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Response surface methodology to extraction of dioscoreae polysaccharides and

Carbohydrate Polymers 83 (2011) 32–37

Contents lists available at ScienceDirect

Carbohydrate

Polymers

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c a r b p o

l

Response surface methodology to extraction of dioscoreae polysaccharides and the effects on rat’s bone quality

Zhong-Lian Huang a ,?,Zhen-Yu Liang b ,Guan-Jia Li c ,Hanbiao Hong c

a

Department of Orthopedics,The First Af?liated Hospital of Shantou University Medical College,Shantou,Guangdong Province 515041,PR China b

Department of Orthopedics,Nanfang Hospital,Southern Medical University,Guangzhou,Guangdong Province 510515,PR China c

Department of Orthopedics,Dafeng Hospital,Shantou,Guangdong Province 515154,PR China

a r t i c l e i n f o Article history:

Received 9June 2010

Received in revised form 3July 2010Accepted 7July 2010

Available online 13 July 2010Keywords:

Response surface methodology Optimization Extraction

Dioscoreae polysaccharides Bone

Osteoporosis

a b s t r a c t

The objective of this study was to determine the optimum extraction conditions for dioscoreae polysac-charides.The optimum conditions were evaluated with fractional factorial design and optimized using response surface methodology.The effects of three independent variables,namely extraction time,extraction temperature and ratio of liquid to solid on the yield of dioscoreae polysaccharides were investi-gated.Results indicated that the data were adequately ?tted into three second-order polynomial models.The independent variables,the linearity of extraction time and temperature,the quadratics of extrac-tion temperature and ratio of liquid to solid had a signi?cant effect on the yield of polysaccharides.The optimal extraction parameters were extraction time of 140min,temperature of 100?C and the ratio of liquid to solid of 3,according to the response surface analysis.Pharmacology experiment showed that dioscoreae polysaccharides decreased serum ALP,IL-6and BGP levels,and enhanced BMD,BMC,bone Hyp,Ca 2+and P 3+levels.

? 2010 Elsevier Ltd. All rights reserved.

1.Introduction

Osteoporosis,a common disease characterized by a decreased bone mineral density (BMD)and a microarchitectural deterio-ration of bone structure,is a chronic condition chie?y affecting postmenopausal women,in whom the skeleton loses a signi?-cant percentage of its mineralized mass and mechanical resiliency,thereby becoming prone to fracture (Deyhim et al.,2006).Plenty of studies around this disease have been carried out in recent years.The etiology of human osteoporosis is multifactorial,resulting from an interplay of genetic components,hormonal excess or de?ciency,dietary factors,and physical activity (Hulley,Conradie,Langeveldt,&Hough,2002;Okumura et al.,1987).As the population ages,the incidence of hip fractures and costs for treatment will rise dramati-cally in the future,unless effective prophylactic measures are taken (Nagareddy &Lakshmana,2005).

Nature medicines have been investigated for some wonder-ful pharmacological activities (Alim et al.,2009;Yongabi,Harris,Lewis,&Agho,2009;Yongabi,Mbacham,Nubia,&Singh,2009;Kim et al.,2009;Rezazadeh et al.,2009).Chinese traditional medicine,Rhizoma dioscoreae,is the root of Dioscorea opposita Thunb (Zhang,Liu,&Chen,2004).It is useful for treating the angio-

?Corresponding author.Tel.:+86135********;fax:+86135********.E-mail address:huangzlst@https://www.sodocs.net/doc/502636564.html, (Z.-L.Huang).cardiopathy,controlling blood pressure and improving immunity ability and also can be used as an anti-decrepitude tonic (McAnuff,Omoruyi,Morrison,&Asemota,2002;McAnuff-Harding,Omoruyi,&Asemota,2006).In China,dioscoreae is also used as tonic nour-ishment and is considered a potential functional food.Effects of dioscoreae on bone quality have not been investigated.

