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Optimizing conditions for DPS separation
38
Feature
Filtration+Separation January/February 2017
Pharma & biotech
Optimizing conditions for DPS separation T
his study explores the optimal extraction technology of polysaccharides from dendrobium officinale (DPS). The best combination of extraction parameters was through response surface methodology with a three-variable-threelevel Box-Behnken design.
The stem of the plant Dendrobium officinale is used in traditional Chinese medicine and has been used as an herbal medicine in many Asian countries for hundreds of years. The major medicinal components include polysaccharides, alkaloids, amino acids, and several trace mineral elements.
anti-cancer, immunological and hypoglycaemic activities [1-4]. Much attention has been paid to the bioactivities of DPS in recent decades and great advances have been made in bioactive and chemical studies [5-6]. The total polysaccharides content of Dendrobium officinale has been measured at 29.38%.
Dendrobium officinale polysaccharide (DPS) is one of the main bioactive components with anti-HIV antioxidant, antiblood coagulation, anti-radiation,
High market demand has led to the excessive harvesting and exploitation of Dendrobium officinale. Therefore, a biotechnology-based approach capable
of producing stable polysaccharides in large quantities is a promising strategy to meet market demand. Response surface methodologies (RSM) was a collection of statistical techniques originally described by Box and Wilson [7] as being effective for desirable responses that are influenced by the factors and their interactions.. In the present study, the research objectives were to determine the optimum
Figures A – C. Response surface plot and contour plot showing the effect of extraction factors on the yield of DPS. www.filtsep.com
0015 1882/17 ©2017 Elsevier Ltd. All rights reserved
40
Feature
extraction factors for the polysaccharides of Dendrobium officinale and to assess their effects using RSM.
Extraction of polysaccharides 1000g dendrobium officinale, ground into powder, were exhaustively extracted with a mixture solvent of ethanol/ethyl acetate (1:1, V/V) for 3 hr to remove some organic substances, such as alkaloids, fat, amino acids, and several trace mineral elements. The residue was extracted with 1% (w/v) aqueous NaCl solution in a designed temperature 55°C, for 1 hr. Then, the combined water solutions extracts was extracted with 2% (w/v) aqueous NaHCO3 solution (pH≈9) in a designed temperature (60-70°C), ratio of water solutions to material (10-14 mL/g) and extraction time (1-2 hr). Finally, crude polysaccharide was obtained by centrifuge at 10000 rpm for 20 min after repeatedly extracted sequentially with ethanol and deproteinized with Sevag regent (CHCl3/n-BuOH with V/V=4:1), decolorize with H2O2 solution, dialyzer, and lyophilized to get the grayish DPS. The yield of polysaccharides was determined by the method of Dubois and Fox [8, 9].
Statistical analysis A statistical analysis system from Design-Expert 8.0.5 Software (Trial version, Stat-Ease Inc., Minneanopolis, MN, USA) was used to predict models through regression analysis and analysis of variance (ANOVA). Optimal extraction conditions for maximizing responses to the yield of DPS were pre-established, by which subsequent confirmatory experiments were carried out to validate the equations. Results of all experimental sets were used for modeling the yield of DPS. Then, the predicted model was determined by multiple regression analysis. The regression equation for the yield of DPS was Y=25.12+0.44×X1+0.22×X2+2.60×X30.92×X1X2-1.80×X1X3+1.04×X2X35.35×X12-3.41×X22+0.10×X32 The model indicated that the extraction temperature had the most linear effect on the yield of DPS as it showed the largest positive linear coefficient. The www.filtsep.com
Filtration+Separation January/February 2017
experimental data were statistically analyzed by analysis of variance (ANOVA). The ANOVA of the quadratic regression model indicated that the model was significant, as the low dispersion (R2=0.9863), high model F-value (55.88) and low P-value (P <0.0001). Meanwhile, the lack of fit F-value of 3.55 and P-value of 0.7742 implied that the lack of fit was insignificant relative to the pure error due to noise. The effect of the interaction of different parameters on extract yield was simulated using Design-Expert software against any two independent variables by plotting the response surface plot. Meanwhile, considering all the possible combinations, three response surfaces were obtained. The interactive roles of extraction temperature, ratio of water solutions to
increase in the yields of DPS was observed when the extraction time and the ratio of water solutions to the raw material were increased. However, with the further increasing of the ratio of water solutions to the raw material, the yield DPS increased to a value and then decreased.
