a function of the independent variables:
126
Y β0 ∑βi X i ∑βii X i2 ∑∑βij X i X j
ip t
111
3
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an
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cr
(2)
Ac
i 1
3
i 1
3
3
(3)
i 1 j 1
127
where Y is the dependent variable (extraction yield), β0 is the constant coefficient, βi,
128
βii and βij are the linear, quadratic and interaction coefficients, respectively. The
129
statistical significance of the terms in the regression equations was examined.
130
2.4. Analysis of polysaccharides characterization
131
The obtained LMPs under the optimum condition was stored in a desiccator prior
132
to analysis. The sugar content was determined by the reaction of sugars with phenol in 6
Page 7 of 36
the presence of sulfuric acid using glucose as a standard [14]. Ash were determined
134
according to AOAC (1990) method [15], while the protein content in the solid
135
polysaccharide was determined using the Kjeldahl method with a conversion factor of
136
6.25 [16]. Relative viscosity (to deionized water) of LMPs was measured in NDJ-1
137
Rotation Viscometer (Jinghai Technology Co. Ltd., Shanghai, China) at a
138
concentration of 10 mg/mL and 25 oC. FT-IR spectrum was obtained using a
139
Spectrum 100 FT-IR spectrophotometer (PerkinElmer, USA). The dried LMPs was
140
grinded with potassium bromide power and pressed into pellet for spectrometric
141
measurement
142
analysis/differential scanning calorimetry (Simultaneous TGA/DSC, STA-499 F3,
143
NETZSCH) was used to determine the thermodynamic characteristics of crude
144
polysaccharides. LMPs was heated from 30 to 600 oC at a heating rate of 10 oC /min
145
under an atmosphere of nitrogen. X-ray diffraction pattern for the polysaccharide was
146
analyzed using a Siemens D5000 (Japan) diffractometer equipped with a Cu Kα target
147
at 40 kV and 30 mA with a scan rate of 4°/min. The diffraction angle ranged from 2θ
148
= 5° to 2θ = 70°. 1H and 13C NMR spectra of isolated polysaccharide were recorded in
149
an NMR spectrophotometer (Bruker ultrashield 300 NMR); chemical shifts are
150
expressed in ppm downfield from tetramethyl silane.
151
2.5. Antioxidant activity assay
152
2.5.1. DPPH free radical (DPPH•) scavenging assay
range
of
4000–450
cm−1.
an
the
Thermal
gravimetric
Ac
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ed
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in
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133
153
The DPPH• scavenging activity of LMPs was carried out according to the method
154
of Liu et al [17] with minor modification. Briefly, 1 mL of DPPH solution (0.1 mM
7
Page 8 of 36
DPPH in 95% ethanol) was added with 3 mL LMPs at the concentration of 0.5, 1.0,
156
1.5, 2.0 and 2.5 mg/mL and reacted at room temperature. The mixture was shaken and
157
the absorbance was measured at 517 nm. Ascorbic acid (Vc) was used as the positive
158
control. The DPPH• scavenging activity was calculated using the following formula:
159
DPPH• scavenging activity (%) = [A0–(A1–A2)] × 100/A0
160
where A0 is the absorbance of the control (water instead of the sample solution), A1 is
161
the absorbance of the sample, and A2 is the absorbance of the sample under identical
162
condition as A1 with ethanol instead of DPPH• solution.
163
2.5.2. Hydroxyl radical (OH•) scavenging assay
ip t
155
an
us
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(4)
Scavenging effects of LMPs on OH• was performed by the method previously
165
described by Halliwell et al [18] with a minor modification. Reaction mixtures in a
166
final volume of 1.0 mL contained deoxyribose (60 mM), phosphate buffer (pH 7.4, 20
167
mM), ferric trichloride (100 μM), EDTA (100 μM), H2O2 (1 mM), ascorbic acid (100
168
μM) and different concentrations of LMPs (0.5–2.5 mg/mL). The reaction solution
169
was incubated for 1 h at 37 oC, and then 1 mL of 1% thiobarbituric acid and 1 mL of
170
20% (v/v) HCl were added to the mixture. The mixture was boiled for 15 min and
171
cooled on ice. Vc was used as a reference material. The absorbance of the mixture
172
was measured at 532 nm. The OH• scavenging activity was calculated according to
173
the following equation:
174
OH• scavenging activity (%) = [A0–A1] × 100/A0
175
where A0 is the absorbance of the control (water instead of the sample) and A1 is the
176
absorbance of the sample.