Response surface methodology (RSM)is a mathematical tool,which can help in arriving at optimum conditions for a reac-tion with minimum number of experiments to obtain statistically acceptable results.RSM enables evaluation of the effects of multi-ple parameters,alone or in combination,on response variables and also predicts their behavior under given sets of conditions (Chiang,Shih,&Chu,2001).The objective of this study was to optimize the extraction time,extraction temperature,and ratio of liquid to solid by RSM for maximum yield of dioscoreae polysaccharides.Then,effects of dioscoreae on bone quality in rats were investigated.2.Materials and method

2.1.Preparation of the dioscoreae polysaccharides

Dioscoreae was collected in November 2004from a local herb market and stored at room temperature in a dry place prior to use.The root parts of dioscoreae were dried at ambient temper-ature.Then,distilled water was added to 100g of dried dioscoreae ?nely powered and the mixture was heated under re?ux for certain

0144-8617/$–see front matter ? 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.carbpol.2010.07.018

Z.-L.Huang et al./Carbohydrate Polymers83 (2011) 32–3733

Table1

Factors and levels.

Factor Low Center High

X1?1(80min)0(110min)1(140min)

X2?1(80?C)0(90?C)1(100?C)

X3?1(2)0(3)1(4)

time and then the decoction was?ltered.The?ltrate was frozen at

?20?C and lyophilized.The crude yield of the lyophilized material

was approximately18%(w/w),it was stored at ambient tempera-

ture until further use.

2.2.Experimental designs

Response surface methodology(RSM)with central composite

design(CCD)was employed to investigate the optimal parame-

ters of dioscoreae polysaccharides.Three independent parameters

namely,extracting time,extracting temperature and ratio of liq-

uid to solid at three different levels each,were employed.The

parameters were chosen and their levels were based on preliminary

experiments carried out in our laboratory.

Table1gives the range of variables employed.The actual set

of experiments performed(experimental runs1–20)and the yield

of dioscoreae polysaccharides obtained are shown in Table2.A

second-order polynomial equation was developed to study the

effects of variables on the yield.The equation indicates the effect

of variables in terms of linear,quadratic,and cross-product terms:

Y=ˇ0±

ˇi X i±

ˇii X2

i

±

ˇij X i X j(1)

where Y is the yield of dioscoreae polysaccharides(%),X i and X j are the levels of variables,ˇ0the constant term,ˇi the coef?cient of the linear terms,ˇii the coef?cient of the quadratic terms,and ˇij the coef?cient of the cross-product terms.All the experimen-tal data were statistically analyzed by the SAS statistical package (version8.1,SAS Institute,Cary,NC,USA).The graphical representa-tions of the above equation in the form of surface plots were used to describe the individual and cumulative effects of the test variables on the response.

Table2

Experimental design and responses of the dependent variables to the extract parameters.

Run X1X2X3Y1

1?1.00000?1.00000?1.000000.9 2?1.00000?1.00000 1.00000 1.0 3?1.00000 1.00000?1.00000 1.2 4?1.00000 1.00000 1.00000 1.3

5 1.00000?1.00000?1.00000 1.2

6 1.00000?1.00000 1.00000 1.4

7 1.00000 1.00000?1.00000 1.5

8 1.00000 1.00000 1.00000 1.4

9?1.681790.000000.00000 1.2 10 1.681790.000000.00000 1.5 110.00000?1.681790.00000 1.2 120.00000 1.681790.00000 1.4 130.000000.00000?1.68179 1.2 140.000000.00000 1.68179 1.3 150.000000.000000.00000 1.4 160.000000.000000.00000 1.4 170.000000.000000.00000 1.4 180.000000.000000.00000 1.4 190.000000.000000.00000 1.4 200.000000.000000.00000 1.42.3.Fourier transform infrared spectroscopy(FT-IR)

FT-IR spectra of polysaccharides KBr pellet were obtained with a FT-IR spectrometer(Spectrum One Version B,Perkin Elmer,Amer-ica)(Huang,Wu,Wei,Liao,&Chen,2010).

2.4.Animal group and treatment

Thirty-two adult male Wister rats,weighing about170g,were bred in the animal research center of our university,China.Ani-mals were handled in accordance with laboratory animal welfare guidelines.Animals were housed in4groups of8animals in a controlled environment of24?C and12h light(dark cycle from 7pm to7am)with food and water freely available and randomly assigned to the normal control group(1),model control(2),and two polysaccharides-treatment groups(3and4).The acclimatized rats were bilaterally ovariectomized using the dorsal approach. The rats were anaesthetized with ether.A single longitudinal skin incision was made on the dorsal midline at the level of the kid-neys.Both ovaries were ligated and removed in the model control and two polysaccharides-treatment groups.Success of ovariectomy was con?rmed at necropsy by failure to detect ovarian tissue and by observation of marked atrophy of uterine horns.During the surgery, aseptic techniques were used to avoid the potential infection of pathogens.

