Studies On The Structure Of Red Carotenoids In Potamogeton Crispus L.and Its Biological Activity Of Carotenoids Extracts

Potamogeton crispus L.(abbrev. P. crispus) is potamogetonaceae submerged vascular macrophyte, which is distributed all around the world and widely grown in lake, reservoir, trench and pond. The appearance that ducks fed with P. crispus could lay natural red-yolk eggs with good quality and price had been known in lake areas long ago, but the reason is an unsolved riddle up to now. The quantity of P. crispus fed by ducks is restricted because that this macrophyte has a short growing period every year. Many researches have proved that the egg yolk color is closely related with the type and concentration of feed carotenoids. Carotenoid not only is a kind of important natural pigment, but also plays an important role in health care such as anti-oxidation, immunity-regulation, inhibiting tumor and anti-cancer etc. Our lab has proved that the carotenoids extracts of red-yolk eggs had a marked inhibition on the proliferation of S180 tumour, so it has very important significance to make comprehensive studies on carotenoids of P. crispus. In this study, the chemical analysis, spectral analysis, chromatographic analysis and mass spectrographic analysis are used to research the relations of carotenoids between P. crispus and red-yolk duck eggs, especially the methods on the isolation, purification, characteristic and structure of red carotenoids in P. crispus. The experimental technologies of cellular and molecular biology are used to study the effect of carotenoids extracts of P. crispus (CEPC) on the growth inhibition and apoptosis of human hepatoma cells line QGY-7703 in vitro. The food and feed processing technologies are used to realize the exploitation and industrialization of functional P. crispus feed for red-yolk egg duck. The main results are as follows: 1. Preliminarily comparing P. crispus with the red-yolk duck eggs in carotenoids characteristics. Establishing the methods of isolation, purification and preparationof the red carotenoids from P. crispus.1.1 The optimum conditions for extraction of total carotenoids from freeze dried P. crispus powder are studied. The results are as follows: petroleum ether-acetone(1:3,v/v) as extracting solvent, the ratio of solvent volume to the material amount is 90 mL/g, extracting 3 times at room temperature, total extracting time is 1.5h. The extraction ratio of total carotenoids is 95.5%, the content of total carotenoids in freeze dried P. crispus powder is 552.57mg/kg.1.2 The red carotenoids and part yellow carotenoids contained in P. crispus have well coincidence relation with red-yolk duck eggs. The red carotenoids in red-yolk duck eggs stemmed from P. crispus.1.3 Three red carotenoids, i.e. r1, r2 and r3 are isolated and purified from P. crispus by means of MgO-Diatomaceous Earth (l:2,w/w) column chromatography and silica-gel G TLC, the developing solvent for the silica G plate are: hexane-ethyl acetate-acetone-methanol=27:4:2:2 (v/v/v/v), the Rf of r1, r2 and r3 on the TLC are 0.32,0.34 and 0.38, respectively.2. Characterization on the structure of red carotenoids rl, r2 and r3 from P. crispusThe structure of red carotenoids rl, r2 and r3 are identified and characterized by UV-VIS, FT-ER, Raman spectroscopy, HPLC-ESI-MS, Low resolution EI-MS (LR-EI-MS) and High resolution EI-MS (HR-EI-MS) respectively.2.1 All of UV-VIS spectra for rl, r2 and r3 have a peak with somewhat of smooth and symmetrical profile in solvents, such as acetone, methanol, ethanol, chloroform and carbon disulphide, but a "three-peak" shaped asymmetrical profile in hexane. Comparing with the spectra in hexane, the λ max of r1, r2 and r3 increase with the strengthening of solvent polarity. The λ max trend of r1, r2 and r3 is from large to small (hypsochromic shift). The FT-IR spectra of r1, r2 and r3 show that they are the polyene compound with carbonyl group. The Raman spectra of rl, r2 and r3 show that they have the basic structure of carotenoids. The qualitative test show that r1, r2 and r3 contain carbonyl group (not 5, 6 or 5, 8-Epoxides). These are important evidence of r1, r2 and r3 as carotenoids containing carbonyl group, and they differ from astaxanthin, canthaxanthin and β -carotene based on these analyses as above. There are small difference in the structure of r1, r2 and r3 each other.2.2 The molecular weight of r1, r2 and r3 are 562 by HPLC-ESI-MS and LR-EI-MS, the precise molecular weight of r1, r2 and r3 are 562.38164,56238146 and 562.38002by HR-EI-MS, respectively. The analysis results show