Studies On The Structure Identification, Classification And Bioactivities Of Carotenoids In Potamogeton Crispus L.

Potamogeton crispus L. (P. crispus) is a submersed herbaceous perennial plant, which is distributed very widely. It was found that ducks fed with P. crispus could lay natural red-yolk eggs with good quality in vast lake areas. It has been proved that the color of egg yolk is closely related with the type and concentration of carotenoids in feed. The studies in our lab showed that there are abundant carotenoids in the P. crispus. Carotenoids not only can enhance the color of egg yolk, but also play an important role in health care such as antioxidation, immunity-regulation and antieaneer etc.. These bioaetivities have become one of Hotpoint in today's researches. P. crispus is a new resource of carotenoids, while there are few reprots about the systematic researches of structure indentifieation, classification, bioaetivities and deep exploitation and use of its carotenoids at home and abroad. So it has very important strategic significance to make comprehensive studies on carotenoids of P. crispus for its exploitation and use.In this study, modern apparatus means of chromatogram and spectrum and the chemical analysis are used to research the structures and classification of carotenoids in P. crispus. And some advanced technologies of cellular and molecular biology were used to study the bioactivities (including antioxidation activity in vitro, anticancer activity both in vitro and in vivo) and anticancer mechartism of carotenoids. The results are as follows:1 Structure identification and Classification of carotenoids in P. crispusCarotenoids in P. crispus, were extracted with a mixture of acetone and petroleum ether (1:1, v/v). Total. carotenoids from P. crispus (TCPC) were obtained after complete saponification by 40 % (w/v) KOH in methanol. Then fractions of y₁, y₂, y₃ and y₄ were separated and purified by column chromatography and thin layer chromatography (TLC). In order to identify the structures of y₁, y₂, y₃ and y₄, technologies of chromatogram and spectrum combining with the chemical analysis were systemically used. The results showed that y₁, y₂, y₃ and y₄ were neoxanthin, violaxanthin, lutein andβ-carotene, respectively. (The red pigments which were identified by a graduated Ph.D. from our lab were rhodoxanthin and its cis-trans isomers.) So P. crispus contains two kinds of carotenoids, i.e. carotenes and xanthophylls, which include neoxanthin, violaxanthin, lutein, rhodoxanthin andβ-carotene.The content of total carotenoids in P crispus which was 230.8μg/g (dry weight) by spectrophotometer. The relative contents of various carotenoids in P. crispus (dry weight) were examined by means of HPLC. The results showed that there were 80.2μg ofβ-carotene, 2.4μg of neoxanthin, 24.8μg of violaxanthin, 84.4μg of lutein and 36.0μg of rhodoxanthin in per gram of P crispus respectively.2 Bioactivities of carotenoids in P. crispusTo clarify the bioactivities of total carotenoids (TCPC) and/or rhodoxanthin (RPC) from P. crispus, the test samples, animals and cells were exposed to TCPC and/or RPC respectively and the effects in antioxidation, antitumor activities and apoptosis in Hela cells were examined.(1) Antioxidation activities of carotenoids in P. crispus in vitroThe effects of TCPC on·OH, red blood cells (RBC) hemolysis, liver mitochondria swelling and lipid peroxidation in liver homogenate and liver mitochondria were studied in this part. The results showed that TCPC could eliminate .OH effectively and inhibit lipid peroxidation induced by·OH. It could protect the biomembrane, reduce RBC hemolysis and lighten the degree of liver mitochondria swelling. So TCPC had very strong antioxidation activities.The effects of TCPC and RPC on lipid peroxidation in oil were also studied. Only RPC had little antioxidation in both bean oil and rape oil, while TCPC could protect lipid against peroxidation in both bean oil and rape oil. Both TCPC and RPC had antioxidation effects in both oils when cooperating with VC. And the effect of TCPC was better than RPC. In the test of photosensitive oxidation of both oils, TCPC and RPC could inhibit the photosensitive oxidation induced by methylene blue. And the antioxidation effects increased with the increase of the concentration of TCPC and RPC.(2) Antitumor activities of TCPC on hepatocarcinoma H₂₂ tumor-bearing miceIn this research, the hepatocarcinoma H₂₂ tumor-beating mice were used as the model. After the mice were fed with TCPC at doses of 10mg/kg.d, 20mg/kg.d and 30mg/kg.d for 10d respectively, it was found that TCPC could obviously inhibit the growth of transplanted hepatoearcinoma H₂₂ tumor and inhibition rates of tumor are 37.4%, 50.7% and 52.3% respectively. TCPC of each group all increased the delayed-type hypersensitivity (DTH) response, promoted the T lymphocyte proliferation and NK cell activity significantly. The antitumor activities of TCPC were accompanied by the significant increase in SOD activity and decrease in MDA content. H₂₂ tumor cell apoptosis induced by TCPC and the change of tumor cell cycle were detected by FCM. The antitumor mechanism of TCPC might involve the enhanced cellular immune function, the improved antioxidant capability, apoptosis induced by TCPC and the change of tumor cell cycle in H₂₂ tumor-bearing mice.(3) Apoptosis induced by TCPC and RPC in Hela cellsIt was found for the first time that the apoptosis induced by TCPC and RPC in Hela cells (human cervical carcinoma) was one of anticancer mechanisms of carotenoids. In addition, the change of cell cycle, the decrease of mitochondrial transmembrane potential (MTP), the increase of intracellular Ca²⁺ level and activity or content of NOS and NO might participate in the process of apoptosis induced by TCPC and RPC. 2.5-10μg/mL TCPC and 5-20μg/mL RPC could inhibit cell proliferation of Hela cells in a dose and time-dependent manner by MTT assay. After Hela cells were treated with TCPC and RPC for 4d, typical apoptotic morphological: changes could be observed by inverted microscope, fluorescence microscope, transmission electron microscope (TEM)and LSCM. After treatment of TCPC and RPC for 4d to Hela cells, compared with the control group, the change of cell cycle distribution and loss of mitoehondrial transmembrane potential were detected by FCM; intracellular Ca²⁺ level examined under LSCM increased obviously; NO content and NOS and iNOS activity in the culture supernatant of Hela cells increased and SA content in the cell membrane also increased.There are few reports about structure identification and classification of carotenoids in P. crispus, bioactivities including antioxidation and anticancer activities of TCPC and RPC until now.