Food Chemistry 168 (2015) 538–545

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Absorption and desorption behaviour of the flavonoids from Glycyrrhiza glabra L. leaf on macroporous adsorption resins Yi Dong a, Mouming Zhao a,b, Dongxiao Sun-Waterhouse a, Mingzhu Zhuang a, Huiping Chen a, Mengying Feng a, Lianzhu Lin a,⇑ a b

College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China Pulp & Paper Engineering State Key Laboratory, South China University of Technology, Guangzhou 510640, China

a r t i c l e

i n f o

Article history: Received 7 April 2014 Received in revised form 28 June 2014 Accepted 22 July 2014 Available online 31 July 2014 Keywords: Macroporous adsorption resins Flavonoids Pinocembrin Adsorption behaviours

a b s t r a c t The kinetics of adsorption and desorption behaviours of five macroporous resins for enriching flavonoids from Glycyrrhiza glabra L. leaf were investigated. All five resins showed similar and effective adsorption and desorption properties. A pseudo-second-order kinetics model was suitable for evaluating the whole adsorption process. Additionally, two representative resins (XAD-16 and SP825) were chosen for adsorption thermodynamics study. The adsorption of the representative resins was an exothermic and physical adsorption process. Further column chromatography of XAD-16 and SP825 showed that the total flavonoids (from 16.8% to 55.6% by XAD-16 and to 53.9% by SP825) and pinocembrin (from 5.49% to 15.2% by XAD-16 and to 19.8% by SP825) were enriched in 90% ethanol fractions. Meanwhile, the antioxidant capacities and nitrite-scavenging capacities were 2–3 times higher than those of the crude extract. The fractions with high flavonoid and pinocembrin contents could be used as biologically active ingredients in functional food. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Licorice, the root of Glycyrrhiza glabra L., is one of the most extensively used herbal medicines and natural sweeteners in ancient China, Span, Persia, India and Russia (Seo et al., 2010). Licorice is named as ‘‘guo lao’’ and used in prescriptions of Traditional Chinese Medicine in quantity. Years of clinical and laboratory researches proved that licorice was effective in treating some diseases, such as chronic hepatitis, hyperlipaemia, atopic dermatitis, and other ailments (Vaya, Belinky, & Aviram, 1997). Triterpene glycyrrhetic acid (GA) and flavonoids (LF) are two groups of bioactive compounds in licorice root (Fu et al., 2005). Licorice flavonoids have been permitted for use as antioxidants in food, skin-whitening agents in cosmetics and main constituents in stomach medicine. However, our previous work found that the leaf of G. glabra L., which is used as the feed for cattle and flock or burnt into fertilizer (as fuel), was rich in flavonoids, especially pinocembrin. Pinocembrin is one of the significant flavonoids in propolis and royal jelly, possessing good antimicrobial, antiinflammatory, antioxidative, and pharmacological effects (Rasul et al., 2013). Thus, licorice-derived products, with high bioactive ⇑ Corresponding author. Tel./fax: +86 20 87113914. E-mail address: [email protected] (L. Lin). http://dx.doi.org/10.1016/j.foodchem.2014.07.109 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

contents, including flavonoids, are highly desired. Macroporous adsorption resins have been widely used in chemical and medicinal industries, especially for extraction, separation and purification of biochemical products. These uses include enrichment of active ingredients in natural plants, such as flavonoids in Houttuynia cordata Thunb. (Zhang, Li, Wu, & Zhao, 2007), madecassoside and asiaticoside from Centella asiatica (Jia & Lu, 2008), hesperidin (Scordino, Di Mauro, Passerini, & Maccarone, 2003), flavone C-glycosides from trollflower (Sun et al., 2013), rutin and quercetin from Euonymus alatus (Thunb.) Siebold (Zhao, Dong, Wu, & Lin, 2011). Macroporous adsorption resins were also then used for environmental management (Liu et al., 2010). Moreover, the adsorption and desorption conditions and behaviours of different macroporous adsorption resins were investigated, and their thermodynamic and kinetic models were constructed (Ayranci & Hoda, 2005; Gao, Yu, Yue, & Quek, 2013; Scordino et al., 2003). Previous researches certified that the polarity, surface area and average pore diameter strongly contributed to the adsorption capacity of macroporous resins (Scordino et al., 2003; Serarols, Poch, & Villaescusa, 2001). In order to improve the concentration and purity of bioactive compounds, macroporous resins were often used along with some high efficient separation techniques, such as high-speed counter-current chromatography, high-performance liquid chromatography and

