Extraction and deglycosylation of flavonoids from sumac fruits using steam explosion

Food Chemistry 126 (2011) 1934–1938

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Analytical Methods

Extraction and deglycosylation of flavonoids from sumac fruits using steam explosion Guozhong Chen, Hongzhang Chen ⇑ National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100190, China

a r t i c l e

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Article history: Received 19 April 2010 Received in revised form 1 September 2010 Accepted 6 December 2010 Available online 21 December 2010 Keywords: Steam explosion Sumac fruits Flavonoid extraction Kinetics Deglycosylation

a b s t r a c t The effects of steam explosion on the extraction and conversion of flavonoids from sumac fruits were studied. Steam explosion caused the reduction of particle size and led to the formation of large fissures and micropores on the sumac fruit coat. However, there was little change in total flavonoid content. A study of the process kinetics showed that the flavonoid yield of sumac fruits steam-exploded at 200 °C for 5 min reached the maximum of 19.65 mg/g dry weight at 20 min, which was about 8-fold higher than that of the raw sample. In addition, quercitrin (quercetin-3-O-rhamnoside), the dominant flavonoid in sumac fruits, was deglycosylated and converted into quercetin by steam explosion. The conversion ratio was 84.51% under the steam explosion condition of 200 °C for 5 min. It can be concluded that steam explosion is a promising process for application in flavonoid extraction and conversion in the food industry. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Sumac is the common name for the genus Rhus, which contains over 250 individual species in the family Anacardiaceae. These plants are wild arbors that grow in temperate and tropical regions worldwide. In general, sumac can grow in non-agriculturally viable regions, and has a long history of use by indigenous people for medicinal and other purposes. This suggests potential for commercialising these plants without competing for food production land uses (Wyk & Wink, 2004). Sumac contains flavonoid compounds (Lee, Lim, & Jang, 2002) and the extracts of the plant have been shown to have antioxidant (Bozan, Kosar, Tunalier, Ozturk, & Baser, 2003), free radical scavenging (Candan & Sokmen, 2004), antimicrobial (Nasar-Abbas, Halkman, & Al-Haq, 2004) and hypoglycemic (Giancarlo, Rosa, Nadjafi, & Francesco, 2006) biological activities. These studies have shown that this genus offers promise as a natural source of commercial flavonoids. The traditional techniques of solvent extraction for flavonoids from plant materials usually require longer extraction time, thereby running the severe risk of thermal degradation for most of the flavonoids (Zhang, Shan, Tang, & Putheti, 2009). Hence, improving extraction efficiency and decreasing extraction time in order to reduce the loss of flavonoids is highly important. Ultrasonic, microwave and supercritical fluid technologies have exhibited certain advantages for flavonoid extraction (Bimakr et al., 2009; Gao & Liu, 2005). However, there are difficulties involved in applying ⇑ Corresponding author. Tel.: +86 10 82627067; fax: +86 10 82627071. E-mail address: [email protected] (H. Chen). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.025

these technologies to industrial production, including complex operations and high costs. Steam explosion is an economical and environment-friendly processing method, which is widely used for the pretreatment of lignocellulosic materials (Cara et al., 2008; Chen & Liu, 2007; Chen, Liu, Yang, & Li, 2005; Shevchenko, Chang, Robinson, & Saddler, 2000). The physico-chemical treatment crushes the samples through the sudden reduction of high to atmospheric pressure after steaming the samples for a certain amount of time in a reactor. Kurosumi et al. (2007) has reported that steam explosion followed by hot water and methanol extractions is an effective method for extracting and separating antioxidant compounds from tissues of Sasa palmata. However, application of steam explosion in flavonoid extraction has not yet been reported. In this research, sumac fruits were steam-exploded before ethanol extraction, and the effects of steam explosion on particle size, microstructure, total flavonoid content, and extraction efficiency were investigated. Considering that high temperature – a characteristic of steam explosion – may influence the chemical structure of flavonoids in sumac fruits, extracts from steam-exploded samples were analysed by HPLC–PDA–ESI/MS/MS. This study should be helpful in understanding the application prospect of steam explosion in flavonoid extraction for industrial development. 2. Materials and methods 2.1. Materials Fresh sumac (Rhus chinensis Mill.) fruits from Anhui Province in China were hand-cleaned and dried in a shaded, well-aired