After the operation,8plastic cages(400mm×240mm×240mm)were used and4rats from the same group were housed in each cage.Each cage was held up by a plastic support with four legs of which height could be freely adjusted.

The rats of the groups1(normal control)and2(model control) were treated with vehicle alone for7weeks.

The rats of the group3received dioscoreae polysaccharides by gavage at a dose of100mg/kg bw for7weeks.

The rats of the group4received dioscoreae polysaccharides by gavage at a dose of200mg/kg bw for7weeks.

At the end of experimental period,rat body weights were recorded.The rats were fasted for16h.Animals were subjected to light ether anaesthesia and killed by cervical dislocation.

Blood sample was collected via abdominal aorta puncture, serum was then prepared by centrifugation of the collected blood (2000rpm for20min).Serum samples were then stored at?80?C for biochemical determinations.Femurs were dissected and?lled in physiological saline and stored at?20?C for measurement.

2.5.Assay for BMD,BMC and serum chemistry

Bone hydroxyproline was analyzed as described previously (Delport,Maas,van der Merwe,&Laurens,2004).

The level of IL-6in serum was measured with the commer-cially available enzyme-linked immunosorbent assay kit(Catalog #KAC1261;Biosource International,Inc,Camarillo,CA)according to the manufacturer’s instructions.

The level of BGP in serum was measured with a competitive enzyme immune assay method,according to the manufacturer’s instructions(NovoCalcin;Metra Biosystems).

The BMD and BMC of the femurs were measured using DXA equipped with appropriate software for bone density assessment in small laboratory animals as reported elsewhere(Beaupied et al., 2009).

Serum alkaline phosphatase(ALP),bone calcium(Ca),and phosphorus(P)concentrations were measured by standard col-orimetric methods using commercial kits(ZhongSheng BeiKong Bio-technology and Science,PRC)and analyzed by a Cobas Inte-gra400Plus automatic biochemical analyzer(Roche Diagnostics, Switzerland).

34

Z.-L.Huang et al./Carbohydrate Polymers 83 (2011) 32–37

Table 3

Effect estimate.Effect Estimate Std error t ratio P -value X 10.117490.016829 6.9815<0.0001X 20.090530.016829 5.37950.0003X 3

0.0342820.016829 2.03710.0690X 1×X 1?0.0295870.016382?1.8060.1011X 1×X 2?0.03750.021988?1.70550.1189X 1×X 3?0.01250.021988?0.56850.5822X 2×X 2?0.0472640.016382?2.88510.0162X 2×X 3?0.03750.021988?1.70550.1189X 3×X 3

?0.064942

0.016382

?3.9642

0.0027

2.6.Statistical analysis

Data were presented as mean ±S.D.and analyzed by one-way analysis of variance followed by Student’s t -test for comparison of 2groups using SPSS software Version 12.0.P -values of less than 0.05were considered statistically signi?cant.3.Result and discussion 3.1.Fitting the models

The experimental conditions and the corresponding response values from the experimental design are given in Table 2.As observed in Table 2,treatment runs 6,7,8,10,12,15,16,17,18,19and 20showed high levels of the dioscoreae polysaccharides at 1.4%,1.5%,1.4%,1.5%,1.4%,1.4%,1.4%,1.4%,1.4%,1.4%,and 1.4%,respectively.The maximum extraction ef?ciency was observed in run number 7and 10,while the minimum extraction ef?ciency was achieved in run number 1.The values of the coef?cients in the second-order polynomial equation are presented in Table 3.

The regression equation for the extraction ef?ciency of dioscoreae polysaccharides (Y )was as follows:

Y 1=1.401822+0.11749×X 1+0.09053×X 2+0.034282×X 3

?0.029587×X 1×X 1?0.0375×X 1×X 2?0.0125×X 1×X 3?0.047264×X 2×X 2?0.0375×X 2×X 3?0.064942×X 3×X 3

(2)

The signi?cance of each coef?cient was determined by F -value and P -value.Corresponding P -values suggest that,among the test variables used in this study,X 1(extraction time),X 2(extraction

Table 4

Fit statistics for Y 1.

