| Rhodococcus sp.B7740?Rh sp.B7740?were isolated from 25 meters deep seawater at the arctic B77 site?49.28‘49.28‘146?W and 76?N?by Chinese national arctic expedition team during the third arctic scientific research.The bacteria had been determined using sequence analysis of the 16S r RNA gene.Bacteria colony is bright orange red and the pigment produced by this new strain in its special living environment has never been reported before.In order to explore and understand the special nutrition and functionality of the red pigment produced by Rhodococcus marine,we conducted relevant preliminary studies?including high-speed counter current chromatography-HSCCC,multi-spectroscopic analysis and LS-MS/MS?and found that they belong to a mixture of carotenoids and isoprenoids?B7CIQE?,rich in special structure aromatic carotenoids such as isorenieratene,chlorobactene and synechoxanthin.These three carotenoids are very rare and novel in nature,which was the first report finding in Rhodococcus.In addition,the presence of high-yield?approximately 1 mg/g bacterial wet weight?,expensive,long-chain protonated methyl naphthoquinone MK8?H2?provides an extremely important material basis and meaning for this study of valuable isoprene compounds.On this basis,the potential biological activities of B7CIQE were studied.Through experiments,we obtained an unreported efficient HSCCC method for the separation and purification of B7CIQE;multi-spectroscopic analysis and biotech experiments were conducted to investigate the biological activity of isorenieratene.This research not only shows a new research field for exploring the nature pigment resources beneficial to human in the novel microorganisms of the arctic ocean,but also enriches the diversity of Marine microbial carotenoids.Meanwhile,this paper provides scientific basis and research basis for the development of B7CIQE and isorenieratene as natural dietary nutritional agents.The main results are as follows:1. The main components of B7CIQEB7CIQE was firstly extracted from Rhodococcus sp.B7740 with lysozyme and surfactant.Through HPLC-DAD-MS/MS analysis,the main components of B7CIQE were determined.The results showed that B7CIQE contained 14 different kinds of isoprenoid quinones and 9 kinds of carotenoids.Among that,B7CIQE contained 18%all-trans-synechoxanthin?24.18–31.65?g/g?,11%chlorobactene?14.78–19.34?g/g?,9%isorenieratene?12.09–15.82?g/g?,which never been reported before.It has provided a new research direction and field for the arctic Marine rhodococcus,enriched the diversity and novelty of carotenoids derived from polar Marine microorganisms,and obtained new resources of novel aromatic carotenoids.These results make Rh sp.B7740 a possible engineering bacterium with great potential to produce carotenoids.2. Anti-oxidation and anti-proliferation activity of B7CIQEThe antioxidant activity of B7CIQE and the proliferation activity of human liver cancer cells Hep G2 and human oral cancer cells KB were studied in vitro and at the cell level by means of?-carotene bleaching and so on.The inhibition rate of?-carotene oxidation by different antioxidants was in the decreasing order of B7CIQE?70.20%?>butylated hydroxytoluene?66.70%?>epigallocatechin gallate?17.80%?>lycopene?1.90%?.Peanut oil with the addition of B7CIQE had the lowest initial oxidation temperature?175??followed by?-carotene?165??,lutein?162??and lycopene?160??.The inhibition rate of protein oxidation was in the decreasing order of B7CIQE?25.75%?>?-carotene?24.97%?>lutein?17.94%?>lycopene?10.40%?.The median effective concentration?EC50?against Hep G2 cells wasin the decreasing order of lutein?20.86?g/m L?<?-carotene?124.88?g/m L?<B7CIQE?126.34?g/m L?<lycopene?139.24?g/m L?,whereas the EC50 against KB cells decreased in the following order:B7CIQE?25.14?g/m L?<lutein?64.29?g/m L?<lycopene?69.87?g/m L?<?-carotene?149.16?g/m L?.The experiment proved that B7CIQE had excellent antioxidant activity and anti-proliferation effect on liver cancer cells and oral cancer cells,providing a preliminary basis for further research on its bioactivity in human body.3. Isolation,purification and identification of aromatic carotenoids from B7CIQEExperiments show that HSCCC is an efficient method to isolate and purify B7CIQE,and also other kinds of caroteoids.When the solvent system was n-hexane-acetonitrile-dichloromethane and the volume ratio was fixed at 10:6:75:3.25,most kinds of carotene in B7CIQE had better separation and purification effect.The purity of isorenieratene and chlorobactene collected after purification could reach above90%?isorenieratene's purity was about 92.87%,chlrobactene's purity was about 96.39%?,which demonstrated the high efficiency of this method.At the same time,three aromatic carotenoids isolated and purified by HSCCC were further identified using ultra-precision mass spectrum.Results showed that the differences pf the test values and theoretical calculated values of three aromatic carotenoids?synechoxanthin,isorenieratene and chlorobactene?were all within 10ppm,which further confirmed the molecular structure of these three carotenoids.4. Study on the stability of isorenieratene??,?