G. Chen, H. Chen / Food Chemistry 126 (2011) 1934–1938

environment for 15 days; these were packed in paper bags and stored in a dark and dry place at room temperature (25 °C) until the experiment (after one week). The prepared fruits contained 5% moisture content. About 500 g of sumac fruits were placed inside a steam explosion vessel (Weihai Automatic Control Reactor Ltd., China), treated at 190 and 200 °C for 3 and 5 min, respectively, by saturated steam in a 4.5 L reactor, and then terminated by explosive decompression (Chen et al., 2005; Chen & Liu, 2007). The steam-exploded fruits were dried in a forced-draught oven at 60 °C for 12 h, ground with a mortar and pestle, and then sieved into four different size ranges (1.70–3.00 mm, 0.83–1.70 mm, 0.25–0.83 mm and <0.25 mm) before being weighed. These particles were considered to be spherical, and the average particle size was calculated by selecting 100 particles at random using a microscope (Olympus BX41, Olympus Corp., Japan). Quercetin, used as a standard sample, was purchased from the National Institute for the Control of Pharmaceutical and Biological Products.

(mg/g dry weight) and k is the specific rate of the flavonoid yield (min1). Flavonoid extraction from sumac fruits should be considered to be two parallel diffusion processes: one faster and one slower (Herode, Hadolin, Kerget, & Knez, 2003; Osburn & Katz, 1944). Generally, most of the flavonoid compounds are derived quickly from broken cells, while flavonoid removal from the intact cells affected by osmosis requires a much longer time. Therefore, the equation needs to be modified to the following form:

C t ¼ C 1  ð1  expðk1 tÞÞ þ C 2  ð1  expðk2 tÞÞ

Microstructural observations of sumac fruits were respectively carried out using scanning electron microscopy (SEM, JSM– 6700F, JEOL, Japan) operated under vacuum at an accelerating voltage of 5.0 kV after dehydration, critical-point drying and sputter-coating with gold. 2.3. Flavonoid extraction

2.4. Determination of total flavonoid content Total flavonoid content was measured using a modified colorimetric method (Zhishen, Mengcheng, & Jianming, 1999). Extract solution (0.5 mL) was added to a test tube containing 4.5 mL of distiled water. Sodium nitrite solution (5%, 0.5 mL) was added to the mixture and maintained for 5 min, after which 0.5 mL of 10% aluminum chloride was added. After 6 min, 4 mL of 1 M sodium hydroxide was finally added. The absorbance of the mixture at 510 nm was measured after 15 min in comparison to a standard curve that was prepared using quercetin. The flavonoid contents were expressed as mg quercetin equivalent/g dry weight. 2.5. Kinetics of flavonoid extraction

ð1Þ

where Ct is the extracted flavonoids at time t (mg/g dry weight), C1 is the maximum flavonoid yield when time approaches infinity

ð3Þ

where E is the residual flavonoids in sample (% total flavonoids), Ct is the extracted flavonoids at time t (mg/g dry weight), C0 is the total flavonoid content of material (mg/g dry weight), a is the model constant, and b is the diffusion rate constant. The values of parameters for different steam explosion conditions were determined from flavonoid extraction data by performing a linear regression of Eq. (3) in its linearised form expressed by:

