Isolation And Identification Of Endophytic Fungi From Noni And Their Bioactivities Of Secondary Metabolites

Morinda citrifolia Linn.is a traditional medicinal plant of rubiaceae morinda.It originates from the tropical islands of the South Pacific,and it also has a small amount in Hainan,Xisha and Taiwan.The roots,skins,fruits and leaves of Noni are all used medicinally.Noni fruits are rich in various glycosides,terpenoids,iridoids,vitamins and other chemical ingredients or effective nutrients,having significant effects to defend cancer,hypertension,tuberculosis,hepatotoxicity,and anti-oxidation diseases.Although Noni has special anti-cancer and anti-oxidant effects,it is still in the process of research.Due to the limitations of its growth conditions and population distribution and mad people's pursuit,the germplasm resources of the species are not abundant.Debris felling and destruction make it on the verge of endangered extinction.Therefore,it is very urgent to find new sources of anti-oxidant and anti-tumor active substances.Because of a tropical coastal climate and soil conditions which are suitable for the growth of Noni,Hainan has a geographical advantage for the development and research of Noni.In this study,not full maturity healthy fruits and leaves of Noni were disinfected on the surface,then the endophytic fungi were isolated and extracted.The bacterial species were identified using the characteristics of colonies,26s rDNA D1/D2 region and ITS rDNA sequencing.Using the "Further Neighbor" principle of Mothur software to divide an operation taxonomic unit(OTU)to acquired sequences at a level of 0.03 difference,and MEGA 5.2.1 software was used to construct a cluster analysis tree map according to the N-J method.The antioxidant capacity of the secondary metabolites was analyzed by DPPH radical scavenging,ABTS radical scavenging and total reduction of FRAP.The secondary metabolites including total polyphenols(TPC)and total flavonoid(TFC)were determined by the Folin-Shoka method and AIC13 color method.,and antioxidant active ingredient content of them are analyzed at the same time.Using hepatoma cell line SMMC-7721,Lewis lung cancer,and prostate cancer cell line PC-3 as indicator cancer cells to test the antitumor activity of their secondary metabolites by MTT assay.The main results are as follows:(1)Three blank control experiments(environmental control,surface sterilization detection,and tissue blotting)were designed in this experiment.The results showed that the surface of noni fruits and leaves were completely disinfected and the obtained fungi were endophytic fungi.The surface disinfection reagents used in this test were 75%alcohol and 2.6%sodium hypochlorite and tested according to the time gradient.The experimental results showed that the 180 s+30 s,180 s+60 s treatment had the best bacterial effect after 8 days,having 7-10 colonies and 4-5 strain species;120 s+30 s,120 s+60 s,240 s+30 s,240 s+60 s,300 s+30 s,300 s+60 treatments showing good results were obtained in about 8 days with 4-5 colonies and 2-3 strain species;there was no colony on the treatments 80 s+180 s,240 s+150 s,240 s+180 s,300 s+90 s,300 s+120 s,300 s+150 s,and 300 s+180 s.(2)In this experiment,a total of 102 endophytic fungi were isolated and purified,and the colony morphological characteristics were initially identified as 63 species.Sequences were obtained by PCR amplification.Based on sequence similarity(with a threshold of 97%),the obtained OTUs were divided into 36 OTUs,and their phylogenetic trees were constructed for cluster analysis.The results showed that 63 endophytic fungi are classified into Basidiomycota and Ascomycota.Some of them belong to Didymosaphaeriaceae,Capnodiaceae,and Magnaporthaceae,and the other belong to Cldosporium,Aspergillus,Colletotrichum,Diaporthe,Hypoxylon,Fusarium,Phyllosticta,Gibberella,Aureobasidium,Rhodotorula,Cryptococcus.The dominant groups were Aspergillus and Cladosporium,which accounted for 28.57%and 15.87%of the total number of isolates,respectively,and the rest of the isolates were less than 10%.(3)The secondary metabolites of all noni endophytic fungi in this experiment had different degrees of antioxidant activity,and showed a clear dose-effect relationship within a certain range.The DPPH antioxidant activity of the extracts(Hh-1,Hs-2,Hd-3)ranged from 23.93 to 31.27 mmol TE/g DW,of which Hd-3 secondary metabolites had the strongest antioxidant activity;the order of ABTS antioxidant capacity based on the average TE value was:Hh-1(11.04 mmol TE/g DW)>Hd-3(9.64 mmol TE/g DW)>Hs-2(6.08 mmol TE/g DW);the highest ability of FRAP iron reduction antioxidant capacity is Hh-1 showing 8.22 mmol TE/g DW,while the lowest ability of Hs-2 was 5.68 mmol TE/g DW;the content of TPC is in the range of(52.29-72.81),and the highest value was found in Hh-1 extract(72.81);the TFC content was between(74.34-107.95)and the highest value was found in the Hd-3 extract(107.95).(4)Endophytic fungi Hh-1,Hs-2 and Hd-3 inhibited the proliferation of SMMC-7721(liver),Lewis(lung)and PC-3(prostate)tumor cells in different degrees showing significant dose-dependent effect and extremely significant differences(P<0.05).The IC 50 ranged from 45.46 to 108.99?g/mL,and the IC 50 of Hd-3 to SMMC-7721,Lewis and PC-3 tumor cells were 45.46 ?g/mL,53.33 ?g/mL and 80.48 ?g/mL,respectively showing spectral antitumor activity;IC50 of Hh-1 to SMMC-7721,Lewis and PC-3 tumor cells were 106.18 ?g/mL,46.08 ?g/mL and 79.71 ?g/mL,respectively,and the anti-tumor activity of some types of cells was better.Fungi Leptoxyphium(Hd-3)may produce chlorinated peroxidase and exhibit extensive catalytic activities on various organic substrates.In summary,noni,the unique traditional medicinal plants in Hainan,are rich in various endophytic fungi,and their secondary metabolites have strong anti-oxidant and anti-tumor activity.Endophytes and host plants have long-term symbiosis relationship,and the co-evolution may lead to exchange of genes and information between plants and their host,resulting in similar metabolic pathways and the same or similar secondary metabolites of biological activity,from which it is deduced that the biological activity of Noni may be related to its endogenous fungi,providing certain theoretical basis for development and utilization of Noni.