Ac
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164
(5)
8
Page 9 of 36
177
2.5.3. Superoxide anion radical (O2•–) scavenging assay O2•– were generated by pyrogallic acid method [19] with a minor modification.
179
The system contained 2.5 mL of phosphate buffer solution (PBS) (0.1 M, pH 8.2), 4
180
mL of LMPs at the concentration of 0.5, 1.0, 1.5, 2.0 and 2.5 mg/mL, 2.5 mL of
181
pyrogallic acid (6.0 mM), and 0.5 mL of thick hydrochloric acid for termination the
182
reaction. The solution was incubated at 25 oC and determined at 299 nm. Vc was used
183
as the positive control. The O2•– scavenging activity was calculated as follows:
184
O2•– scavenging activity (%) = [A0–(A1–A2)] × 100/A0
185
where A0, with the presence of pyrogallic acid but without LMPs; A1, with the
186
presence of pyrogallic acid and LMPs; and A2, with the presence of LMPs but without
187
pyrogallic acid.
188
2.6. Statistical analyses
(6)
ed
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ip t
178
The significant terms in the model (Eq. (3)) were found by analysis of variance
190
(ANOVA) for each response. The adequacy of the model was checked accounting for
191
2 R2 (Coefficient of determination), Radj (the adjusted R2) and PRESS in Eqs. (7)–(9),
192
respectively [20]:
193
R 1
194
2 Radj 1
195
PRESS
196 197
Ac
ce pt
189
SS Residual SS Residual SS Model
(7)
SS Residual / DFResidual ( SS Residual SS Model ) /( DFResidual DFModel )
N
i
(YPred, i YExp,i )2
(8)
(9)
“Adequate precision” compares the range of the predicted values at the design points to the average prediction error. The definition of “Adequate precision” is in Eqs.
9
Page 10 of 36
198
(10) and (11):
199
Adequate precision
Max( y ) Min ( y )
(10)
v( y ) v( y )
1 N Nσ 2 v( y ) n i1 n
(11)
ip t
200
In Eqs. (7)–(11), SS is the sum of squares, DF is the degrees of freedom, YExp, i is
202
the experimental responses, YPred, i is the predicted responses, y is the predicted
203
value, N is the number of model parameters,
204
ANOVA table, and n is the number of experiments.
is the residual mean square from
us
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201
Statistical analysis was performed using Design-Expert Software Version 7.0
206
(STAT-EASE Inc., Minneapolis, USA) and SPSS (Version 15, SPSS Chicago IL)
207
statistical software. Comparison of means was performed by one-way analysis of
208
variance (ANOVA) followed by Duncan’s test.
209
3. Results and discussion
210
3.1. Optimization of LMPs extraction process
211
3.1.1. Fitting of second order polynomial equation
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an
205
By applying multiple regression analysis on the experimental data, the
213
Design-Expert software generated a second-order polynomial equation that can
214
express the relationship between process variables and the response. The final
215
equation obtained in terms of coded factors is given below:
216
Extraction yield = 4.69–0.43X1 +0.26X2 –0.16X3 +0.27X4 –0.038X1 X2 –0.033X1 X3
217
+0.36X 1 X 4 –0.25X 2 X 3 –0.34X 2 X 4 –0.30X 3 X 4 –0.60 X 12 –0.47 X 22
218
–0.51 X 32 –0.47 X 42
Ac
212
(12)
10
Page 11 of 36
219
where X1 is ultrasonic power (W), X2 is extraction temperature (oC), X3 is extraction
220
time (min), and X4 is W/M ratio (mL/g).
221
3.1.2. Model analysis Figure 1.
223
Table 2.
224
Table 3.
225
Table 2 summarized the results of ANOVA, goodness-of-fit and the adequacy of
226
the model. The R2 for Eq. (12) was 0.9925, which was relatively high (close to unity),
227
indicating a close agreement between experimental and predicted values of LMPs
228
yield. This can be further evidenced by plotting the predicted values against the
229
experimental values of LMPs yield as shown in Fig. 1, where the line of the best fit
230
with the slope of 0.9929 and R2 = 0.9925 were obtained. This demonstrates that the
231
2 established model is very suitable to explain the experimental range studied. The Radj
232
is the correlation measure for testing the goodness-of-fit of the regression equation [6].