-carotene?in model gastric condition with iron and its interaction with HSAThe diaromatic ring and symmetrical structure of?,?-carotene was first found in European cheese.In this study,the pigment was found and isolated from Rhodococcus for the first time and studies on its structure and biological activity are very rare.This compound has higher stability than plant derived carotenoids.In order to understand the stability of isorenieratene with transition metal ions and low p H during human digestion,a simulated gastric environment was chosen to investigate its stability and retention rate.Studies using the stomach model showed that isorenieratene purified from B7CIQE could resist different forms of iron catalysis and oxygen attack in low p H environment,and had a significantly higher stability?the retention rate is above 95%?than beta-carotene and lutein in the presence of iron ions and oxygen.At the same time,the interaction between isorenieratene and human serum albumin?HSA?was explored by using multiple spectral methods and simulated docking.Results showed that isorenieratene,similar to dietary carotenoids beta-carotene and lutein from higher plants,could bind statically to HSA and inhibit its fluorescence spectrum intensity.The binding constants of isorenieratene to HSA?Ksv and Ka?were all in the appropriate range,slightly different from that of non-aromatic carotene,and the value was slightly higher.This indicates that isorenieratene is in a good state in the storage and distribution of blood circulation.The experimental results also showed that the binding process of Isorenieratene and HSA was spontaneous exothermicity,which was consistent with the beta-carotene-HSA and lutein-HSA systems.And the interaction between isorenieratene and HSA is mainly dominated by hydrophobicity and electrostatic attraction.Computer simulated docking experiments show that isorenieratene may attach to both site I and site II of HSA,but site II may be the main binding site,which is consistent with the results of fluorescence competition experiments.Synchronous fluorescence spectroscopy showed that the interaction between isorenieratene and HSA enhanced the polarity of Trp and Tyr micro-environment in HSA.The results of infrared spectrum showed that,compared with HSA,the alpha-helix structure in isorenieratene-has system showed a downward trend,and the beta-turn increased,indicating that part of the protein structure was unfolded.Surface enhanced Raman results showed that compared with free HSA system,isorenieratene-HSA interaction reduced the protein alpha-helix structure,and the Phe micro-environment in isorenieratene-HSA system was more hydrophobic.In addition,in the surface enhanced Raman spectrum,the carotenoid-characteristic peaks of isorenieratene-HSA system were stronger than those of non-aromatic carotenoids-HSA systems,indicating that isorenieratene had higher stability in the binding process.This is due to its high polyunsaturated symmetry,namely,the double aromatic ring and the symmetrical isoprene structure,which allows the electrons to be distributed evenly across the large?bond,improving its stability.5. Inhibitory effect of isorenieratene on ultraviolet-induced retinal injury and its mechanismThe potential effects of isorenieratene on UVB-induced damage were analyzed using the multi-layer phospholipid capsule model and the human retinal cell ARPE-19 model.Experimental data showed that isorenieratene had better antioxidant capacity and anti-uvb radiation effect than the two macular xanthophylls?MXs,lutein and zeaxanthin?.The results of EPR spectrum showed that isorenieratene could effectively reduce the formation of single oxygen radical and hydroxyl radical in the model lipid system.Compared with lutein?88.84%Q1-LL?and zeaxanthin?77.67%Q1-LL?,MTT and flow cytometry results proved that isorenieratene?90.94%Q1-LL?had better protective effect on ARPE-19?75.36%Q1-LL?in UVB irradiation model.The molecular mechanism by which isorenieratene protects ARPE19 and reduces uv damage was explained by ROS,RT-PCR and WB analysis,which mainly includes two aspects:one is its excellent intracellular antioxidant effect;the other is the up-regulation of tspo gene expression and increased TSPO protein content in cells by isorenieratene.In addition,molecular docking confirmed that isorenieratene could bind TSPO protein well.Therefore,we believe that isorenieratene,as an aromatic carotenoid,may have multiple active functions,including protecting the retina and inhibiting uv-induced injury. |
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