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preparative high performance liquid chromatography (Kuang et al., 2013; Sun, Sun, & Liu, 2007; Yue, Zhao, Mei, & Shao, 2013; Zhang, Jiao, Liu, & Wu, 2007; Zhang, Liang, Kuang, Yuan, & Wang, 2012). The aim of this research was to investigate the adsorption and desorption behaviours of five types of macroporous resins, namely XAD-4, XAD-16, HP-2MGL, SP-825, and SP207. Meanwhile, the static adsorption kinetics of the flavonoids of G. glabra L. leaf extract on these resins and the thermodynamics of the flavonoids on two selected resins are investigated. 2. Materials and methods 2.1. Licorice leaf powder The leaves of licorice (G. glabra L.) were supplied by Xinjiang Tianshan Pharmaceutical Industry Co. Ltd. (Xinjiang, China), separated carefully from the stem, sun-dried, pulverized by a laboratory knife mill (FW100, Taisite Instrument Co., Ltd., Tianjin, China) and sieved. Particles sized 0.3 mm (mean diameter) were collected in sealed bags, and stored in a glass desiccator over silica at room temperature before further processing. 2.2. Materials and chemicals Macroporous resins (XAD-4, XAD-16, HP-2MGL, SP207 and SP825) were purchased from H&G Co., Ltd., Beijing, China. 2,20 Azobis (2-methylpropionamidine) dihydrochloride (AAPH), trolox and fluorescein sodium salt were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile (CH3CN), of HPLC grade, was obtained from Merck (Darmstadt, Germany). All other chemicals were of analytical (AR) grade. Standard pinocembrin was obtained by column chromatography in our previous work. 2.3. Preparation of crude extract from licorice leaf powder Licorice (G. glabra L.) leaf powder (100 g) was extracted with 80% (v/v) aqueous ethanol in an ultrasonic cleaner (KQ-800KDE, Kunshan Ultrasonic Equipment Co., Ltd., Jiangsu Kunshan, China) with power of 800 W for 90 min at 60 °C. The solvent to powder mass ratio was 20:1. The supernatant was obtained by filtering the extracting solution and the residue was used for further extraction. The extraction process was repeated 3 times and all the supernatants were combined and condensed by using a rotary evaporator (RE52AA, Yarong Equipment Co. Ltd., Shanghai, China) under reduced pressure at 50 °C to complete removal of ethanol. Then, the obtained crude extract was stored in a refrigerator at 4 °C prior to further use. 2.4. Pretreatment of macroporous resins Specifications of the five macroporous resins used in this study are summarized in Table 1. All the resins were first treated in absolute ethanol for 24 h, and then washed with deionized water (to complete removal of ethanol), soaked in 5% (m/m) NaOH for 6 h, washed with deionized water (until the pH of filtrate was 7),

soaked in 5% (v/v) HCl for 6 h, and washed with deionized water (until the pH of filtrate was 7) in sequence. Finally, all the resins were dried at 60 °C in a blast drying oven (DHG-9070A, Shanghai Shenxian Thermostatic Equipment Co., Ltd., Shanghai, China) to reach a constant weight. 2.5. Static adsorption and desorption experiments 2.5.1. Adsorption kinetics of macroporous resins on the flavonoids from G. glabra L. leaf Each resin was accurately weighed (5 g) and carefully transferred into a 250 ml flask. The resins were activated through soaking in 98% ethanol (with the ratio of 20:1) overnight. Then the resins were washed with deionized water until the ethanol was completely removed. The crude liquid extracts (100 ml with a total flavonoid concentration of 0.983 mg/ml) were put into different conical flasks. These flasks were sealed with stoppers and placed in a thermostatic oscillator (THZ-82A, Changzhou Aohua Instrument Co., Ltd., Jiangsu, China). The whole process of adsorption was done at 30 °C with a shaking speed of 150 rpm for 1080 min. One millilitre of liquid extract was withdrawn at time points of 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 180, 240, 360 and 1080 min. The total flavonoid content of each sample was determined and the adsorption kinetics equation of each resin was separately established. 2.5.2. Adsorption thermodynamics of macroporous resins on the flavonoids from G. glabra L. leaf Four aliquots (accurately weighed 0.5 g) of each resin were transferred, individually, into four 100 ml flasks, which had the same constitution of macroporous resins and were named as G1, G2, G3 and G4, respectively. The resins were activated overnight with 98% ethanol before, and then the ethanol was removed through washing with deionized water. Resins in each flask were soaked in 10 ml flavonoid solutions of different concentrations (0.076, 0.153, 0.354, 0.981 and 1.801 mg/ml, respectively) and the four groups flasks were shaken, using four thermostatic oscillators at the same speed of 150 rpm but at four different temperatures (25, 35, 45 and 55 °C, respectively). The absorption time was determined by the adsorption kinetics. The total flavonoid contents of the supernatants were also determined. The adsorption thermodynamics equation for each resin was established accordingly. 2.5.3. Desorption of macroporous resins on the flavonoids from G. glabra L. leaf Six aliquots (accurately weighed 0.5 g) of each resin were placed into six 100 ml flasks, respectively, activated with 98% ethanol, soaked in aqueous solutions containing flavonoids at the optimized concentration and the optimized temperature for an optimized time period. The solutions were removed and the residues were retained in the flasks when the absorption equilibrium was reached. Then aqueous ethanol solutions of different concentrations (0%, 20%, 40%, 60%, 80% and 100% ethanol content) were mixed with the six groups of resins in the flasks. All these flasks were shaken in the thermostatic oscillator at 45 °C with a shaking