233
The higher it is the better degree of correlation between the actual and predicted
234
2 values of the yield of LMPs. The value Radj for Eq. (12) was 0.9854, which indicates
235
that 98.54% of the total variation in the yield was attributed to the experimental
236
variables studied. Moreover, a low value of coefficient of the variation (C.V.) (4.39%)
237
clearly indicated high degree of precision and good deal of reliability for the
238
experimental values [6]. Besides, “Adequate precision” measured the signal to noise
239
ratio. A ratio greater than 4 was desirable [21]. The “Adequate precision” of 42.5691
240
indicated that this model could be used to navigate the design space.
Ac
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222
11
Page 12 of 36
The adequacy of the fitted quadratic polynomial model of the LMPs yield was
242
further justified through ANOVA (Table 2). The “Model F-Value” of 141.0251
243
implied the model was significant. There was only a 0.01% (p < 0.0001) chance that a
244
“Model F-Value” this large could occur due to noise. At the same time, the “Lack of
245
Fit F-Value” of 2.1785 implied the Lack of Fit was not significant relative to the pure
246
error, meaning that all models accurately predicted the related responses. The p-values
247
were used as a tool to check the significance of each coefficient and indicated the
248
pattern of interactions between variables [22]. As shown in Table 3, the independent
249
variables (X1, X2, X3, and X4), the interaction terms (X1X4, X2X3, X2X4, and X3X4), and
250
all two quadratic terms ( X 12 , X 22 , X 32 , and X 42 ) significantly affected the yield of
251
LMPs (p < 0.05).
252
3.1.3. Analysis of contour and response surface plots
ed
M
an
us
cr
ip t
241
Figure 2.
254
Figure 3.
255
In order to gain a better understanding of the results, the predicted models are
256
presented in Figs. 2 and 3 as the 3-D response surface plot and contour plot. From
257
Figs. 2a and 3a, it can be seen that the maximum extraction yield of LMPs could be
258
achieved when X1 and X2 were 112.8 W and 71.5 oC, respectively.
Ac
ce pt
253
259
Figs. 2b and 3b showed the effects of X1 and X3 on the yield of LMPs. With X2
260
set at 70 oC and X4 at 30.0 mL/g, it indicated that the maximum extraction yield of
261
LMPs can be achieved when X1 and X3 were at the threshold level of 115.6 W and
262
57.7 min, respectively.
12
Page 13 of 36
Figs. 2c and 3c showed the 3-D response surface plot and the contour plot at
264
varying X1 and X4 at fixed X2 70 oC and X3 60 min. It can be seen that the maximum
265
extraction yield of LMPs could be achieved when X1 and X4 were 112.4 W and 30.9
266
mL/g, respectively.
ip t
263
In Figs. 2d and 3d, when the 3-D response surface plot and the contour plot were
268
developed for the extraction yield of LMPs with varying X2 and X3 at fixed X1 120 W
269
and X4 30 mL/g. It indicated that the maximum extraction yield of LMPs can be
270
achieved when X2 and X3 at the threshold level of 71.3 oC and 57.0 min, respectively.
us
cr
267
In Figs. 2e and 3e, when the 3-D response surface plot and the contour plot were
272
developed for the extraction yield of LMPs with varying X2 and X4 at fixed X1 120 W
273
and X3 60 min. It indicated that the maximum extraction yield of LMPs can be
274
achieved when X2 and X4 at the threshold level of 70.9 oC and 31.2 mL/g,
275
respectively.
ed
M
an
271
When X1 and X2 was fixed at 120 W and 70 oC, the 3-D response surface plot and
277
the contour plot based on independent variables X3 and X4 were shown in Figs. 2f and
278
3f. It can be seen that the yield of LMPs increased with the increase of X3 from 40 to
279
56.1 min, then dropped slightly from 56.1 to 80 min, and the yield of LMPs increased
280
rapidly with the increase of X4 from 25 to 32.3 mL/g, but when beyond 32.3 mL/g, the
281
yield of LMPs did not further increase.
