Effects Of Black Rice Extract Against Hepatotoxicity And Nephrotoxicity Induced By Stress And Studies On Anthocyanins In Fruit Of Kadsura Coccinea (Lem.) A.C. Smith

Anthocyanins, a group of phenolic compounds, are responsible for the attractivecolours of many plants in nature. They have a lot of pharmacological activities such asantioxidant, antitumor and antiinflammatory. In this study, anthocyanins from black riceand Kadsura coccinea fruit were both investigated. In part one, effects of anthocyanin-richblack rice extract against restraint-stress induced hepatotoxicity and KBrO3-inducednephrotoxicity were firstly studied. In part two, anthocyanins in K. coccinea fruit werequalitatively and quantitatively analyzed for the first time. In addition, antioxidant activity,pH and thermal stabilities of the major anthocyanin in K. coccinea fruit were also studied.In first part, UPLC/Q-TOF-MS method was used to qualitatively analyze the blackrice anthocyanin extract. Contents of anthocyanins in black rice extract were determinedby HPLC method. In addition, DPPH and ABTS radical scavenging assays were used toevaluate the in vitro antioxidant activity of anthocyanin-rich black rice extract.Intraperitoneal injection of KBrO3could lead to nephrotoxicity in mice. HPLC method wasused to determine the serum Cr level. The levels of serum BUN, renal XOD, MDA and NOwere measured by spectrophotometric method. The changes of above markers weredetermined after treatment with anthocyanin-rich black rice extract. Restraint stress couldresult in hepatotoxicity in mice. The Reitman method was used to determine the serumGPT level. The GSH level was measured by spectrophotometric method. What’s more, thechanges of above markers were also determined after treatment with anthocyanin-richblack rice extract. As a result, UPLC/Q-TOF-MS analysis indicated that there were9anthocyanins in black rice extract, of which8were identified includingcyanidin-3,5-diglucoside, cyanidin-3-gentiobioside, cyanidin-3-glucoside,cyanidin-3-sambubioside, cyanidin-3-rutinoside, peonidin-3-glucoside, cyanidin andpeonidin. By HPLC analysis, each gram of black rice extract contained416.9mg of anthocyanins expressed as cyanidin-3-glucoside. The DPPH and ABTS radical scavengingactivities of anthocyanin-rich black rice extract were comparable to Vc. Treatment withKBrO3caused seriously nephrotoxicity in mice, which happened9h later. The levels ofserum Cr and BUN increased significantly. The levels of renal XOD, MDA and NO alsoincreased. Pretreatment with anthocyanin-rich black rice extract remarkably reduced theserum Cr and BUN levels and also reduced the renal XOD, MDA and NO levels.Twelve-hours restraint stress led to seriously hepatotoxicity in mice with increasing atserum GPT level. Pretreatment with anthocyanin-rich black rice extract remarkablyreduced the GPT, XOD, MDA and NO levels and raised the GSH level. Hence, wespeculate that the protective effects of anthocyanin-rich black rice extract againstKBrO3-induced nephrotoxicity and restraint stress-induced hepatotoxicity were by meansof scavenging excessive free radicals and alleviating the state of oxidative stress in body.In second part, anthocyanins in K. coccinea fruit were extracted with1%HCl-methanol. Anthocyanins were identified by UPLC/Q-TOF-MS. Quantitative analysisof anthocyanins was performed by HPLC. The major anthocyanin in K. coccinea fruit waspurified for further analyses of pH, thermal stabilities and antioxidant activity. pH andthermal stabilities of the major anthocyanin were respectively analyzed byspectrophotometric method and HPLC method. The in vitro antioxidant activity of themajor anthocyanin was carried out by DPPH and ABTS radical scavenging assays. As aresult,4anthocyanins were detected in K. coccinea fruit and they were identified asdelphinidin-3-xylosylrutinoside, cyanidin-3-glucosylrutinoside,cyanidin-3-xylosylrutinoside and cyanidin-3-rutinoside. The contens of aboveanthocyanins decreased in the following order: cyanidin-3-xylosylrutinoside (83.74%)>>delphinidin-3-xylosylrutinoside (7.30%)> cyanidin-3-glucosylrutinoside (5.03%)>cyanidin-3-rutinoside (3.91%). Cyanidin-3-xylosylrutinoside, as the major anthocyanin inK. coccinea fruit, was stable and maintained its original properties at pH around1.Moreover, cyanidin-3-xylosylrutinoside was very sensitive to temperature changes and itwas even unstable at room temperature. Cyanidin-3-xylosylrutinoside tended to degradeinto cyanidin-3-sambubioside, cyanidin-3-glucoside, cyanidin-3-rutinoside and cyanidin athigh temperatures (60,90°C). In addition, the DPPH and ABTS radical scavengingactivities of cyanidin-3-xylosylrutinoside were better than those of BHT.