Optimal extraction By employing the software Design-Expert, optimal conditions predicted for the corresponding response variable of the extraction temperature, ratio of water solutions to raw material and extraction time were 64.28, 12.41 g/mL and 120 min for the yield of DPS. Under the optimal conditions, the maximum predicted yield of DPS was 28.05%. Verification experiments, carried out at the predicted conditions, showed values reasonably close to those predicted and further confirmed the adequacy of predicted models. The verification experimental value of the yield of DPS
"Response surface methodology (RSM) produced much higher polysaccaride and more environmentally-friendly extraction and solvent systems over a shorter period." material and extraction time are illustrated in the three-dimensional curves of the calculated response surface shown in Figure. Figure A demonstrated the effects of the ratio of water solutions to the raw material and extracting temperature on the yields of DPS. An increase in the yields of DPS was observed when the ratio of water solutions to raw material and extraction temperature was increased, and a highest value was obtained. Based on varying extraction temperature and extraction time, the 3D response surface plots of the yields were developed and showed in Figure B. An increase in the yields of DPS was observed when the extraction time is kept at a higher level and then decreased with the increase of extraction temperature. Figure C demonstrated the effects of the ratio of water solutions to the raw material and extraction time on the yield of DPS. An
was 27.93%. The results indicated that the model was satisfactory and could considerably enhance the yield of DPS. To further increase the extraction yields of DPS in this design, other variables such as pH and ratio of ethanol to ethyl acetate may be studied.
References 1, 2, 3, 4, 5, 6, 7, 8, 9 Please contact the author directly for full references.
•
Contact
Feng Jin, Xin Sui, Fu-Shuang Ma, Shu-Heng Wang College of Biological and Food Engineering, Jilin Institute of Chemical Technology, No. 45 Chengde road, Longtan District, Jilin 132022, China [email protected]
Feature
Filtration+Separation January/February 2017
Pharma & biotech
Optimizing conditions for DPS separation T
his study explores the optimal extraction technology of polysaccharides from dendrobium officinale (DPS). The best combination of extraction parameters was through response surface methodology with a three-variable-threelevel Box-Behnken design.
The stem of the plant Dendrobium officinale is used in traditional Chinese medicine and has been used as an herbal medicine in many Asian countries for hundreds of years. The major medicinal components include polysaccharides, alkaloids, amino acids, and several trace mineral elements.
anti-cancer, immunological and hypoglycaemic activities [1-4]. Much attention has been paid to the bioactivities of DPS in recent decades and great advances have been made in bioactive and chemical studies [5-6]. The total polysaccharides content of Dendrobium officinale has been measured at 29.38%.
Dendrobium officinale polysaccharide (DPS) is one of the main bioactive components with anti-HIV antioxidant, antiblood coagulation, anti-radiation,
High market demand has led to the excessive harvesting and exploitation of Dendrobium officinale. Therefore, a biotechnology-based approach capable
of producing stable polysaccharides in large quantities is a promising strategy to meet market demand. Response surface methodologies (RSM) was a collection of statistical techniques originally described by Box and Wilson [7] as being effective for desirable responses that are influenced by the factors and their interactions.. In the present study, the research objectives were to determine the optimum
Figures A – C. Response surface plot and contour plot showing the effect of extraction factors on the yield of DPS. www.filtsep.com
0015 1882/17 ©2017 Elsevier Ltd. All rights reserved
40
Feature
extraction factors for the polysaccharides of Dendrobium officinale and to assess their effects using RSM.