Table 1 Specifications of the five macroporous resins in this study. Macroporous resin

Polarity

Material

Particle size (mm)

Specific surface area (m2/g)

Pore diameter (Å)

XAD-4 XAD-16 HP-2MGL SP207 SP825

Non-polar Non-polar Moderately-polar Non-polar Weak polar

Polystyrene Polystyrene Methacrylate Brominated polystyrene Polystyrene

0.64 0.7 >0.35 0.25 >0.25

750 800 470 630 1050

100 150 340 210 114

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speed of 150 rpm for 120 min. Then 1 ml of liquid was withdrawn from each flask for TPC analysis and subsequent determinations of desorption ratio and optimal desorption concentration for each resin. In parallel, four groups of flasks containing resins were prepared as described above. However, the thermostatic oscillation was conducted at four different temperatures 25, 35, 45 and 55 °C for 120 min. The corresponding desorption ratio and optimal desorption temperature for each resin were also determined. 2.5.4. The equations used in this study The adsorption/desorption, kinetics and thermodynamic model equations used in this study were the same as in our previous research (Lin, Zhao, Dong, Yang, & Zhao, 2012): The adsorption content:

qt ¼

ðC 0  C t ÞV i W

ð1Þ

The adsorption ratio:

A ð%Þ ¼

C0  Ce  100% C0

ð2Þ

The desorption ratio:

D ð%Þ ¼

Cd V d  100% ðC 0  C e ÞV i

ð3Þ

temperature (K) and A was a constant. The Langmuir and Freundlich equations were used to describe the adsorption equilibrium and linearity fitting of each equation (Fu et al., 2008; Jin et al., 2008). 2.6. Column chromatography experiments The glass column (2.5 cm  60 cm) was wet-packed with selected resins (bed volumes 200 ml). Two grams of crude extract from licorice leaf were dissolved in 100 ml of distilled water (which gives a final concentration of 20 mg/ml) and the solution was carefully loaded onto the top of the column. The column was kept in a chromatography cabinet (CXG-1, Huxi analysis instrument factory Co., Ltd., Shanghai, China) at a constant temperature (4 °C) overnight until the adsorption equilibrium was reached. An aliquot (800 ml) of distilled water, 10% (v/v) ethanol, 30% (v/v) ethanol, 50% (v/v) ethanol, 70% (v/v) ethanol, 90% (v/v) ethanol and 100% (v/v) ethanol was loaded sequentially into the column at 20 °C and eluted with a constant flow rate of 10 ml/min. Each fraction was separately condensed at reduced pressure and 50 °C, using a rotary evaporator (RE52AA, Yarong Equipment Co., Shanghai, China). All the obtained fractions after evaporation were lyophilized in a freeze-dryer (Marin Christ, Osterode, Germany), weighed and stored at 20 °C prior to further analysis. The recovery ratios of the leaf extract were calculated as follow:

The pseudo-first-order kinetics model was expressed as:

lnðqe  qt Þ ¼ k1 t þ ln qe

ð4Þ

The pseudo-second-order kinetics model was expressed as:

1 1 1 1 ¼  þ qt k2 q2e t qe

ð5Þ

The particle diffusion kinetics model was expressed as:

qt ¼ kd  t 1=2 þ C

ð6Þ

The Langmuir equation and its variable form:

qe ¼

qm K L C e 1 þ K LCe

Ce 1 1 ¼  Ce þ qm K L qe qm

ð7Þ

The Freundlich equation and its variable form:

qe ¼ K F C e1=n

ln qe ¼

1  ln C e þ ln K F n

ð8Þ

The enthalpy changes (DH) were calculated by the Van’t Hoff equation:

ln K ¼ 

DH þA RT

ð9Þ

In these equations, qt, qe and qm stood for the total flavonoid adsorption capacity (mg/g dry resin) at time t, the equilibrium adsorption capacity and the maximum adsorption capacity to form monolayer (mg/g dry resin), respectively. C0, Ce, and Cd stood for the initial concentration, the equilibrium concentration, and the concentration of the total flavonoid (mg/ml) in desorption solution, respectively. Vi stood for the volume (ml) of flavonoids solutions used and Vd stood for the volume of the desorption solution (ml), whilst W stood for the dry weight (g) of the resin used. A was the adsorption ratio (%) and D was the desorption ratio (%). k1, k2 and kd refer to the rate constants of pseudo-first-order, pseudosecond-order and particle diffusion kinetics models of adsorption process, respectively. C was the constant in the particle diffusion kinetics model. KL was the affinity parameter between resins and flavonoids (ml/mg). KF was the adsorption capacity of the resins and 1/n was the adsorption intensity of the resins. R was the universal gas constant (8.314 J mol1 K1). T was the absolute