Ac
ce pt
276
282
In general, efficiency of UAE is influenced by multiple parameters such as
283
ultrasonic power, extraction temperature, extraction time, and W/M ratio, among
284
others, and their effects may be either independent or interactive [10, 23]. Ultrasonic
13
Page 14 of 36
enhancement of extraction was attributed to disruption of cell walls, particle-size
286
reduction, and enhanced mass transfer of the cell contents as a result of cavitation
287
bubble collapse [24, 25]. Our studies have shown that UAE with water is an
288
alternative means of increasing the speed of polysaccharide extraction.
289
3.1.4. Model adequacy checking
ip t
285
Figure 4.
291
Generally, it is necessary to check that the fitted quadratic polynomial model
292
gives a sufficient approximation to the actual values. Unless the model shows an
293
adequate fit, proceeding with an investigation and optimization of the fitted response
294
surface likely gives poor or misleading results [26]. In addition to determination
295
coefficient, the adequacy of the models was also evaluated by the residuals [23]. As
296
shown in Fig. 4(a), the normal probability plot is a suitable graphical method for
297
judging residuals normality. The normality assumption was satisfied as the residual
298
plot approximated along a straight line. Fig. 4(b) shows that the residuals scatter
299
randomly on the display, suggesting that the variance of the original observation is
300
constant for all values of Y. Hence, trends observed in Fig. 4 revealed that, no obvious
301
patterns were found and residuals appeared to be randomly scattered.
302
3.1.5. Experimental validation of the optimized condition
Ac
ce pt
ed
M
an
us
cr
290
303
Table 4.
304
Optimization of the extraction procedure was based upon higher extraction yield
305
[7]. The optimal values of the selected variables were obtained by solving the
306
regression equation using the Design-Expert software. The suitability of the model
14
Page 15 of 36
equation for predicting optimum response value was investigated under the following
308
optimal conditions: ultrasonic power 113.6 W, extraction temperature 71.5 oC,
309
extraction time 54.7 min, and W/M ratio 30.7 mL/g (Table 4). The conditions were
310
determined to be optimum by RSM optimization process and were also used to
311
predict the values of the response. Under these conditions, the experimental extraction
312
yield of LMPs was (4.81 ± 0.12)%, which was agreed with predicted value 4.84%. No
313
significant difference (p > 0.05) was found between the experimental and the
314
predicted value. Therefore, the results indicated the suitability of the model employed
315
and the success of RSM in optimizing the extraction conditions.
316
3.2. Preliminary characterization of LMPs
M
an
us
cr
ip t
307
In the present study, LMPs was prepared through a series procedure of UAE
318
based on the optimal extraction conditions, centrifugation, ethanol precipitation and
319
drying. Then, LMPs was preliminary characterized by physicochemical analysis. The
320
contents of total sugar, protein, and ash in LMPs were 80.61 ± 2.03, 2.31 ± 0.22 and
321
4.33 ± 0.14%, respectively. Notably, the relative viscosity (to deionized water) was
322
2.74 ± 0.21.
324
ce pt
Ac
323
ed
317
Figure 5.
FT-IR spectroscopy of LMPs is shown in Fig. 5a. A strong and broad absorption
325
peak at 3380 cm−1 for O−H stretching vibrations, a peak at 2930 cm−1 for C−H
326
stretching vibrations, and a strong extensive absorption in the region of 900–1200
327
cm−1 for coupled C−O and C−C stretching and C–OH bending vibrations were
328
observed in LMPs, indicating the characteristic absorptions of polysaccharides [27].
15
Page 16 of 36
Furthermore, an asymmetrical stretching peak at 1610 cm−1 and a weak symmetrical
330
stretching peak near 1400 cm−1 were assigned to the absorbance of the deprotonated
331
carboxylic group (COO−), indicating LMPs be acidic polysaccharides [28]. In
332
addition, the absorption at 1265 cm−1 was related to S＝O stretching vibration of the
333
sulfate group.
ip t
329
TGA and DSC curves of LMPs are shown in Fig. 5b. Three different stages were
335
well defined during TGA and DSC analysis. The first one was basically associated
336
with the weight loss (moisture) due to dehydration, which covered a temperature
337
range between 25 oC and 120 oC. Subsequently, pyrolysis reactions of LMPs started at
338
120 oC. The second stage started at 185 oC and consisted in the devolatilization of the
339
sample, with evolution of the volatile matter mainly occurring between 220 oC and
340
540 oC. Finally, the third stage began close to 540 oC and was maintained up to 600
341
o
ed
M
an
us
cr
334
C.
