Elsevier

Carbohydrate Polymers

Volume 147, 20 August 2016, Pages 354-364
Carbohydrate Polymers

Box–Behnken design for extraction optimization, characterization and in vitro antioxidant activity of Cicer arietinum L. hull polysaccharides

https://doi.org/10.1016/j.carbpol.2016.03.092Get rights and content

Highlights

  • The hot water extraction of CHPS was optimized by a Box–Behnken design.

  • The maximum extraction yield of CHPS was 5.37 ± 0.15%.

  • Three fractions were purified from CHPS by using the method of chromatography.

  • Crude CHPS and its purified fractions showed excellent antioxidant activity in vitro and in cellular level.

Abstract

The optimal extraction conditions with a yield of 5.37 ± 0.15% for extraction of polysaccharides from chickpea (Cicer arietinum L.) hull (CHPS) were determined as extraction temperature 99 °C, extraction time 2.8 h and ratio of water to raw material 24 mL/g. Three fractions of CHPS-1, CHPS-2 and CHPS-3, with average molecular weight of 3.1 × 106, 1.5 × 106 and 7.8 × 105 Da, respectively, were obtained from crude CHPS by chromatography of DEAE Fast Flow and Sephadex G-100. CHPS-1 was composed of mannose, rhamnose, galactose, galacturonic acid, glucose and arabinose, CHPS-2 was composed of mannose, rhamnose, galacturonic acid, galactose, xylose and arabinose, CHPS-3 was composed of galacturonic acid, galactose and rhamnose. CHPS-3 showed the strongest reducing power and protective effect on H2O2-induced oxidative injury in PC12 cells and highest scavenging activities against DPPH and ABTS radicals, while CHPS-2 showed the highest scavenging activity against superoxide anion radical.

Introduction

Legumes play an important role in human diet due to their cheap price and abundant nutrition (rich in protein and carbohydrates). In developing countries, they are even considered as poor man’s meat for their high contents of protein and slow released carbohydrates (Tharanathan & Mahadevamma, 2003). The seed coat (hull) of legumes is often indigestible and may have a bitter taste. Accordingly, dehulling is one of the most important operations in post-harvest handling of legumes. It has been reported that the total hull generated is about 20% of the total quantity legumes processed (Kanatt & Sharma, 2011). The industry of post-harvest handling of legumes, therefore, generates a large amount of bio-waste, which is rich in bioactive compounds that can be potential source of antioxidant, antimicrobial or other bioactive compounds. Chickpea (Cicer arietinum L.), a typical and popular legume in North Africa, Middle East, Southern Europe, America and Australia, is the third important pulse crop in the world and rich in starch (37.5–50.8%) and protein (Jukanti, Gaur, Gowda, & Chibbar, 2012). Based on seed morphology and cultivation area, chickpea can be grouped into two types: Desi (C. arietinum ssp. Orientale and C. arietinum ssp. Asiatinum) and Kabuli (C. arietinum ssp. Mediterraneum and C. arietinum ssp. Eurasiaticum). Desi chickpeas have small and dark seeds and a thick coat, cultivated mostly in Asia and Africa; while Kabuli chickpeas have large and light color seeds and a thin and smooth coat, cultivated mostly in Europe, North America, West Asia and North Africa (Jukanti et al., 2012; Wood, Knights, & Choct, 2011).

Free radicals are generated from normal metabolism and can also be stimulated by some diseases or some xenobiotics (Osinska-Jaroszuk et al., 2015). Although they play an important role in many normal functions of living organisms, their uncontrolled production will damage the cells and cause undesirable effects on cell functions (Li, Liu, Fan, Ai, & Shan, 2011). Free radicals fall into three main types: reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive sulfur species (RSS) (Carocho & Ferreira, 2013). Different types of free radicals may have different reaction mechanisms, but all have relationship with many kinds of diseases such as cancer (Bennett, Rojas, & Seefeldt, 2012), diabetes (Cao, Xie, & Chen, 2015), fatty liver disease (Serviddio, Bellanti, & Vendemiale, 2013), inflammation (Li, Wang, Zhao, Wu, & Peng, 2015) and so on. In order to reduce the damage of free radicals, many kinds of antioxidants are utilized widely at present. They can interact with free radicals and terminate the chain reaction before vital molecules are damaged (Oroian & Escriche, 2015). However, synthetic antioxidants are considered to be responsible for liver damage and carcinogenesis (Carocho, Morales, & Ferreira, 2015; Grice, 1988). Therefore, it is essential to develop natural nontoxic antioxidants to protect human body from free radicals and retard the progress of many chronic diseases. In the search of new natural antioxidants, a number of polysaccharides obtained from plants, animals and microorganisms have been demonstrated to possess potent antioxidant activities and potential applications as natural antioxidants (Liu, Willfor, & Xu, 2015; Osinska-Jaroszuk et al., 2015; Qiao et al., 2009; Xu, Ye, Sun, Tu, & Zeng, 2012). Considering the excellent nutritional value and biological functions of natural polysaccharides, some researches about the dietary fibers from cereals and legumes have been reported (Dodevska et al., 2013). However, the information on the extraction and antioxidant activities of polysaccharides from chickpea hull (CHPS) is not available to date.

In this study, the extraction of CHPS was firstly optimized by using response surface methodology (RSM) based on a Box-Behnken design (BBD). Then, the crude CHPS was purified by chromatography of DEAE Fast Flow column and Sephadex G-100 column, and the crude CHPS and its purified fractions were partially characterized via chemical analysis, high performance liquid chromatography (HPLC) and Fourier transform-infrared spectroscopy (FT-IR). Finally, the antioxidant activities of crude CHPS and its purified fractions were investigated by several chemical assays and an in vitro cellular assay. To the best of our knowledge, it is the first report on the extraction and antioxidant activities of CHPS.

Section snippets

Materials and chemicals

Chickpea seeds (Kabuli type) were purchased from a local market in Urumchi, Xinjiang Uygur Autonomous Region of China. DEAE Fast Flow, Sephadex G-100, ascorbic acid (vitamin C, VC), [2,2′-azinobis-(3-ethyl-benzothiazolin-6-sulfonic acid)] diammonium salt (ABTS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferrozine, nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium (NBT), phenazine methosulphate (PMS),

Effect of extraction temperature on extraction yield of CHPS

To investigate the effect of temperature on extraction yield of CHPS, the extraction was carried out at different temperature (60, 70, 80, 90 and 100 °C), while the extraction time and ratio of water to raw material were fixed at 2 h and 20 mL/g, respectively. As shown in Fig. 1A, the extraction yield increased from 1.73% to 5.13% accompanying the increase of extraction temperature from 60 °C to 100 °C, which may be interpreted that the polysaccharide diffusion coefficient increases with the

Conclusion

RSM was used to optimize the extraction conditions of CHPS extraction, and the optimal extraction conditions were obtained: extraction time 2.8 h, extraction temperature 99 °C, ratio of water to raw material 24. Under these conditions, the extraction yield of CHPS was 5.37 ± 0.15%, which is in good agreement with the predicted value of 5.81%. Crude CHPS mainly contained three fractions (CHPS-1, CHPS-2 and CHPS-3) and the contents of carbohydrate, protein, uronic acid and sulfuric radical in CHPS-1,

Acknowledgements

This work was supported by a grant-in-aid for scientific research from the National Natural Science Foundation of China (No.31201454), a grant from Specialized Research Fund for the Doctoral Program of Higher Education (20130097110021) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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