Recovery ratio ð%Þ ¼

X

weight of each fraction=  weight of crude extract 100%

2.7. Determination of total flavonoid content A colorimetric aluminium chloride method was used after some modifications (Chang, Yang, Wen, & Chern, 2002). An aliquot (0.25 ml) of the diluted solution of testing sample was added to a glass test tube, followed by addition of 4.75 ml of aluminium chloride in methanol (0.01 M). The resultant mixture was allowed to stand for 10 min at room temperature before its absorbance was measured at 314 nm, using an ultraviolet visible spectrophotometer (UV-2550, Shimadzu Corporation, Kyoto, Japan). A blank, consisting of all reagents and solvents but the testing sample, was also established for absorbance measurement. The total flavonoid content was determined, using a quercetin calibration curve with concentrations ranging from 0.05 to 0.5 mg/ml in 80% ethanol. The results were expressed as the mass percentage of the total flavonoid in dried testing sample. The calibration curve, containing seven data points, was linear, with R2 being 0.9999. 2.8. Oxygen radical absorbance capacity assay The oxygen radical absorbance capacity (ORAC) of the testing sample was determined by using the same method as that reported in our previous study (Lin et al., 2012). 2.9. Nitrite-scavenging capacity assay The nitrite-scavenging capacity assay was performed, following the method of (Liu et al., 2011) with some modifications. The testing sample was dissolved in DMSO with a concentration of 10 mg/ml and used as the stock solution. Each stock solution was diluted with distilled water to 1 mg/ml. Each diluted sample (1 ml) was first mixed with 1 ml of sodium nitrite solution (1 mM) and topped up to 5 ml with citrate buffer (0.2 M) at pH

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2.0. The resultant reaction mixture was incubated in a water bath at 37 °C for 1 h. Then, an aliquot (1 ml) of the reaction mixture was further mixed with 5 ml of 2% acetic acid and 0.4 ml of Griess reagent (1% sulfanilic acid in 30% acetic acid and 1% naphthylamine in 30% acetic acid with the ratio of 1:1) before incubation at room temperature for 15 min. Subsequently, the absorbance was measured at 520 nm. The nitrite-scavenging capacity (%) was calculated as follows:

Nitrite-scavenging capacity ð%Þ ¼ ½1  ðAS  AS0 Þ=A0   100% where A0, AS0 and AS were the absorbances of the control without testing sample, the sample blank without Griess reagent and the testing sample, respectively. 2.10. High performance liquid chromatography (HPLC) analysis HPLC analysis was performed, using the Waters 600 HPLC system (Waters Co. Milford, MA, USA) equipped with the Waters RP-C18 column (150  4.6 mm i.d., 5 lm; Waters Co. Milford, MA, USA). A gradient mobile phase consisted of distilled water (A) and acetonitrile (B) with a flow rate of 1 ml/min and temperature at 25 °C, 0–5 min: 5% B; 5–10 min: 5–10% B; 10–30 min: 10–30% B; 30–50 min: 30–50% B; 50–70 min: 50–60% B; 70–80 min: 60–100% B; 80–85 min: 100% B; 85–90 min: 100–50% B; 90–95 min: 50–5% B; 95–100 min: 5% B. The injection volume was 20 ll. The absorbance wavelengths for detection were selected at 280 and 310 nm. The testing samples and standard (pinocembrin) were dissolved with DMSO to a concentration of 5 mg/ml (filtered through 0.45 lm filters). 2.11. Statistical analysis All tests were performed in triplicate and the results were reported as means ± standard deviation of three parallel measurements. Significance of the differences between variables was tested

A

by one-way ANOVA, using SPSS 11 (SPSS Inc., Chicago, IL, USA). The mean values were considered significantly different at p < 0.05.

3. Results and discussion 3.1. Adsorption kinetics of five resins on the flavonoids from the G. glabra L. leaf The adsorption kinetic curves of five resins are shown in Fig. 1A. The adsorption processes of all the resins exhibited three-stage changes and reached equilibrium after 30 min. At the first stage, the adsorption capacity of SP-207 and SP-825 resins showed a linear and rapid increased trend over the first 4 min whilst the adsorption capacity of XAD-4, XAD-16 and HP-2MGL resins showed a linear and rapid increased trend over the first 8 min. At the second stage, the adsorption capacity of all these resins increased more slowly and the adsorption equilibrium was reached at 30 min to 1080 min. This result suggested that all of these five resins were rapid adsorption resins. In order to elucidate the adsorption behaviours and mechanisms of all resins, pseudo-first-order, pseudo-second-order and particle diffusion kinetics models were chosen to evaluate the adsorption processes. All these equations, including derived parameters such as correlation coefficient and dynamic parameters, are summarized in Table 2. The pseudo-second-order kinetics model was chosen as the most favourable model for exhibiting the adsorption processes of the flavonoids from the G. glabra L. leaf on the five resins, due to the good correlation obtained. The diffusion curves of five resins (the plots (qt versus t1/2)) are shown in Fig. 1B. The plots exhibit weak linear trends over the time period selected. Thus the whole process was divided into three stages. This result suggested that the adsorption of the flavonoids from G. glabra L. leaf, on all five resins, might contain multiple processes. The three diffusion stages include the boundary layer diffusion (0–4 or 8 min), gradual adsorption stage (4 or 8–30 min) and