that r1, r2 and r3 contain carbon, hydrogen, and oxygen in the same proportion 40 C:50 H:2 O. They have the same molecular formula C40H50O2 and the molecular weight 562.2.3 Comparing red carotenoids from P. crispus with from the fruit of Taxus-media by TLC, UV-VIS and HPLC-ESI-MS, the results show that the rl, r2 and r3 are corresponding to Tr1,Tr2 and Tr3, respectively.We conclude that rl, r2 and r3 have the same molecular structure as Rhodoxanthin, and infer that they are isomers of Rhodoxanthin. Probably rl is all-trans-Rhodoxanthin, r2 and r3 are its cis isomers.3. Effects of carotenoids extracts of P. crispus (CEPC) on growth inhibition and apoptosis of human hepatoma cells line QGY-7703 in vitroTo study the effects of CEPC on growth inhibition and apoptosis of human hepatoma cells line QGY-7703 in vitro, the QGY-7703 cells are treated with culture medium containing 10, 20, 40 and 60 μ mol/L CEPC. We adopted a lot of analysis methods, i.e. MTT(for inhibition rates), visible light, fluorescence and laser scanning confocal microscope (LSCM) and flow cytometer (FCM) etc. To observe the change of human hepatoma cells in morphology and inspect the change of Ca2+ concentration in cytoplasm. The results show that the CEPC inhibit the growth of hepatoma cells QGY-7703 significantly in a dose and time-dependent manner in vitro. The apoptosis of hepatoma cells are induced effectively by low concentration of CEPC (10 or 20 μ mol/L) and in short time(48 h). The hepatoma cells treated with CEPC are evidently arrested at G0/G1 phase in the cell cycle. The increase of concentration of Ca2+ in the cytoplasm of test group cells is probable one of the important factors that CEPC cause the apoptosis of hepatoma cells QGY-7703.The results infer that the CEPC has the potentiality to anti-hepatoma.4. The exploitation of pellet P. crispus feed (PPC) and the dietary-supplementing experiments with PPC to the ducksThe pellet P. crispus feed (PPC) are manufactured successfully. The main technological process of PPC are drying, crushing, pelleting and packing. The effects are evaluated and compared for dietary supplementation with PPC to ducks. 4.1 The results indicated that there is a quantity-effect relationship between the supplementing levels of PPC, the Roche color fan (RCF) score and the percentage of over 15-grade of egg yolks. The mixed feed made up with 20% PPC and 80% common egg-duck feed is the best in comprehensive effect. The average color grade of egg-yolkincreases to 15-grade from 7-grade (CK) in the fourth day. It has significant difference compared with CK (P<0.01). The percentage of over 15-grade red-yolk eggs reaches 80% of total eggs in 13 and 14 days, and all eggs reaches over 14-grade. The average production egg rate in trial group is similar with CK. The average egg weight is significant increased compared with CK (P<0.05).4.2 The chemical and nutritional composition of P. crispus powder(PCP), PPC and the best mixed feed of red-yolk ducks are determined. The results show that the crude protein and total carotenoids contents of PCP are 13.36% and 463.1mg/kg respectively. They are greatly higher than maize, and its crude protein content approaches the wheat bran. The main nutritional composition of PPC are as follow: gross dry matter 90.05%, crude protein 12.96%, crude fat 4.36%, crude fiber 4.75%, nitrogen-free extract 54.16%, crude ash 13.82%, Ca 1.72% and total P 0.24%. The main composition of red-yolk ducks feed accords with the ducks feed standard issued by Chinese Commercial Ministry (SB/T 10262-1996) on the whole. The important cause of the good effects on increasing color grade of yolks, promoting growth of ducks, increasing egg weight, keeping a higher production egg rate by the P. crispus feed might be the many nutritional composition and good nutritional value, especially the higher contents of protein, essential amino acids, carotenoids and other vitamins. The PPC will have a good market prospects in the foreseeable future.This work are supported by grants from the Doctor Foundation of Chinese Education Ministry (No. 20020504008), Plans for Tackling Key Problems in Science and Technology of Hubei Province (No. 2003AA204B07). The results on the research of P. crispus carotenoids provided scientific basis for further studying and developing the P. crispus resources and functional food of red-yolk egg.Any literature of scientific and technological relative to the P. crispus research about red carotenoids structure, the anti-hepatoma function by its carotenoids extracts and the functional pellet feed that it is not-found until now.