Effect of seed germination of Cajanus cajan on pinostrobin and cajaninstilbene acid quality

1. 6th Thailand-Taiwan Academic Cooperation Conference on “Food and Agricultural Innovation: Going Global” 15 November 2010 at Kasetsart University, Bangkok, Thailand Effect of seed germination of Cajanus cajan on pinostrobin and cajaninstilbene acid quality Chen Chih-Yuan aLai Chun-Yen bHuang Tzou-Chi b* aGraduate Institute of Bioresources , National Pingtung University, 912, Pingtung, Taiwan b Department of Food Science, National Pingtung University, 912, Pingtung, Taiwan Corresponding author:tchuang@mail.npust.edu.tw ABSTRACT The guandu bean or pigeon pea (Cajanus cajan (L.) Millsp.) is a kind of leguminous plant of the Fabbaceae family and is grown in Asia, Africa and South America. With the improvement of the life quality, disease prevention and health care, Cajanus cajan has been paid more attentions. Seeds were soaked in water for 24 hours and germinated. The sprouts of Cajanus cajan were freeze-dried and divided into root, stem and leave. Microwave-assisted extraction using ethanol-water (60:40) as solvent was applied to extract the major components from the dried samples. LC-MS were used to characterize the chemical structure. Two major compounds, pinostrobin and cajaninstilbene acid were found in the leave of spouts but not in the seeds. The concentration of these two compounds increased with increased cultivation time. Keywords: Cajanus cajan, seed germination, microwave-assisted extraction, LC-MS, pinostrobin, cajaninstilbene acid Introduction Using standards for pinostrobin and cajaninstilbene acid, retention times, UV–Vis spectra, and MS–MS spectra matched pinostrobin and cajaninstilbene acid from Pigeon pea extract. The variation in contents of pinostrobin and cajaninstilbene acid compounds in pigeon pea during seedling growth period was shown in Fig. 2. Two major compounds, pinostrobin and cajaninstilbene acid were found in the leave of spouts but not in the seeds. The concentration of these two compounds increased with increased cultivation time. It was observed that Seedlings are vulnerable to pathogen attacks and for efficient protection, chemical defenses must be deployed and accumulated very early in the seedling development (Ceballos et al., 1998), hence, much secondary metabolites are generated. The variation in contents of phenolic compounds correlates with the metabolic balance of plants and external environmental disturbances. Pigeon pea (Cajanus cajan (L.) Millsp.) is a famous and multi-use grain crop in semi-tropical and tropical developing countries. Pigeon pea is an ideal source of protein and Vitamin B in human diet, especially in the vegetarian population. It has been used for many years for treating diabetes, hepatitis, measles, jaundice and dysentery, expelling bladder stones and stabilizing the menstrual period (Fu et al., 2006). Chemical composition investigations revealed that there are a number of phenolic compounds present in pigeon pea leaves, stems and roots, including flavonoids, stilbenes, etc.(Duker-Eshun et al., 2004). In previous investigation, the ethanol extracts of pigeon pea leaves as well as the main constituents vitexin, orientin, pinostrobin and cajaninstilbene acid exhibited moderate antioxidant activity (Wu et al., 2009). Therefore, pigeon pea is a beneficial food and potential source of natural antioxidants. A Materia and Method Pinostrobin 41.67 min Cajaninstilbene acid 47.45 min Germination procedure Matured pigeon pea seeds were bought from JIANSHIH township, HSINCHU county, TAIWAN.After immersion in water for 24 h, the pigeon pea seeds were planted in the same laboratory. Extraction of compounds Pigeon pea seedlings were separated into leaves, stems and roots, respectively. After sampling, two samples of fresh pigeon pea were weighed, one was used to determine the moisture content; the other was used for extraction. Sample (1 g) was homogenised for 2 min with 20 ml of aqueous-ethanol (40:60, v/v) solution, the mixture was subjected to solvent extraction in a microwave-assisted extraction system (Microwave Start E, Milestone, USA) for 10 min. The irradiation power was 700W and the extraction temperature was 75 C. After vacuum filtered. All solutions and samples prepared for LC–MS were filtered through 0.45m nylon membranes (Millipore, MA, USA) prior to use. LC-MS conditions Chromatographic analyses were performed by using LCQ series LC-MS System(Thermo Fisher Scientific ,USA). Separation of the analytes was achieved on a C18 reversed-phase column(2.0 mmX150 mm , 5μm, Phenomenex,USA). The mobile phase consisted of methanol (A) and 0.08% formic acidaqueous solution (B) using the following gradient elution programfor separation: 0–30 min, 33–65% (A); 30–40 min, 65–90% (A); 40–50 min, 90% (A);50–60min, 90–33% (A). The column temperature was maintained at 30℃, the flow rate 200 μl/min and the injection volume was 10μl . The detection wavelength was 280 nm B C Results and discussion To further characterize individual components of the extract, MS and MS–MS spectra were obtained. The pinostrobin and cajaninstilbene acid of ion scans are shown in Fig. 2. The MS–MS spectrum of Fig. 1A matched the fragmentation spectrum of Cajaninstilbene acid (Fig.1C). The Fig. 1B matched the fragmentation spectrum of Cajaninstilbene acid (Fig.1D). D A B Fig. 2. Variation in contents of pinostrobin and cajaninstilbene acid in (A)leaves(B)seeds(C)roots(D)stems of pigeon pea during seedling growth period. References Ceballos, L., Hossaert-McKey, M., McKey, D., & Andary, C. (1998). Rapid deployment of allelochemicals in exudates of germinating seeds of Sesbania (Fabaceae): roles of seed anatomy and histolocalization of polyphenolic compounds in antipathogen defense of seedlings. Chemoecology, 8, 141–151. Duker-Eshun, G., Jaroszewski, J. W., Asomaning, W. A., Oppong-Boachie, F., & Christensen, S. B. (2004). Antiplasmodial constituents of Cajanus cajan. Phytotherapy Research, 18, 128–130. Xin Huaa, Yu-Jie Fua, Yuan-Gang Zu, Nan Wu, Yu Kong, Ji Li, Xiao Pengb, Thomas Efferth (2010) Plasma pharmacokinetics and tissue distribution study of cajaninstilbene acid in rats by liquid chromatography with tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 52: 273–279. Wei Liua, Yu Konga, Yuangang Zua, Yujie Fua, Meng Luoa, Lin Zhanga, Ji Lia (2010) Determination and quantification of active phenolic compounds in pigeon pea leaves and its medicinal product using liquid chromatography–tandem mass spectrometry. Journal of Chromatography A 1217: 4723–4731. Wei Liu, Yuan-Gang Zu, Yu-Jie Fu, Yu Kong, Wei Mac, Mei Yang, Ji Li, Nan Wua (2010) Variation in contents of phenolic compounds during growth and post-harvest storage of pigeon pea seedlings. Food Chemistry 121: 732–739. Yu-Jie Fu , Wei Liu, Yuan-Gang Zu, Mei-Hong Tong, Shuang-Ming Li, Ming-Ming Yan, Thomas Efferth, Hao Luo (2008) Enzyme assisted extraction of luteolin and apigenin from pigeonpea [Cajanus cajan (L.) Millsp.] leaves. Food Chemistry 111: 508–512. D C Fig. 1. Ion mass spectrum of (A) pinostrobin in leaves of pigeon pea, (B) Cajaninstilbene acid in leaves of pigeon pea, (C)Cajaninstilbene acid was purchased from Sigma,(D) Cajaninstilbene acid quote fromWei et al.,(2010).