B

40 39

39

38

38

37

XAD-16

37

36

XAD-4

36

35

HP-2MGL

34

SP207

33

q t (mg/g)

q t (mg/g)

40

SP825

XAD-16 XAD-4

35

HP-2MGL

34

SP207

33

32

32

31

31

30

30

29

SP825

29

2

4

6

8

10

15

20

25

30

40

50

60

90

120 180 240 360 1080

1.0

2.0

3.0

4.0

Time (min)

t

D XAD-16

80

XAD-4 60

HP-2MGL SP207

40

SP825

1/2

6.0 (min

1/2

7.0

8.0

10.0

100

90

XAD-16

85

XAD-4

80

HP-2MGL

75

SP207 SP825

70

20

9.0

)

95 desorption ratio (%)

C 100 desorption ratio (%)

5.0

65 0 0

20%

40%

60%

ethanol concentration

80%

100%

60 25

35

45 desorption temperature

55

Fig. 1. Adsorption and desorption behaviours of the flavonoids from G. glabra L. leaf on five resins. (A) Adsorption curves. (B) Diffusion curves. The black lines in B stand for the different adsorption stages. (C) Effect of ethanol concentration on desorption. (D) Effect of temperature on desorption.

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Table 2 Pseudo-first-order or pseudo-second-order kinetics equations and associated model parameters. Resins XAD-4

