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Reverse-phase high performance liquid chromatography for the quantification and optimization for extracting ten kinds of carotenoids in pepper (Capsicum annuum L.) leaves Jing Li, Jianming Jian Xie, hua Ji Yu, Jian Lv, feng Jun Zhang, long Xiao Wang, Cheng Wang, Nan Chao Tang, Chun Ying Zhang, Mohammed Mujitaba Dawuda, Qiang Dai Zhu, and Li Guo Ma J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02440 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on August 26, 2017
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Journal of Agricultural and Food Chemistry
Reverse-phase high performance liquid chromatography for the
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quantification and optimization for extracting ten kinds of carotenoids in pepper
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(Capsicum annuum L.) leaves
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Jing Lia, Jianming Xiea*, Jihua Yua, Jian Lva, Junfeng Zhanga,b, Xiaolong Wanga,
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Cheng Wanga, Chaonan Tanga, Yingchun Zhanga, Mohammed Mujitaba Dawudaa,c
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Daiqiang Zhua, and Guoli Maa
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Anning District, Lanzhou, 730070, P. R. China. b
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Vegetable Institution of Gansu Academy of Agricultural Science, Anning
District, Lanzhou, 730070, P. R. China. c
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College of Horticulture, Gansu Agricultural University, Yingmen Village,
Department of Horticulture, FoA, University for Development Studies, P. O.
Box TL 1882, Tamale, Ghana
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*Corresponding Author
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Xie Jianming, Phone: + 86 13893335780. E-mail:
[email protected] 16 17
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ABSTRACT
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Carotenoids are considered to be the crucial elements in many fields, furthermore,
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the significant factor in pepper leaves under low light and chilling temperature.
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However, little literates focused on the method to determinate and extract the contents
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of carotenoid compositions in pepper leaves. Therefore, a time-saving and highly
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sensitive reverse-phase high performance liquid chromatography method for
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separation and quantification of ten carotenoids was developed and an optimized
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technological process for carotenoid compositions extraction in pepper leaves was
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established for the first time. Our final method concluded that six xanthophylls eluted
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after about 9–26 min. By contrast, four carotenes showed higher retention times
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nearly after 28–40 min, which significantly shortened time and improved efficiency.
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Meanwhile, we suggested that 8 mL of 20% KOH–methanol solution should be added
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to perform saponification at 60 °C for 30 min. The ratio of solid–liquid was 1:8, and
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the ultrasound–assisted extraction time was 40 min.
KEY
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WORDS:
pepper
leaves;
carotenoid;
high
performance
liquid
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chromatography; separation and quantification; optimization; ultrasound assisted
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extraction
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INTRODUCTION
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INTRODUCTION
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Carotenoids, a large group of natural pigments with colors ranging from yellow
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to red, are widely distributed in higher plants, algae and microbes, especially in all
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photosynthetic organisms, and also present in some non-photosynthetic bacteria and
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fungi1. A number of researches demonstrated that carotenoids are responsible for the
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abundance of bright colors; are in close correlation to preventing an age-related
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macular degeneration of the eye2; can reduce the occurrence of cancers3, 4,
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cardiovascular diseases5, and diseases related to low immune function6, 7. Meanwhile,
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carotenoids are thought to play two major roles in photosynthesis, namely,
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light-harvesting and photoprotection8, accounting for being complex II antenna
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proteins (LHC- II) associated with photosystem II (PSII)9 and dissipating excess
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energy through quenching triplet state chlorophyll10. In addition, carotenoids behave
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as important antioxidants11, 12. Traditional methods for the detection of carotenoids include spectrophotometry13
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paper
chromatography14
(PC),
column
chromatography15,
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(SP),
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chromatography16 (TLC), and high performance liquid chromatography (HPLC).
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HPLC is commonly used as a method of separating and determining photosynthetic
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pigments such as chlorophylls and carotenoids because of its speed and high
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sensitivity. The HPLC methods and extraction methods are constantly being modified
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and improved. Due to the different carotenoid compositions, numbers, species, and 3
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thin-layer
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different purposes of separation and detection in various materials, the methods
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applied by researchers are diverse. Previous researches have reported on the
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separation and quantification of carotenoids using HPLC17-23. Daood and Biacs (2005)
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developed an HPLC method for simultaneously detecting of lycoxanthin, lycopene
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epoxide, lycopene and β-carotene in red pepper fruits. This method was performed on
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Nucleosil C18 column with a gradient elution program consisting of three mobile
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phases, namely, 9% water in methanol, methanol-acetonitrile-isopropanol (10:35:55),
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and
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all-trans-β-cryptoxanthin, all-trans-α-carotene, and all-trans-β-carotene in buriti,
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mamey, marimari, peach palm, physalis, and tucuma were separated and detected on
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C30 YMC column with 0.9 mL/min flow rate at 22 °C and a linear gradient of MeOH
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with 0.1% triethylamine/ methyl tertiary butyl ether from 95:5 to 70:30 in 30 min, to
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50:50 in 20 min, and then maintained for 35 min. Meanwhile, the content ranged from
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38 µg/g to 514 µg/g of the total detected carotenoid25. The contents of β-carotene,
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lycopene, phytoene, and phytofluene in 50 watermelon cultivars were separated and
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quantified by HPLC with C30 YMC carotenoid column and the gradient elution
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consisted of three mobile phases, namely: A 90% methanol and 10% ddi water
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containing 0.5% trithylamine and 150mM ammonium acetate; B 99.5% 2-propanol
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and 0.5% triethylamine and C 99.95% tetrahydrofuran and 0.05% triethylamine26. The
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process of carotenoid extraction is sophisticated, and it is affected by extraction
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solvent, time, temperature and pre-treatment processes. The research performed by
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Mezzoso et al (2011) indicated that the procedure that combined cooking, drying, and
methanol24.
The
carotenoids
of
all-trans-lutein,
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all-trans-zeaxanthin,
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milling was the best pre-treatment for carotenoid extraction from P. brasiliensis and P.
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paulensis using acetone and hexane: isopropanol (50:50,v/v). The concentration of
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astaxanthin reached up to 252 µg/g27. The yield of carotenoid from shrimp waste was
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significantly affected by the sunflower oil to waste (P