The XRD pattern of LMPs is shown in Fig. 5c. The sample shows peaks at
343
approximately 28o, 31o, 33o and 42o 2θ. However, other peaks are very weak and
344
unresolved or are shoulders on more intense peaks. The result of the XRD confirms
345
that of the DSC, which shows that LMPs exhibits both crystalline and amorphous
346
portions [29].
Ac
ce pt
342
347
The signals of 1H NMR were 4.91 ppm (α-C-1), 4.76 ppm (β-C-1), 3.70 ppm
348
(C-5), 3.52 ppm (C-4), 3.65 ppm (C-3), and 3.37 ppm (C-2) are shown in Fig. 5d. The
349
anomeric protons have been assigned to β-sugar and α-sugar residues due to presence
350
of signals between 4.47–4.91 ppm and 5.08–5.09 ppm, respectively [30]. The signals
16
Page 17 of 36
351
at 60.74 ppm can be attributed to an O–methyl group attached to the 4-position of the
352
D-glucuronic
353
β-D-galactopyranose 103.84 ppm (C-1), 30.42 ppm (C-3), 69.71 ppm (C-4), 75.28
354
ppm (C-5), and 62.59 ppm (C-6) [31].
355
3.3. Antioxidant activity analysis
ip t
acid [29, 30]. Additionally, signals in Fig. 5e were also observed for
Table 5.
357
The result of antioxidant activities of LMPs are shown in Table 5 and compared
358
with Vc as control standards. The DPPH• scavenging ability increased from 10.94 to
359
75.69%, when the concentration of the polysaccharides increased from 0.50 to 2.5
360
mg/mL. The scavenging ability was lower than that of Vc. Similar results have been
361
reported in other plant polysaccharides [7, 21].
M
an
us
cr
356
The OH• scavenging ability increased from 13.06 to 69.39%, when the
363
concentration of LMPs increased from 0.50 to 2.50 mg/mL (Table 5). This shows
364
LMPs exhibited scavenging activity towards OH• in a concentration-dependent
365
manner and the scavenging effect increased based on the concentration of LMPs.
366
However, the antioxidant activity of LMPs was detected to be lower than that of Vc at
367
each concentration point. Further, O2•– scavenging activity of LMPs followed a
368
dose-dependent manner at all tested concentrations (Table 5). O2•– scavenging effects
369
of LMPs and Vc were 61.18% and 80.63%, respectively, at the concentration of 2.50
370
mg/mL.
Ac
ce pt
ed
362
371
As known to all, free radicals, chemical reactions and several redox reactions of
372
various compounds may cause protein oxidation, DNA damage, and lipid
17
Page 18 of 36
peroxidation in living cells [32]. In order to reduce damage to the human body and
374
prolong the storage stability of foods, synthetic antioxidants are used widely at
375
present. However, recent research suggested that synthetic antioxidants were
376
responsible for liver damage and carcinogenesis [33-35]. Our data indicate that
377
polysaccharides isolated from L. macranthoides have high antioxidant activities and
378
can be explored as a novel and potential natural antioxidant and anticancer agent for
379
use in functional or medicinal foods.
380
4. Conclusion
us
cr
ip t
373
RSM was used to determine the optimal process parameters that gave a high
382
extraction yield. ANOVA showed that the effects of ultrasonic power, temperature,
383
time, and W/M ratio were significant and quadratic models were obtained for
384
predicting the response. The optimal conditions were: ultrasonic power 113.6 W,
385
temperature 71.5 oC, time 54.7 min, and W/M ratio 30.7 mL/g. The extraction
386
information on L. macranthoides obtained in this work should also be helpful in other
387
species. The polysaccharides were characterized by FT-IR, DSC, TGA/XRD, 1H and
388
13
389
antioxidant activities assays demonstrated that LMPs had strong scavenging activities
390
in vitro on DPPH•, OH• and O2•–. LMPs should be explored as a novel potential
391
antioxidant, and further studies are essential to evaluate antioxidant activities in vivo
392
and elucidate the antioxidant mechanism.