y = 0.0803x + 2.1323 y = 0.0141x + 0.0259

R = 0.9317 R2 = 0.9383

qt ¼ kd  t1=2 þ C

y = 1.7919x + 29.775

R2 = 0.8944

lnðqe  qt Þ ¼ k1 t þ ln qe 1 1 1 1 q ¼ k q2  t þ q

y = 0.0876x + 2.2073 y = 0.0160x + 0.0257

R2 = 0.9424 R2 = 0.9453

qt ¼ kd  t1=2 þ C

y = 1.9967x + 29.102

R2 = 0.8905

lnðqe  qt Þ ¼ k1 t þ ln qe ¼ k 1q2  1t þ q1

y = 0.0891x + 2.2101 y = 0.0166x + 0.0258

R2 = 0.9214 R2 = 0.9839

qt ¼ kd  t1=2 þ C

y = 1.8815x + 29.145

R2 = 0.8385

lnðqe  qt Þ ¼ k1 t þ ln qe 1 1 1 1 q ¼ k q2  t þ q

y = 0.108x + 2.0619 y = 0.0089x + 0.0259

R2 = 0.7607 R2 = 0.9046

qt ¼ kd  t1=2 þ C

y = 1.2311x + 32.713

R2 = 0.8824

lnðqe  qt Þ ¼ k1 t þ ln qe ¼ k 1q2  1t þ q1

y = 0.0503x + 1.5991 y = 0.0086x + 0.026

R2 = 0.7562 R2 = 0.8803

qt ¼ kd  t1=2 þ C

y = 1.1842x + 32.713

R2 = 0.8483

t

HP-2MGL

1 qt

SP207

t

SP825

2

lnðqe  qt Þ ¼ k1 t þ ln qe ¼ k 1q2  1t þ q1

1 qt

XAD-16

Correlation coefficient R2

Dynamic equations

1 qt

2 e

2 e

2 e

2 e

2 e

e

e

e

e

e

equilibrium stage (30–1080 min) (as the three straight black lines with different slopes show in Fig. 1B). The plots do not pass through the origin which indicates that both boundary layer diffusion and intraparticle diffusion were the rate-controlling factors of adsorption (Lorenc-Grabowska & Gryglewicz, 2005). The particle diffusion kinetics models could not represent the whole adsorption process well because of the weak correlation coefficients (Ayranci & Hoda, 2005). However, it could describe a definite mechanism of adsorption in a particular stage. 3.2. Adsorption and desorption capacity of five resins on the flavonoids from G. glabra L. leaf The adsorption of flavonoids reached equilibrium on the five resins after 30 min. The adsorption capacities of the five resins were 39.6 mg/g dry resin for XAD-4, 39.8 mg/g dry resin for XAD-16, 39.3 mg/g dry resin for HP-2MGL, 39.5 mg/g dry resin for SP207 and 39.5 mg/g dry resin for SP825, respectively. In comparison, according to the pseudo-second-order kinetics model, the theoretical adsorption capacities of five resins calculated were 38.6 mg/g dry resin for XAD-4, 38.9 mg/g dry resin for XAD-16, 38.8 mg/g dry resin for HP-2MGL, 38.6 mg/g dry resin for SP207 and 38.5 mg/g dry resin for SP825, respectively. The calculated values fit well with the experimental results. This result suggested that the pseudo-second-order kinetics model was suitable for evaluating the adsorption capacity of the five resins on the flavonoids from G. glabra L. leaf and all five resins had comparable adsorption capacities. The effects of ethanol concentration (Fig. 1C) and desorption temperature (Fig. 1D) on desorption capacities of the five resins were also evaluated. The results showed that the ethanol concentration and desorption temperature significantly influenced desorption capacity of the five resins. With the increase of ethanol concentration, the desorption ratios of the flavonoids from G. glabra L. leaf on all five resins, improved from 3.56% to 91.2% for XAD-16, from 3.93% to 87.2% for XAD-4, from 3.25% to 86.2% for HP-2MGL, from 3.71% to 85.1% for SP207 and from 4.03% to 86.7% for SP825. In fact, most of the flavonoids were desorbed with 80% ethanol, and this result was consistent with some studies about desorption of some resins on phenolic or flavonoids (Silva, Rogez, da Silva, & Larondelle, 2012). However, minimal difference was found in the desorption ratios of flavonoids amongst the five resins at the same ethanol concentration. Meanwhile, higher desorption temperature led to a greater desorption ratio. The

Dynamic parameters k2 = 0.0476 qe = 38.6100

k2 = 0.0413 qe = 38.9105

k2 = 0.0401 qe = 38.7597

k2 = 0.0754 qe = 38.6100

k2 = 0.0786 qe = 38.4615

desorption ratios of five resins improved significantly from 66.1% to 89.8% for XAD-16, from 63.8% to 87.3% for XAD-4, from 65.2% to 86.5% for HP-2MGL, from 63.0% to 88.6% for SP207 and from 64.8% to 90.7% for SP825 when the temperature rose from 25 °C to 45 °C, respectively. However, desorption ratios did not increase with further increase in desorption temperature. In summary, all the above results showed that the five resins exhibited similar adsorption behaviours and possessed good adsorption capacities. This suggested that all the chemical constitutions of five resins (polystyrene, methacrylate and brominated polystyrene) were suitable for adsorption of the flavonoids from G. glabra L. leaf. This result was also consistent with the conclusion of Tomás-Barberán, Blázquez, Garcia-Viguera, Ferreres, and Tomás-Lorente (1992) that polystyrene resins could achieve a good separation effect on flavonoids. Meanwhile, the similar adsorption capacities of the five resins indicated that both the chemical constituents (polystyrene, methacrylate and brominated polystyrene) and the physical structures (particle size, specific surface area and pore diameter) were important for the adsorption of the resins (Lin et al., 2012). Additionally, this study showed that flavonoids possessing two benzene rings could be adsorbed by non-polar, weakly-polar or moderately-polar chemical constituents with suitable particle size, specific surface area and pore diameter. The p–p conjugation between flavonoids and the benzene ring of resins might be one of the forces in the adsorption of flavonoids on these five resins (Liu, Liu, Chen, Liu, & Di, 2010). Therefore, the adsorption performance of macroporous resins on the flavonoids was associated with the synergistic effects of their chemical composition and physical properties (Fu et al., 2005). The XAD-16 and SP825 resins with larger specific surface area possessed a little stronger desorption capacity than did the other three resins, suggesting that the specific surface area might be one of the critical factors that affect the desorption process (Scordino et al., 2003). 3.3. Adsorption thermodynamics of resins on the flavonoids from G. glabra L. leaf The effects of adsorption temperature on the adsorption capacity of all five resins are shown in Fig. 2A. With increase of adsorption temperature, the adsorption ratios of all five resins decreased from 91.8% to 79.1% for XAD-16, from 90.3% to 73.8% for XAD-4, from 90.3% to 76.2% for HP-2MGL, from 91.6% to 80.0% for SP207 and from 91.7% to 80.8% for SP825, respectively. Based on the advantages of desorption capacities (showed in Fig. 1C and D)

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A

95

adsorption ratio (%)

90

XAD-16 XAD-4

85

HP-2MGL SP207

80

SP825 75

70

25

35

45

55

adsorption temperature

B 40

C

30

XAD-16

25

25

20

35

15

45

10

55

q e(mg/g)

q e(mg/g)

40 35

35

30

SP-825

25

25

20

35

15

45

10

55

5

5

0

0 0

0.2 Ce

0.4 mg/ml

0.6

0

0.8

0.2 Ce

0.4 mg/ml

0.6

0.8

Fig. 2. Adsorption thermodynamics on five resins (A), and adsorption isotherm of XAD-16 resins (B) or SP825 (C) for the flavonoids from G. glabra L. leaf.