Master model Predictive model Mean 1.305 1.305R 2

91.76%91.76%Adj.R 284.35%84.35%RMSE 0.0621910.062191CV

4.765572

4.765572

temperature),(X 2)2(extraction temperature ×extraction temper-ature),and (X 3)2(ratio of liquid to solid ×ratio of liquid to solid)are signi?cant model terms with P -values of less than 0.05.Other terms are insigni?cant.Here,a lower value of CV (4.76)indicated a better precision and reliability of the experiments was carried out (Chen et al.,2010;Gan,Abdul Manaf,&Latiff,2010;Kuo &Lai,2009;Sabzali,Gholami,&Sadati,2009).For the model ?tted,the coef?cient of determination (R 2),which can check the good-ness of a model,was 0.92.This implied that the sample variation of 92%for the extraction ef?ciency of dioscoreae polysaccharides was attributed to the independent variables,and only 8%of the total variation cannot be explained by the model.From the above,it can be concluded that the developed model could adequately represent the real relationship among the parameters chosen (Table 4).

Figs.1A and 2A show the effects of the extraction time and temperature on the extraction yield of dioscoreae polysaccharides when the ratio of liquid to solid was ?xed at 3.At this ?xed ratio of liquid to solid,the maximal polysaccharides yield (1.53%)was obtained when a time of 140min and a temperature of 100?C were used.In Figs.1A and 2A ,increasing the extraction temperature or time within the ranges tested in this study led to an increase in the yield of polysaccharides.Therefore,the higher temperature and longer extraction time were advantageous to the extraction of dioscoreae polysaccharides.The higher mass transfer rate and solubility and the increased extraction time could be the primary factors responsible for these changes.

In Figs.1B and 2B ,when

the extraction temperature was ?xed at 100?C,it was revealed that the maximum yield (1.51%)of polysac-charides was achieved when the extraction time 140min and ratio of liquid to solid 3were used.At all extraction times,the maximal yield of polysaccharides was attained when a extraction tempera-ture of 100?C was used,while ratio of liquid to solid greater than 3caused a decrease in the extraction yield.These results imply that the quantity of the water needs to be optimized to ensure effective extraction of polysaccharides.

Fig.1.(A)Three-dimensional response surface plot for the effects of extraction time (X 1)and extraction temperature (X 2)on the yield of dioscoreae polysaccharides at central level of ratio of liquid to solid (X 3);(B)three-dimensional response surface plot for the effects of extraction time (X 1)and ratio of liquid to solid (X 3)on the yield of dioscoreae polysaccharides at central level of extraction temperature (X 2);(C)three-dimensional response surface plot for the effects of extraction temperature (X 2)and ratio of liquid to solid (X 3)on the yield of dioscoreae polysaccharides at central level of extraction time (X 1).

Z.-L.Huang et al./Carbohydrate Polymers 83 (2011) 32–37

35

Fig.2.(A)The contour plots of the effects of extraction time (X 1)and extraction temperature (X 2)on the yield of dioscoreae polysaccharides at central level of ratio of liquid to solid (X 3);(B)the contour plots of the effects of extraction time (X 1)and ratio of liquid to solid (X 3)on the yield of dioscoreae polysaccharides at central level of extraction temperature (X 2);(C)the contour plots of the effects of extraction temperature (X 2)and ratio of liquid to solid (X 3)on the yield of dioscoreae polysaccharides at central level of extraction time (X 1).

As shown in Figs.1C and 2C ,when a ?xed extraction time of 180min was used,the extraction yield of polysaccharides increased as the extraction temperature increased,regardless of the ratio of liquid to solid.Although the maximal extraction yield of polysac-charides for each extraction time was obtained when the ratio of liquid to solid was 3,at the concentration of the ratio greater than 3,the extraction yield of polysaccharides decreased.These ?ndings con?rmed that there is an optimal quantity of water for the effective

extraction of polysaccharides.As shown in Figs.1C and 2C ,the max-imum extraction of polysaccharides (1.58%)was obtained when the ratio of liquid to solid was 3and the temperature was 100?C.3.2.FT-IR spectroscopy

The main absorbance bands were a broad band around 3400–3300cm ?1(H bonds,OH groups),a distinct peaks

at

Fig.3.Fourier transform infrared spectra of dioscoreae polysaccharides.

36Z.-L.Huang et al./Carbohydrate Polymers83 (2011) 32–37

Table5

Effect of dioscoreae polysaccharides on serum ALP,IL-6and BGP levels.