393
Acknowledgements
394
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ed
M
an
381
Ac
C NMR, which showed typical chemical structure of polysaccharides. The results of
This work was financially supported by the Chongqing Health Bureau for
18
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Traditional Chinese Medicine (No. zy20132075).
396 397
ip t
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us
401 402
an
403
M
404 405
ed
406
409 410 411 412
Ac
408
ce pt
407
413 414 415 416
19
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477 478
cr
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an
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ce pt
476
Ac
475
ip t
461
479 480 481 482
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Figure captions
484
Fig. 1. Correlation between the predicted and experimental yield of L. macranthoides
485
polysaccharides (LMPs).
486
Fig. 2. Response surface plots (a–f) showing the interactive effects of ultrasonic
487
power (X1), extraction temperature (X2), extraction time (X3), and W/M ratio (X4) on
488
the extraction yield of L. macranthoides polysaccharides (LMPs). Experimental data
489
and conditions are shown in Table 1.
490
Fig. 3. Contour plots (a–f) showing the interactive effects of ultrasonic power (X1),
491
extraction temperature (X2), extraction time (X3), and W/M ratio (X4) on the extraction
492
yield of L. macranthoides polysaccharides (LMPs). Experimental data and conditions
493
are shown in Table 1.
494
Fig. 4. (a) Normal probability of internally studentized residuals. (b) Plot of internally
495
studentized residuals vs. predicted response.
496
Fig. 5. Preliminary characterization of L. macranthoides polysaccharides (LMPs)
497
obtained under the optimum UAE conditions. (a) Fourier transform infrared spectrum
498
(FT-IR). (b) Thermal gravimetric analysis and differential scanning calorimetry (TGA
499
and DSC) thermograms. (c) X-ray diffraction (XRD) pattern. (d) 1 H NMR spectra
500
and (e) 13 C NMR spectra.
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Table 1
Table 1 Factors and levels for response surface methodology, central composite design matrix, experimental data and predicted values for five-level-four-factor response surface analysis. Variable levelsb
LMPs yield (%)c
ip t
Numbera
X2
X3
X4
Observed
Predicted
1
-1(100)
-1(65)
-1(40)
-1(25)
1.98±0.09
2.08
2
1(140)
-1
-1
-1
0.78±0.12
0.66
3
-1
1(75)
-1
4
1
1
-1
5
-1
-1
1(80)
6
1
-1
7
-1
1
8
1
9
-1
us
3.87
-1
2.28±0.08
2.29
-1
3.08±0.16
2.93
1
-1
1.46±0.17
1.37
1
-1
3.77±0.08
3.71
ed
M
an
3.89±0.11
1
1
-1
1.94±0.12
2.00
-1
-1
1(35)
3.27±0.07
3.21
1
-1
-1
1
3.15±0.11
3.21
-1
1
-1
1
3.53±0.21
3.62
Ac
11
-1
ce pt
10
cr
X1
12
1
1
-1
1
3.31±0.18
3.46
13
-1
-1
1
1
2.86±0.09
2.85
14
1
-1
1
1
2.69±0.15
2.72
15
-1
1
1
1
2.13±0.11
2.25
16
1
1
1
1
2.07±0.07
1.97
17
-2(80)
0
0
0
3.17±0.21
3.16
Page 25 of 36
0
0
0
1.45±0.15
1.45
19
0(120)
-2(60)
0
0
2.17±0.17
2.29
20
0
2(80)
0
0
3.45±0.16
3.32
21
0
0(70)
-2(20)
0
3.07±0.11
2.96
22
0
0
2(100)
0
2.21±0.07
2.31
23
0
0
0(60)
-2(20)
2.12±0.12
2.25
24
0
0
0
2(40)
3.49±0.21
3.35
25
0
0
0
0(30)
4.71±0.21
26
0
0
0
us
4.69
0
4.75±0.18
4.69
27
0
0
0
0
4.78±0.17
4.69
28
0
0
0
0
4.55±0.16
4.69
29
0
0
0
0
4.58±0.17
4.69
30
0
ed
0
4.77±0.20
4.69
0
M 0
cr
ip t
2(160)
an
18
Experiments were conducted in a random order.
b
X1: ultrasonic power (W), X2: extraction temperature (oC), X3: extraction time (min),
ce pt
a
c
Ac
X4: W/M ratio (mL/g).