Table 3 Freundlich model and thermodynamic parameters of the flavonoids from G. glabra L. leaf on the XAD-16 and SP-825 resins. Resins

Temperature (K)

Freundlich model

DH (kJ/mol)

KF

1/n

R2

XAD-16

298 308 318 328

329 254 78.7 33.0

0.946 0.912 1.06 0.886

0.9832 0.9966 0.9792 0.9925

20.3

SP825

298 308 318 328

373 236 72.1 28.4

0.580 0.890 0.970 0.949

0.9903 0.9954 0.9736 0.9971

23.0

and similar good adsorption capacities, XAD-16 and SP825 resins were selected for thermodynamics research. The adsorption isotherms of XAD-16 and SP825 resins are shown in Fig. 2B and C, respectively. With increase of temperature, the slope of the Qe  Ce plots declined markedly for both XAD-16 and SP825 resins. After the fitting of Freundlich and Langmuir models, it was found that the Langmuir model was not suitable for the adsorption behaviours of XAD-16 and SP825 resins (evidenced by the weak correlation coefficients for the Langmuir model on XAD-16 and SP825 resins at different temperatures) (Ayranci & Hoda, 2005). The Freundlich model and thermodynamic parameters of the flavonoids from G. glabra L. leaf on the XAD-16 and SP825 resins are shown in Table 3. The Freundlich model, with good correlation coefficients at different temperatures, seemed to be a good model for reflecting the adsorption equilibrium of XAD-16 and SP-825 resins. According to the Van’t Hoff equation, the enthalpy changes (DH) were calculated as 20.3 kJ/mol (XAD-16 resin)

and 23.0 kJ/mol (SP825 resin), respectively. The negative values of enthalpy changes (DH) for both resins suggested that the adsorption process was exothermic and low temperature was good for the adsorption process (Gökmen & Serpen, 2002). Meanwhile, the absolute values of enthalpy changes (DH), for both XAD-16 and SP825 resins, were less than 43 kJ/mol, indicating that the adsorption of the flavonoids from G. glabra L. leaf onto the resin surface was governed by physical mechanisms rather than chemical mechanisms (Lin et al., 2012). 3.4. Enrichment of the flavonoids from G. glabra L. leaf by resin column XAD-16 and SP825 resins were chosen for the column chromatography experiment and a gradient elution process was used to obtain different constituents from crude extract. Both resins had good mass recovery ratios of dried residue (90.9% for XAD-16 and 90.5% for SP825), total flavonoid (89.9% for XAD-16 and

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Y. Dong et al. / Food Chemistry 168 (2015) 538–545

Table 4 Analyses of the fractions eluted from columns packed with the XAD-16 and SP-825 resins. Resin

Ethanol concentration (%)

Mass of dried residue (mg)

Total flavonoid content (%)

Pinocembrin content (%)

ORAC value (lmol trolox equivalent/g)

Nitrite-scavenging capacity (%)

XAD-16

Water fraction 10% 30% 50% 70% 90% Recovery (%)

763 ± 0.33 128 ± 0.71 149 ± 0.75 252 ± 1.32 200 ± 0.43 325 ± 0.66 90.9

2.16 ± 0.01 4.90 ± 0.10 7.49 ± 0.05 14.7 ± 0.09 33.6 ± 0.51 55.6 ± 0.28 89.9

– 1.15 ± 0.17 0.99 ± 0.24 1.03 ± 0.11 2.78 ± 0.33 15.2 ± 0.26 80.3

741 ± 32.4 1927 ± 258 9056 ± 204 7973 ± 142 5823 ± 188 5059 ± 82.9 –

17.3 ± 0.69 36.0 ± 0.42 76.2 ± 0.97 62.2 ± 0.28 47.4 ± 0.08 20.5 ± 0.42 –

SP-825

Water fraction 10% 30% 50% 70% 90% Recovery (%) Original crude extract

741 ± 0.51 109 ± 0.91 167 ± 0.75 273 ± 0.62 189 ± 1.94 330 ± 0.11 90.5

1.73 ± 0.01 4.49 ± 0.13 6.18 ± 0.15 12.2 ± 0.09 32.5 ± 0.13 53.9 ± 0.38 89.4 16.8 ± 0.53

– 1.25 ± 0.15 0.98 ± 0.09 1.01 ± 0.15 1.62 ± 0.46 19.8 ± 1.09 76.9 5.49 ± 0.22

688 ± 79.5 3142 ± 47.1 6401 ± 196 8543 ± 558 10117 ± 66.3 7973 ± 53.1 – 4190 ± 117

19.6 ± 0.54 32.0 ± 0.61 53.2 ± 1.07 64.7 ± 0.26 71.3 ± 0.88 51.3 ± 1.12 – 51.7 ± 1.72