ð4Þ

The values of the above parameters were respectively calculated numerically with linear regression and non-linear regression procedures using the program ‘‘Origin 8.0’’ (OriginLab Corporation, Northampton, USA). 2.6. HPLC–PDA–ESI/MS/MS analyses of isolated peaks from fruit extracts Flavonoid analyses were carried out on an Agilent 1100 liquid chromatography system equipped with a PDA detector and then interfaced with a LCQ Deca XP Thermo Finngan (San Jose, CA) mass spectrometer equipped with an electrospray ionisation source. An Intersil ODS-3 column (4.6  250 mm, particle size of 5 lm) was used to separate components in the crude extracts. Mobile phase was 0.3% aqueous acetic acid-acetonitrile 90:10 (A) and 0.3% aqueous acetic acid–acetonitrile 40:60 (B). The gradient was 20% B to 50% B with flow rate 0.5 mL/min. The first detection was conducted with the PDA detector in a wavelength range of 190–800 nm, followed by a second detection in the mass spectrometer. Tandem scan (MS/MS) were performed in selective mode. Mass spectrometric condition, capillary temperature, sheath gas and aux gas flow rate, ionisation spray voltage, and collision energy were all optimised to achieve optimal signal. The conversion ratio of flavonoid in sumac fruits after steam explosion was calculated as follows:

Conversion ratio ð%Þ ¼ ðQ SE  Q Raw Þ=QRRaw  100

The mechanism of flavonoid extraction has been described by using the exponential Eq. (1) (Wardhani, Vázquez, & Pandiella, 2010; Xu, Huang, & He, 2008), based on Fick’s law expressed by:

C t ¼ C 1  ð1  expðktÞÞ

Ct ¼ aebt C0

ln E ¼ ln a  bt

Prior to conducting flavonoid extraction, the pigments and oil were removed from sumac fruits through repeated extractions in a Soxhlet extractor using petroleum ether (boiling point range of 30–60 °C). The residue was extracted with 70% (v/v) ethanol solution at a solvent-to-solid ratio of 10 (mL/g) at 80 °C for 5, 10, 20, 30, 40, 50, 60, 90, 120 and 180 min, respectively. The solution was filtered through a 0.2 lm membrane filter, after which the amount of total flavonoids was determined using a spectrophotometer. In order to determine the total flavonoid content of sumac fruits, the fruits were ground through a 1 mm screen, degreased using the above method, and then extracted repeatedly with 70% ethanol at 80 °C for 3 times (2 h each time).

ð2Þ

where C1 and C2 are the flavonoid yields after infinite time for the faster and slower diffusion processes (mg/g dry weight), respectively. In addition, k1 and k2 refer to the parameters of extraction rate for the two processes (min1), respectively. Another model (Eq. (3)) was used to investigate the diffusion rate of flavonoids before reaching the equilibrium state. This model has been reported in kinetic studies of oil extraction (Franco, Pinelo, Sineiro, & Núnez, 2007; Kashyap et al., 2007) and is expressed by:

E¼1 2.2. Structural observation

1935

ð5Þ

where QSE and QRaw refer to the contents of quercetin (by area normalisation method, HPLC) in steam-exploded and raw samples, respectively, and QRRaw refers to the content of quercitrin in the raw sample. The contents of peaks were calculated by area normalisation method. The change of the total flavonoid content was negligible, and all the reduced quercitrin was considered to be converted into quercetin.

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Table 1 Particle size and total flavonoid content of sumac fruits steam-exploded under different conditions. Treatment condition

Steam-explosion intensity log R0A

1.70–3.00 mm (%)

0.83–1.70 mm (%)

0.25–0.83 mm (%)

<0.25 mm (%)

Average particle size (mm)

Flavonoid content (mg/g dry weight)B

Raw 190 °C, 190 °C, 200 °C, 200 °C,

– 3.13 3.35 3.42 3.64

100 54.64 33.35 27.32 0.35

0 10.08 9.10 18.86 18.28

0 20.29 34.03 32.48 46.58

0 14.99 23.52 21.34 34.78

2.66 ± 0.25 1.64 ± 1.13 1.18 ± 1.14 1.15 ± 1.06 0.56 ± 0.50

18.49 ± 0.18a 19.17 ± 0.51a 21.40 ± 0.24b 21.11 ± 0.22b 20.89 ± 0.48b

3 min 5 min 3 min 5 min

Mean values in a column with different letters are significantly different at P < 0.05. A R0 = t  exp((T  100)/14.75), where t is residence time (min) and T is steam temperature (°C). B The values are expressed as means ± SD of triplicate tests.