Extraction of polysaccharides 1000g dendrobium officinale, ground into powder, were exhaustively extracted with a mixture solvent of ethanol/ethyl acetate (1:1, V/V) for 3 hr to remove some organic substances, such as alkaloids, fat, amino acids, and several trace mineral elements. The residue was extracted with 1% (w/v) aqueous NaCl solution in a designed temperature 55°C, for 1 hr. Then, the combined water solutions extracts was extracted with 2% (w/v) aqueous NaHCO3 solution (pH≈9) in a designed temperature (60-70°C), ratio of water solutions to material (10-14 mL/g) and extraction time (1-2 hr). Finally, crude polysaccharide was obtained by centrifuge at 10000 rpm for 20 min after repeatedly extracted sequentially with ethanol and deproteinized with Sevag regent (CHCl3/n-BuOH with V/V=4:1), decolorize with H2O2 solution, dialyzer, and lyophilized to get the grayish DPS. The yield of polysaccharides was determined by the method of Dubois and Fox [8, 9].
Statistical analysis A statistical analysis system from Design-Expert 8.0.5 Software (Trial version, Stat-Ease Inc., Minneanopolis, MN, USA) was used to predict models through regression analysis and analysis of variance (ANOVA). Optimal extraction conditions for maximizing responses to the yield of DPS were pre-established, by which subsequent confirmatory experiments were carried out to validate the equations. Results of all experimental sets were used for modeling the yield of DPS. Then, the predicted model was determined by multiple regression analysis. The regression equation for the yield of DPS was Y=25.12+0.44×X1+0.22×X2+2.60×X30.92×X1X2-1.80×X1X3+1.04×X2X35.35×X12-3.41×X22+0.10×X32 The model indicated that the extraction temperature had the most linear effect on the yield of DPS as it showed the largest positive linear coefficient. The www.filtsep.com
Filtration+Separation January/February 2017
experimental data were statistically analyzed by analysis of variance (ANOVA). The ANOVA of the quadratic regression model indicated that the model was significant, as the low dispersion (R2=0.9863), high model F-value (55.88) and low P-value (P <0.0001). Meanwhile, the lack of fit F-value of 3.55 and P-value of 0.7742 implied that the lack of fit was insignificant relative to the pure error due to noise. The effect of the interaction of different parameters on extract yield was simulated using Design-Expert software against any two independent variables by plotting the response surface plot. Meanwhile, considering all the possible combinations, three response surfaces were obtained. The interactive roles of extraction temperature, ratio of water solutions to
increase in the yields of DPS was observed when the extraction time and the ratio of water solutions to the raw material were increased. However, with the further increasing of the ratio of water solutions to the raw material, the yield DPS increased to a value and then decreased.
Optimal extraction By employing the software Design-Expert, optimal conditions predicted for the corresponding response variable of the extraction temperature, ratio of water solutions to raw material and extraction time were 64.28, 12.41 g/mL and 120 min for the yield of DPS. Under the optimal conditions, the maximum predicted yield of DPS was 28.05%. Verification experiments, carried out at the predicted conditions, showed values reasonably close to those predicted and further confirmed the adequacy of predicted models. The verification experimental value of the yield of DPS
"Response surface methodology (RSM) produced much higher polysaccaride and more environmentally-friendly extraction and solvent systems over a shorter period." material and extraction time are illustrated in the three-dimensional curves of the calculated response surface shown in Figure. Figure A demonstrated the effects of the ratio of water solutions to the raw material and extracting temperature on the yields of DPS. An increase in the yields of DPS was observed when the ratio of water solutions to raw material and extraction temperature was increased, and a highest value was obtained. Based on varying extraction temperature and extraction time, the 3D response surface plots of the yields were developed and showed in Figure B. An increase in the yields of DPS was observed when the extraction time is kept at a higher level and then decreased with the increase of extraction temperature. Figure C demonstrated the effects of the ratio of water solutions to the raw material and extraction time on the yield of DPS. An
was 27.93%. The results indicated that the model was satisfactory and could considerably enhance the yield of DPS. To further increase the extraction yields of DPS in this design, other variables such as pH and ratio of ethanol to ethyl acetate may be studied.
References 1, 2, 3, 4, 5, 6, 7, 8, 9 Please contact the author directly for full references.
•
Contact
Feng Jin, Xin Sui, Fu-Shuang Ma, Shu-Heng Wang College of Biological and Food Engineering, Jilin Institute of Chemical Technology, No. 45 Chengde road, Longtan District, Jilin 132022, China [email protected]