89.4% for SP825) and pinocembrin (80.3% for XAD-16 and 76.9% for SP825) (Table 4). The total flavonoid content of 70% ethanol fractions from XAD-16 and SP825 resins were both about 16% higher compared to the original crude extract, and the total flavonoid content of 90% ethanol fractions from XAD-16 and SP825 resins were both about 40% higher compared to the original crude extract. About 85% of the flavonoids were present in the 70% and 90% ethanol fractions, indicating good enrichment of the flavonoids by XAD-16 and SP825 resins. Pinocembrin contents in the 90% ethanol fractions of both resins were the highest amongst all the fractions. It was about three times (for XAD-16) and four times (for SP825) as much as that in original crude extract. These results suggested that pinocembrin, as one of the major flavonoids in G. glabra L. leaf, was the major constituent in the 90% ethanol fractions from both resins. Results of the column chromatography experiments showed that there was insignificant difference in the total flavonoid content and pinocembrin content between the same ethanol fractions of the two resins. Both resins could be used as effective agents for the enrichment of flavonoids and pinocembrin from G. glabra L. leaf. Earlier results showed that there was insignificant difference in flavonoids adsorption and desorption behaviours amongst five resins. Thus, all these five resins might be used for enriching flavonoids from G. glabra L. leaf. Comparison could be made of the ORAC values and nitritescavenging capacities amongst all fractions, including the original crude extract (Table 4). Based on the ORAC values, the fractions with high antioxidant capacity were the 30%, 50%, 70% and 90% ethanol fractions derived from both XAD-16 and SP825 resins. The 30% ethanol fraction of XAD-16 resins and the 70% ethanol fraction of SP825 resins had the best antioxidant capacity, respectively. Meanwhile, the antioxidant capacities of the fractions did not correlate well with their total flavonoid and pinocembrin contents. The 30% ethanol fraction of the XAD-16 resins had low total flavonoid and pinocembrin contents but relatively high antioxidant capacity (ORAC value: 9056 ± 204 lmol trolox equivalent/g). These results suggested that the flavonoids, especially the pinocembrin, were not the only antioxidant component that contributed to the antioxidant capacities. However, fractions with good antioxidant capacities from XAD-16 and SP825 resins could be used as food antioxidants whilst fractions with high total flavonoid and pinocembrin contents could be consumed as nutrition supplements or pharmaceutical products. In addition, the changing trend of the nitrite-scavenging capacities showed a good correlation with the ORAC values for all fractions. The fraction with good antioxidant capacity (high

ORAC value) also exhibited great nitrite-scavenging capacity. This suggested that the fractions with good antioxidant capacity and nitrite-scavenging capacity might be applied as effective antioxidants and nitrite-scavenging agents in the food industry. 4. Conclusions This study demonstrated that macroporous resins could significantly enrich the flavonoids from G. glabra L. leaf. Both the chemical constituent and physical structure affected the adsorption/desorption capacities of resins. The kinetic model analyses suggested that the pseudo-second-order kinetic model was the best model to describe the whole adsorption process of the five resins. Further isotherm model analyses and column chromatography research with gradient elute on two representative resins, XAD-16 and SP825, showed that the Freundlich isotherm model could evaluate the adsorption process well. The adsorption was an exothermic and physical adsorption process, and the flavonoids were enriched significantly and the bioactivities were obviously improved. Acknowledgements Authors are grateful for the financial support from Twelfth five-year National Key Technology R&D Program of China (Project No. 2012BAD37B08-01), Science and Technology Planning Project of Guangdong Province, China (Project No. 2011B030500004) and Fundamental Research Funds for the Central Universities (Project No. 2014ZB0010). References Ayranci, E., & Hoda, N. (2005). Adsorption kinetics and isotherms of pesticides onto activated carbon-cloth. Chemosphere, 60(11), 1600–1607. Chang, C., Yang, M., Wen, H., & Chern, J. (2002). Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis, 10(3), 178–182. Fu, B., Liu, J., Li, H., Li, L., Lee, F. S. C., & Wang, X. (2005). The application of macroporous resins in the separation of licorice flavonoids and glycyrrhizic acid. Journal of Chromatography A, 1089(1–2), 18–24. Fu, Y., Zu, Y., Li, S., Sun, R., Efferth, T., Liu, W., et al. (2008). Separation of 7-xylosyl10-deacetyl paclitaxel and 10-deacetylbaccatin III from the remainder extracts free of paclitaxel using macroporous resins. Journal of Chromatography A, 1177(1), 77–86. Gao, Z. P., Yu, Z. F., Yue, T. L., & Quek, S. Y. (2013). Adsorption isotherm, thermodynamics and kinetics studies of polyphenols separation from kiwifruit juice using adsorbent resin. Journal of Food Engineering, 116(1), 195–201.

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