2.7. Statistical analysis All experiments were carried out in triplicate sets. The reported results are the averages values of three sets of experiments with a standard deviation. The differences between variables were tested for significance using ANOVA and Duncan’s multiple range test. Differences between means were considered significantly different at P < 0.05 (SPSS for Windows 16.0). 3. Results and discussion 3.1. Effects of steam explosion on flavonoid content in sumac fruits Various physical and chemical modifications, including reduction of particle size and expansion of micropores on materials, take place during steam explosion (Grous, Converse, & Grethlein, 1986). Table 1 shows that particle size decreased with the increasing steam temperature and prolonging residence time of steam explosion, although it was not uniform under lower intensity. The scanning electron micrographs of steam-exploded and raw sumac fruits are shown in Fig. 1. Steam explosion caused breakage and destruction of cell walls, leading to the formation of large cavities and intercellular spaces (Fig. 1b), but no pores were observed in the raw fruit coat (Fig. 1a). Mass-transfer through the solid matrix is usually the rate-controlling step in food extractions. Obviously, smaller particles and micropores caused by steam explosion were conducive to the flavonoid extraction by increasing the specific surface area of the material. The effect of steam explosion on the flavonoid content is also shown in Table 1. The flavonoid contents in sumac fruits increased significantly after steam explosion (except at 190 °C for 3 min) (P < 0.05). This result may be explained by the decrease in dry weight of sumac fruits resulting from the degradation of some compositions, such as pectin and hemicellulose, during steam explosion (Chen & Liu, 2007). The flavonoids in sumac fruits could endure the high temperature of the steam explosion for 3–5 min, although previous studies have reported that the process of high temperature leads to the reduction of total flavonoid content (Crozier, Lean, McDonald, & Black, 1997; Dietrych-Szostak & Oleszek, 1999; Prommuak, De-Eknamkul, & Shotipruk, 2008).

groups had a longer equilibrium time (more than 180 min) from the gentle gradients in the curve. The continuous curves in Fig. 2a were obtained by fitting the experimental data to Eq. (2). The correlated results are summarised in Table 2. The coefficients of linear correlation (R2) between predicted and observed values were, in all cases, higher than 0.99, indicating that the proposed kinetic models could be used to describe and predict the extraction of total flavonoids from steam-exploded sumac fruits. For the milled sample, the value of k1 was higher than that of k2, but C1 was lower than C2, suggesting that the slower process dominated during the flavonoid extraction. In contrast, the value of C1 of the steam-exploded sample was higher than that of C2, proving that most flavonoids were removed by the faster diffusion process. In addition, C2 at the condition of 200 °C for 5 min showed a negative value, suggesting that flavo-

3.2. Kinetics of flavonoid extraction from steam-exploded sumac fruits To study the effect of steam explosion on the extraction efficiency of flavonoids from sumac fruits, the kinetics of flavonoid extraction was investigated. The flavonoid yield increased and the equilibrium time decreased as the increasing intensity of steam explosion (Fig. 2a). When the steam explosion condition was 200 °C for 5 min, the flavonoid yield reached the maximum of 19.65 mg/g dry weight, which was about 8-fold higher than that of the raw sample at the equilibrium time of 20 min. In contrast, the milled sample with similar particle size as the steam-exploded

Fig. 1. SEM of (a) raw and (b) steam-exploded sumac fruits. Samples are shown on the same scale to allow direct comparisons of the microstructure.

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G. Chen, H. Chen / Food Chemistry 126 (2011) 1934–1938

Flavonoid yield (mg/g dry weight)

a

Peak 1 20

Peak 2 16

Raw

12

190 °C, 3 min 8

200 C, 5 min 200 C, 3 min

4 0

0

30

60

90

190 C, 5 min 190 C, 3 min

120

Milled Raw

150

200 °C, 3 min

190 °C, 5 min

180

Time (min)

b

0

200 °C, 5 min Quercetin

-1

lnE

Standard 10

-2

15

20

min

Fig. 3. HPLC of flavonoids from steam-exploded sumac fruits. Raw sample and quercetin standard are used as comparison groups.