LMPs: L. macranthoides polysaccharides. Each value represented the mean ± SD (n
= 3).
Page 26 of 36
Table 2
Table 2 Analysis of variance (ANOVA) testing the fitness of the regression equation. Sum of squares df
Mean squares F-value
Model
35.3968
14 2.5283
Residual
0.2689
15 0.0179
Lack of fit 0.2187
10 0.0219
Pure error
0.0502
5
Cor total
35.6657
29
Probability prob > F
141.0251 < 0.0001
2.1785
us
cr
0.0100
0.2018
ip t
Source
2 2 R2=0.9925, Radj =0.9854, Rpred =0.9626, Adequate precision=42.5691, C.V.=4.39%,
Ac
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PRESS=1.33.
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Table 3
Table 3 Testing of the significance of the regression coefficients associated with different experimental factors.
Factor
Standard
95% CI
95% CI
df
F-value low
high
prob > F b
0.0547
4.5735
4.8065
–
X1
-0.4279
1
0.0273
-0.4862
-0.3697
245.1262 < 0.0001
X2
0.2588
1
0.0273
0.2005
0.3170
89.6256
< 0.0001
X3
-0.1629
1
0.0273
-0.2212
-0.1047
35.5306
< 0.0001
X4
0.2738
1
0.0273
0.2155
0.3320
100.3182 < 0.0001
X1X2
-0.0381
1
0.0335
-0.1095
0.0332
1.2972
0.2726 (ns)
X1X3
-0.0331
1
0.0335
-0.1045
0.0382
0.9792
0.3381 (ns)
X1X4
0.3556
1
0.0335
0.2843
0.4270
112.8664 < 0.0001
X2X3
-0.2506
1
X2X4
-0.3444
1
X3X4
-0.3019
X 12
-0.5957
ed 0.0335
-0.3220
-0.1793
56.0569
0.0335
-0.4157
-0.2730
105.8384 < 0.0001
1
0.0335
-0.3732
-0.2305
81.3269
1
0.0256
-0.6502
-0.5412
542.9515 < 0.0001
ce pt
Ac
–
cr
1
an
Intercept 4.6900
us
error
M
estimate
Probability
ip t
Coefficient a
< 0.0001
< 0.0001
X 22
-0.4707
1
0.0256
-0.5252
-0.4162
339.0045 < 0.0001
X 32
-0.5132
1
0.0256
-0.5677
-0.4587
402.9823 < 0.0001
X 42
-0.4720
1
0.0256
-0.5265
-0.4175
340.8073 < 0.0001
a
X1: ultrasonic power (W), X2: extraction temperature (oC), X3: extraction time (min),
X4: W/M ratio (mL/g). b
p < 0.05 indicates statistical significance. ns = not significant at p ≤ 0.05.
Page 28 of 36
Table 4
Table 4 Predicted and experimental values of the responses at the optimum and modified conditions. Conditions a
X1 (W) X2 (oC) X3 (min) X4 (mL/g) LMPs yield (%) 54.72
30.72
4.84 (predicted)
Modified conditions
54.7
30.7
4.81 ± 0.12 (actual)
71.5
X1: ultrasonic power (W), X2: extraction temperature (oC), X3: extraction time (min),
cr
a
113.6
ip t
Optimum conditions 113.64 71.53
Ac
ce pt
ed
M
an
us
X4: W/M ratio (mL/g), LMPs: L. macranthoides polysaccharides.
Page 29 of 36
Accepted Manuscript Title: Ultrasonic extraction optimization of L. macranthoides polysaccharides and its physicochemical properties Author: Zhen Wu Hong Li Yong Yang Hongjun Tan PII: DOI: Reference:
S0141-8130(14)00814-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.12.010 BIOMAC 4772
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
29-8-2014 15-11-2014 3-12-2014
Please cite this article as: Z. Wu, H. Li, Y. Yang, H. Tan, Ultrasonic extraction optimization of L. macranthoides polysaccharides and its physicochemical properties, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.12.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Figure 2
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Figure 3
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Figure 4(a)
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Figure 4(b)
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Figure 5
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