Research On Triterpenoids Secondary Metabolites Of Endophytic Fungi From Kadsura Angustifolia

Plant fungal endophyte is an abundant group, which spends the whole or part of its life cycle colonizing inter- and/or intra-cellularly inside the healthy tissue of the host plant, typically causing no apparent symptoms of disease. Endophytic fungi particularly from medicinal plants have proved to be a rich and important source, which can sometimes produce compounds with unique chemical structure and biological activity analogous to their hosts, have recently received more attention. Medicinal plants Kadsura angustifolia(Lem.) A.C.Smith(Schisandraceae) is a Chinese folk medicinal plant, which can promote blood circulation to treat fracture and irregular menstruation. Phytochemical studies have shown that lignans and triterpenoids are the two main kinds of natural products from Schisandraceae family. Recently, series of terpenoids from Schisandraceae which have novel structures or possess various beneficial pharmacological activities, including anti-inflammatory, anti-tumor, anti-HIV activities, aroused people’s new interest. However, little is known about secondary metabolites of endophytes from K. angustifolia.Therefore, in this study we screened and identified the aimed strains, which can produce Schisandraceae triterpenoids — nigranoic acid, from the endophytic fungi obtained from K. angustifolia in previous work. Then, we selected one strain which showed significant biocatalytic activities to ferment K. angustifolia in larger scale. Based on chromatography technology, triterpenoids were isolated and purified.Structures of the compounds were elucidated by detailed analysis of their spectroscopic data aided by the comparison with reported data of related derivatives. These works will analysis the metabolic difference to explore the relationship between the endophytic fungi and host plants. Meanwhile, the studies provided many novel metabolites for measuring their activities, and also provided theoretical basis of the utility of the endophytic fungi resources. The results are concluded as follows:By our previous work, we have obtained 293 strains of endophytic fungi belonging to 22 different genus, from the internal root bark of the plant K. angustifolia. The fungal isolates were fermented in liquid culture medium(PDB) and their metabolites were analyzed by thin layer chromatography(TLC) and high performance liquid Chromatography(HPLC). The obtained potential nigranoic acid-producing fungus, the strain SWUKD3.1601, was identified as Trichoderma harzianum based on morphological characteristics and the internal transcribed spacer(ITS1-5.8S-ITS4). It is worth mentioning that T. harzianum was reported as a fungicide in previous articles, which was used for suppression of various disease causing fungal pathogens. Later, nigranoic acid was successfully isolated from the metabolites and identified by detailed analysis of its spectroscopic data aided by the comparison with literatures. The current research provides a new method to produce nigranoic acid by fungal fermentation, which could overcome the natural resource limitation of isolating from the leaves and barks of the plant K. angustifolia.We selected one strain with obviously biocatalytic activities, and identified it as Penicillium ochrochloron SWUKD 4.1850. Solid state fermentation technology was used to ferment this strain on the K. angustifolia medium in larger scale. The acetone extractions were fractionated using repeated silica gel column chromatography, sephadex LH-20 column chromatography, preparative thin layer chromatography(PTLC) and recrystallization. Finally, twelve Schisandraceae triterpenoids were isolated from the metabolites extract. The structures of these compounds were elucidated by means of spectroscopic methods including UV, IR, ESI-MS, HR-ESI-MS, single crystal X-ray diffraction, 1D NMR(1H NMR, 13 C NMR, DEPT) and 2D NMR(1H-1H COSY, HSQC, HMBC and ROESY) as well as chemical method. Among them, there were seven new compounds, wuweizidilactone Q(1), henrischinins D-I(2-7) and five known compounds henrischinin A(8), henrischinin B(9), schirubridilactone D(10), wuweizidilactone D(11) lancifodilactone D(12). They were classified into four groups: 18-norschiartane-type,(21,24-Cyclo)-3,4;9,10-seco-cycloartane-type, schisanartane-type and 18(13,14)-abeo-schiartane-type according to their structural characteristics. In previous article, henrischinins A-B exhibited anti HSV-2 activity with EC50 values of 0.24 mg/mL and inhibitory activity against HL-60 with an IC50 value of 16.5, 10.5 μM, resperctively. Schirubridilactone D showed anti HIV-1 activity(EC50=80.8 mg/mL). Analyzing to the differences of secondary metabolites between the host plant K. angustifolia and co-cultures of the strain, we found the phenomenon that the triterpenoids from co-cultures always characterized by highly-oxygenated, and presumed that endophytic fungi may biotransform the original triterpenoids to new ones which increased the degree of oxidation. These results indicated that the biosynthesis of terpenoids in plant Schisandraceae may be associated with the endophytic fungi.Summarily, this work obtained one target fungal strain and some novel secondary metabolites. These results imply that plant fungal endophyte, as an important part of plant system, which can produce novel and bioactive secondary metabolisms, could be potential resources for natural medicine.