-3

200 200

C, C,

5 min 3 min

190 190

C, C,

5 min 3 min

3.3. Deglycosylation of flavonoids by steam explosion

Milled Raw

-4 0

20

40

60

80

100

Crude extract fingerprints were obtained by HPLC with a UV/Vis detector at 359 nm. The results are shown in Fig. 3. The structural elucidation of the peaks was conducted by analysing the data from HPLC online-ultraviolet/visible spectrometry (UV/vis) and mass spectrometry (MS) (Table 3). Peaks 1 and 2 were identified as quercitrin (quercetin-3-O-rhamnoside) and quercetin, respectively. Quercitrin accounted for more than 97% of the total flavonoids in raw sumac fruits; however, this amount decreased gradually with increasing steam temperature and residence time. In comparison, quercetin increased and became the dominant flavonoid after steam explosion at 200 °C for 5 min. The results could be interpreted by the report stating that the glycosidic bond of quercitrin is unstable at high temperature, although quercetin is more stable even at temperatures of more than 200 °C (da Costa, Filho, do Nascimento, & Macêdo, 2002). During steam explosion, quercitrin was hydrolysed and converted into quercetin, and the conversion ratio reached 84.51% at 200 °C for 5 min. This suggested that quercetin could be produced from certain quercetin glycosides, such as quercitrin, by steam-explosion, which exhibited the deglycosylation effect without the addition of acid or enzyme. However, a previous study has reported that quercetin glycosides are lost but not translated into quercetin

120

Time (min) Fig. 2. Effect of steam-explosion conditions on flavonoid extraction from sumac fruits. (a) Flavonoid yield versus extraction time. Solvent, 70% (v/v) ethanol; solvent-to-solid ratio, 10 mL/g; temperature, 80 °C. Markers indicate experimental values and continuous curves are the non-linear regression obtained from Eq. (2). (b) Plot resulting from fitting experimental data to Eq. (4).

noid yield decreased after long extraction time. Hence, a faster diffusion process is necessary to decrease extraction time and avoid or reduce the loss of flavonoids. The diffusion rates of flavonoids before equilibrium were also investigated using Eq. (4) (plotted in Fig. 2b). Table 2 shows that there is no variation (P > 0.05) in value of b (diffusion rate constant) between raw and milled samples. However, the steam-exploded sumac fruits (except at 190 °C for 3 min and at 200 °C for 3 min) displayed higher values (P < 0.05) of b, or about 10-fold higher than that of raw sample at 200 °C for 5 min. This suggested that steam explosion evidently enhanced flavonoid diffusion rate. Table 2 Values resulting from fitting kinetic data of flavonoid extraction to Eqs. (2) and (4). Treatment condition

Raw Milled 190 °C, 190 °C, 200 °C, 200 °C,

3 min 5 min 3 min 5 min

Eq. (2)

Eq. (4)

C1

C2

k1

k2

R2

a

b

R2

1.79 1.54 5.23 17.16 13.10 19.61

1.63 12.45 5.19 1.46 4.35 19.61

0.2476a 85.2515d 0.2275ab 0.2028b 0.3329c 0.2700a

0.0179ab 0.0181ab 0.0170a 0.0128c 0.0233b 8.09  1020d

0.9986 0.9946 0.9984 0.9964 0.9967 0.9973

0.5083 0.8832 0.5609 0.6630 0.3693 0.6648

0.0186a 0.0192a 0.0200a 0.1154b 0.0337a 0.1901c

0.9665 0.9925 0.9814 0.9203 0.9475 0.9972

Mean values in a column with different letters are significantly different at P < 0.05.

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Table 3 Identification of two major peaks isolated from fruit extracts at 359 nm. Peak

Retention time (min)

UV kmax

MS m/z

MS/MS m/z

Compound

1 2

12.0 20.5

255, 349 254, 370

449 303

303 –

Quercitrin Quercetin

through various cooking methods (Price, Bacon, & Rhodes, 1997), although aglycone could be released by fermentation, autolysis, and deglycosylation via a b-glucosidase activity (Coward, Barnes, Setchell, & Barnes, 1993; Lambert et al., 1999). Hence, it is important to conduct further investigations on the deglycosylation effect of steam explosion.

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