Group ALP(U/L)IL-6(pg/ml)BGP(ng/ml)

1105.91±2.292216.25±12.5112.12±0.13 2137.12±4.38**2526.31±9.44**16.71±0.19** 3121.63±2.54##2478.92±11.67##14.25±0.17## 4114.12±3.67##2362.13±14.05##12.03±0.24##

**P<0.01,group1vs group2.

##P<0.01,groups3,4vs group2.

Table6

Effect of dioscoreae polysaccharides on BMD and BMC.

Group BMD BMC

10.2497±0.01980.2407±0.0211 20.2108±0.0089**0.2011±0.0108** 30.2313±0.0076##0.2414±0.0104## 40.2472±0.0081##0.2573±0.0214##

**P<0.01,group1vs group2.

##P<0.01,groups3,4vs group2.

Table7

Effect of dioscoreae polysaccharides on bone Hyp,Ca2+and P3+levels.

Group Hyp(mg/g)Ca2+(mg/cm3)P3+(mg/cm3)

122.4±1.6353.3±5.1112.7±6.9 217.3±1.1**305.1±5.3**93.6±4.2** 321.8±1.3##341.7±6.2##109.6±5.1## 423.4±1.1##361.3±4.8##122.5±4.9##

**P<0.01,group1vs group2.

##P<0.01,groups3,4vs group2.

2929cm?1(C–H asymmetric,C–H stretch of–CH),a ris-ing narrow sharp peak at1642cm?1(COO–,CH3),and a broad peak at1371–1418cm?1(C–O stretch of polysaccharide) (Fig.3).

FT-IR spectra in the1700–600cm?1region give information about the main polysaccharides present in the complicated sys-tems of polysaccharide mixture.The region at1700–600cm?1, which is dominated by stretching vibrations of C–O,C–C,ring struc-tures and deformation vibrations of CH2groups,was found to be useful for the identi?cation of polysaccharides(Fig.3).From Fig.3,one can note that the polysaccharide extracts are char-acterized mainly by the absorbances at1642,1418,1371and 708cm?1.

3.3.Effect of dioscoreae polysaccharides on serum ALP,IL-6and BGP levels in control and experimental animals

Table5depicts the effect of dioscoreae polysaccharides serum ALP,IL-6and BGP levels in control and experimental animals. The results of the study showed a marked increase(P<0.01)in serum ALP,IL-6and BGP levels in model control rats(group 2)when compared to normal control group.A marked protec-tion against ovariectomized damage was observed by signi?cant decrease(P<0.05;P<0.01)in serum ALP,IL-6and BGP levels in polysaccharides-treated rats(groups3and4)as compared to model rats(group2).

3.4.Effect of dioscoreae polysaccharides on BMD and BMC

The bone indexes such as BMD and BMC were found to be decreased(P<0.01)signi?cantly in model control rats(group2) when compared to normal control animals(Table6).A signi?cant increase(P<0.01)in the BMD and BMC levels was observed in polysaccharides-treated rats after the administration of dioscoreae polysaccharides in groups3and4rats as compared with model control group.

3.5.Effect of dioscoreae polysaccharides on bone Hyp,Ca2+and

P3+levels

Table7shows the effect of dioscoreae polysaccharides on bone Hyp,Ca2+and P3+levels in the normal control and experimental animals.In model control(group2)animals,the levels of bone Hyp,Ca2+and P3+were signi?cantly(P<0.01)decreased compared to normal control(group1)animals at the end of7weeks.The oral supplementation of dioscoreae polysaccharides(as a100mg and200mg kg?1day?1dose orally for7weeks)to experimental rats(groups3and4)resulted in signi?cant increases in bone Hyp, Ca2+and P3+levels(P<0.05,P<0.01)as compared to model control (Table7).

4.Conclusion

In this study,RSM was employed to investigate the main and the interaction effects of the variables that are important in the extraction of dioscoreae polysaccharides.The extraction time and temperature were the most important factors affecting the perfor-mance of the process.The optimum conditions were found to be extraction time of140min,temperature of100?C and the ratio of liquid to solid of3with the maximum yield of1.56%.FT-IR spec-tra in the1900–600cm?1region give information about the main polysaccharides present in the complicated systems of polysaccha-ride mixture.The pharmacological assay indicated that the extracts could still inhibit bone degeneration.

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