Extraction And Identification Of The Anthocyanins Of Coix L And Physiological And Biochemical Comparison Of The Different Culm-colored Lines Of The Cultivated Coix L.

The Coix lacryma-jobi L.of the family Graminea,originated from Asia,are ancient and important food,herbal medicinal and forage economical crops,which are mainly distributed in China,Burma,India,Japan and other regions of the Southeast Asia.It is one of the most widely cultivated with the largest cultivation area among the cultivated food and medicinal homologous plants in China.Based on our field observations on more than a dozen of cultivated Coix L cultivars and for several years,there are two types of the colors,green and violet,in culms,leafs,and stigmas within each cultivar;and the violet-culmed parts are rich in anthocyanins.We have selected and obtained near-pure green-culmed and violet-culmed lines of several cultivars.And preliminary observations showed that the violet-and the green-culmed lines are different in some important agronomical traits.Anthocyanin are one of the sources of important natural red pigments.Coix L is of high value not only in nutrition,health-protection but also in comprehensive development and utilization.In this research the selected violet-culmed Coix lines were used as the materials to investigate the extraction,identification,the extracting technology and the stability of Coix anthocyanins;and the physiological and biochemical differences of the green-and violet-culmed lines were comparatively studied.The main results are as follows:1.The optimum conditions for extraction and preparation of Coix anthocyaninsThe absorption spectrum of the extracted Coix anthocyanins in the visible lihght region showed that the the maximum absorption wavelength is 517nm.Through the comparison of the effects of ethanol extraction and the ultrasonic extraction on yield by single factor orthogonal tests,it was found that the optimum extraction conditions for Coix anthocyanins were determined to be 60%ethanol at 50℃,pH 2.0 with a solid liquid ratio of 1:20 for 4 hours;and under the same conditions of ethanol concentration,temperature,pH and solid liquid ratio,the optimum soncication conditions for extraction was 350 W ultra-sonication for 50 min,which significantly increased the extracting efficiency.2.The stability of the extracted Coix anthocyaninsResults showed that the ultrasonciation assisted ethanol extracted Coix anthocyanins were stable in acidic conditions especially at pH2.0;and it will be extremely unstable and turn to green at alkali conditions;temperature had a relative strong effect on Coix anthocyanins but still be in red color;higher temperature reduced both the absorption at 515nm and the retention rate of the extracted Coix anthocyanins;and the extracted Coix anthocyanins could be long-term stored in low-temperature conditions.Hydrogen peroxide greatly reduced the stability of Coix anthocyanins and the retention rate decreased with increase of H₂O₂ concentration;it is sensitive to oxidative reagents and should avoid the presence of oxidative agents in storage;Coix anthocyanins were also shown to be sensitive to reducing agents such as vitamin C,should be also avoided in storage.Metal ions such as Na⁺,K⁺,Ca²⁺and Cu²⁺basically had no or little effects on the stability of the extracted Coix anthocyanins,however Fe²⁺was found to be inhibitive to Coix anthocyanins and should not be in contact with in storage.3.The physiological and biochemical differences of the green-and violet-culmed Coix linesMeasurements in the tillering stage showed that the net photosynthetic rates（P_N）,the contents of chlorophyll a,b and a+b were all higher in green-culmed lines compared with the corresponding violet-culmed lines;while in the early and terminal flowering stages,they were all lower in green-culmed lines compared with the corresponding violet-culmed lines;leaf total antioxidant capacities（T-AOC）at the tillering stage were shown to be higher in green-culmed lines compared with the corresponding violet-culmed lines;but at the early flowering stage,it was higher in violet-culmed lines.The enzymatic activities of both the phosphoenol pyruvate carboxylase（PEPCase）and the ribulose diphosphate carboxylase（RUBPCase）at the tillering stage were shown to be higher in green-culmed lines compared with the violet-culmed lines.The activities of peroxidase and catalase at the tillering stage were shown to be higher in violet-culmed lines compared with the corresponding green-culmed lines.The intercellular carbon dioxide concentration（Ci）,transpiration rate（E）and stomatal conductance（Gs）from the tillering to peak flowering stages were all shown to be higher in violet-culmed lines compared with the corresponding green-culmed lines and the contents of carotenoids（Xc）were also shown to be higher in violet-culmed lines compared with the corresponding green-culmed lines except for Guangxi Coix.The ratios of Chla/b in violet culmed lines were shown to be higher in the tillering stage,lower in the early flowering stage and higher again in the peak flowering stage compared with the green-culmed corresponding lines at the respective flowering stages.The P_N and the ratios of Chla/b in most of the tested materials showed a trend:decreased from tillering to early flowering stages,and increased from early to peak flowering stages;the contents of Chla,b,a+b,carotenoids（Xc）in all the tested materials had a trend:increased from tillering to early flowering stages and decreased from early to peak flowering stages.The T-AOC values showed an increase from tillering to early flowering stages;and the contents of anthocyanins were shown to be higher from tillering to peak and terminal flowering stages.In general,P_N showed positive correlation to the ratio of Chla/b and the contents of Chla,b,a+b and Xc were negatively correlated to the contents of anthocyanins.The established optimum conditions for the extraction and preparation of Coix anthocyanins in this research laid a very good basis for further development and utilization of Coix-based health-enhancing products and for the basic researches concerning the expression of anthocyanin biosynthesis-related genes as well as the breeding of novel Coix varieties based on culm color;the systematic comparison of the physiological and biochemical features of the different culm-colored lines will promote the physiology-based breeding of Coix to develop high-yield and quality novel Coix lines.