Antifungal activity of Cynanchum atratum alkaloids against citrus postharvest blue mould

Abstract: 【Objective】Citrus fruits are often subjected to many postharvest diseases caused by various pathogens during picking, storage and transportation.Green and blue molds, caused by Penicillium digitatum and P. italicum, respectively, are two major postharvest citrus diseases and cause significant economic losses in the commercialization phase.Currently, control of postharvest citrus diseases relies mainly on the use of synthetic fungicides, which usually result in fungicide-resistance of the fungi, environment pollution and food safety problem.In recent years, much attention has been given to preventing citrus from the diseases by natural edible plant extracts.Scientists worldwide devote their time and energy to discover the high effect, low toxicity, safe and inexpensive plant-derived fungicides.Our researches also focus on plant-derived fungicides and chemical constituents target to inhibit P.digitatum and P.italicum in vitro and in vivo.Previously we showed the antifungal activities of Cynanchum atratum (CA) against 15 phytopathogenic fungi causing the postharvest diseases in fruits and vegetables.However, antifungal compoments were not clear.The extracts of n-butanol showed the best antifungal activity against P.italicum.In order to illuminate the antifungal constituents of the active fraction from C.atratum (CA) against phytopathogenic fungi P.italicum that cause citrus postharvest blue mould.【Methods】Antifungal bioassay-guided isolation and identification of constituents in the active fractionfollowed by liquid-liquid extraction and various column chromatographies.The preparative HPLC was used finally to get the pure compounds.The isolated compounds were identified by NMR and HR-ITTOF-MS.【Results】Ethyl acetate (CAE) and n-butanol (CAB) liquid-liquid extracts at the concentration of 25 mg· mL^-1 showed antifungal activity with the inhibition zone of 14.3 and 20.5 mm, respectively.Moreover, CAB showed a higher antifungal activity than CAE and CA at the same concentrations.The water extracts showed no antifungal activity.CAB was further isolated to get the antifungal components.D101 macroporous resin column was firstly used to further enrich the antifungal constituents, which yielded four fractions (CAB-A to CAB-D) , and only CAB-C fraction (the third fraction) showed antifungal activity at the concentration of 1.25 mg· mL^-1.CAB-B (the second fraction) and CAB-C fractions showed antifungal activity at the concentration of 2.5 mg· mL^-1.CAB-B, CAB-C and CAB-D (the fourth fraction) fractions showed antifungal activity at the concentration of 5.0 mg· mL^-1.So the CAB-C fraction was further isolated by silica gel chromatography and yielded four fractions (CAB-C1 to CABC4) .Only CAB-C2 fraction (the second fraction) showed antifungal activity at the concentration of 0.625 mg· mL^-1, while CAB-C3 fraction showed antifungal activity at the concentration of 1.25 mg· mL^-1.However, CAB-C1 and CAB-C4 fractions showed no antifungal activity even at 10 mg · mL^-1.The CABC2 was the active fraction with the antifungal inhibition zone 16.25 mm at the concentration of 0.625 mg · mL^-1.CAB-C2 fraction was further isolated by ODS and Sephadex LH-20 column chromatographies.Finally, five pure compounds were isolated using various column chromatographies and preparative HPLC.Their structures were elucidated by NMR and MS and identified as 10β- (-) -antofine N-oxide, 9-dehydroantofine, 9, 14-dehydroantofine, 14-hydroxyantofine N-oxide, and 10α- (-) -antofine N-oxide.【Conclusion】The five alkaloids were isolated for the first time from C. atratum. Alkaloids were the major antifungal constituents in C.atratum against P.italicum.