Separation and purification of amygdalin from thinned bayberry kernels by macroporous adsorption resins
Introduction
Bayberry (Myrica rubra Sieb. et Zucc.) is a fruit tree native to China, mainly south of Yangtze river, and typically in Zhejiang province. The bayberry tree has a high fruit-setting rate and manual thinning (removal of young, immature fruits during the growing season) is commonly practiced to achieve a higher productivity and improve quality of the remaining fruit [1]. The thinned fruits are usually regarded as waste and discarded. Compared with the mature bayberry fruit, thinned fruit has a higher proportion of stones in a range of 19.6–34.2% (g/100 g fresh weight [FW]). Each fruit stone contains one kernel and these kernels from mature fruit stone are reported to be rich in nutrients such as crude fiber, fat, vitamins, proteins and minerals [2]. The kernels may be crushed and used as nutritious supplements for animal feed and also have potential for development into high value-added products such as polyphenols, vegetable fats and vegetable protein [3]. Preliminary investigations on TBKs reported average contents of moisture, protein, fats and carbohydrates of 30.2%, 28.0%, 58.4% and 1.8% respectively. In addition to valuable nutritional components, bayberry kernels have also been reported to contain amygdalin, a cyanogenic glycoside with potential pharmacological and toxicological effects [2].
Amygdalin is found in seeds and leaves of loquat, apricot, peach, plum and other Rosaceae species. Amygdalin is an α-hydroxy benzyl nitrile, consisting of the aglycone mandelonitrile, and the disaccharide gentiobiose (6-o-β-d-glucopyranosyl-d-glucose) (Fig. 1), with a formula of C20H27O11N and molecular weight of 458 [4]. It was reported that amygdalin may play a role in relieving cough and asthma in humans [5]; regulate immune function and demonstrate anti-tumor activity [6]. Cyanogenic glycosides have high toxicity potential upon ingestion, however the toxicity potential for amygdalin was reported as very low [7]. Amygdalin from loquat seeds has been proved to be therapeutically potential in preventing and/or treating of atherosclerosis, gastric ulcer, psoriasis, arthritis and wound healing [8], [9], [10], [11], [12]. However there is little reported research on amygdalin from TBKs, and its phytochemical properties and possible medicinal uses are not fully investigated.
Macroporous adsorption resins (MARs) are efficient matrices for enrichment of bioactive substances from plant resources due to their high selectivity and adsorption capacity, as well as stability and resistance to degradation by osmotic shock and oxidation [13]. They are a group of polymers containing a permanent network of pores independent of the state of swelling of the resin and thus display much better solvent tolerance than gel-type resins. The adsorption performance of MARs is closely related to its polarity. Non-polar resins with strong hydrophobic pore surface, without any functional groups, are suitable for adsorbing non-polar substances; medium-polar resins with both hydrophilic and hydrophobic surface properties, containing an ester group, are suitable for adsorbing both non-polar and polar substances; and polar resins adsorb polar substance mainly through electrostatic interactions [14], [15]. The isolation of plant polyphenolic compounds routinely use MARs such as Amberlite XAD resin or Sephadex [16], [17], [18]. Although solid-phase extraction and purification (e.g. C18 Sep-Pak cartridge) are commonly used before HPLC determination of bioactive compounds [19], MARs are more efficient in enrichment of bioactive compounds if its concentration in plant is relatively low [13]. To our knowledge, there is no published report using MARs for purification of cyanogenic glycosides.
An efficient method for the purification of amygdalin from TBKs might provide value-addition opportunities for the utilization of this low-value thinned fruit waste. Therefore the aim of this study was to determine the static and dynamic adsorption/desorption parameters of amygdalin from TBKs extract using macroporous resin chromatography.
Section snippets
Chemicals, reagents and samples
Ethanol of analytical grade was purchased from East Pharmaceutical Technology Co., Ltd. (Hangzhou, China); Acetonitrile of HPLC grade, and amygdalin were purchased from J&K Scientific Ltd. (Beijing, China). Distilled water was used throughout. The cultivar of thinned bayberry fruits was Ding-ao which was collected from Wenzhou City (Zhejiang Province, China) in June, 2012. When the thinned bayberry was arrived at our laboratory, stones were separated by removing the outer villiform pulp after
Standard curve of amygdalin and the concentration in the crude extract
According to the method in Section 2.2, a very good regression equation for HPLC determination of amygdalin (y = 4.7657x + 8.562, R2 = 0.9997) was obtained, where y was the peak area of amygdalin and x was the amygdalin concentration (mg l−1). The HPLC chromatograms of the crude TBK sample and authentic amygdalin standard are shown in Fig. 2. The purity of amygdalin in the crude TBK sample was determined to be 4.83% (w/w).
Screening of optimum resin
Adsorption/desorption properties of seven MARs from the static
Conclusions
This study developed a method for separation and purification of amygdalin from TBKs. As a result of 82.0% purity and 77.9% recovery of amygdalin in the enriched product, D101 macroporous resin chromatography with the elution by 40% ethanol (aq.) (v/v) at 30 °C was found suitable for the separation of amygdalin from the crude extracts with 4.8% purity. The results evidenced a good adsorption and separation potential of the D101 resin to amygdalin.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgement
This research project was financially supported by the Program for Zhejiang Leading Team of Sci. & Technol. Innovation (2010R50032), Department of Science and Technology, Zhejiang Province, China.
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