Extraction, purification and anti-TMV effects of α (β)-2,7,11-cembratriene-4,6-diol from tobacco leaves

Background Acetone ethanol extracts from tobacco leaves have antiviral activity against TMV, but the antiviral effects of their specialized metabolites have not been systematically studied yet, especially the underlying mechanism is still unexplored. Results The tobacco cembranoids α(β)-2,7,11-cembratriene-4,6-diol (α(β)-CBD) were extracted and purified with an effective and green protocol including innovatively added 5% phosphoric acid for elution, one time silica gel chromatographic column separation and impurity removal and further HPLC purification. The results of antiviral activities against tobacco mosaic virus (TMV) with the local lesion counting method showed that α(β)-CBD have in vivo higher protective effects of 73.2% and 71.6%, at 75.0 μM, respectively, than control agent Ningnanmycin (53.1%). Notably, The results of ELISA and and TMV-GFP fluorescent optical imaging assay indicated a obviously reduced viral protein and weaker GFP fluorescence signal and smaller infection area, which confirmed their anti-TMV activities at protein level. Furthermore, the enhanced production of SA and JA and the significantly increased transcription of of JA signaling pathway (COI1 and PDF1.2) and SA signaling pathway genes (PR1, NPR1 and EDS1) in treated plants further conformed that exogenously applied α(β)-CBD can effectively elicit the tobacco plant immunity against TMV. Conclusions The α(β)-CBD mainly stimulates disease resistance of tobacco plants to resist TMV and it can be used as bioagents to control TMV in the future.

Proteus vulgaris) [15,16]. Interestingly, in response to tobacco mosaic virus infection, a 68 substantial increase in cembranoids, in particular α/β-CBD, has been achieved with systemic 69 acquired resistance (SAR) leaves [17,18]. However, it is scarcely examined whether the 70 exogenous application of α(β)-CBD has inhibitory effects on tobacco virus infection or not. 71 In continuing efforts to identify natural product-derived antiviral agents against plant 72 virus, we herein report the simultaneous extraction and identification of α-CBD and β-CBD

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The UPLC conditions for detecting CBD after purification were: capillary column: 104 ACQUITY UPLC BEH C18 column (Waters, Milford, USA), 1.7 μm (2.1 × 50 mm); 105 injection volume was 1 μL; the mobile phase was a mixture of acetonitrile and water in a 106 ratio of (60:40) at a flow rate of 0.5 mL/min; column temperature was 40 °C, and the UV 107 wavelength was set at 210 nm.

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Purified α(β)-CBD was dissolved in DMSO and diluted with distilled H 2 O containing  Elkhart, IN, USA) following the user guide. was conducted to quantify in TMV infected 176 tobacco leaves using an assay following the manufacturer's instructions .

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The inhibition level of viral proliferation was recorded and calculated according to the  abundance around 50 min. However, there were still many other components ( Fig. 2A).

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When the ratio was changed to 3:1, many CBTs were found, and the other components were 212 decreased in quantity and abundance (Fig. 2B). The fractions eluted by a 3:1 ratio of 213 petroleum ether/ethyl acetate had the largest number of CBDs than the other ratios (5:1 and 214 7:1), while the slightest number of other components was obtained in quantity and abundance 215 (Fig. 2 C-D). 216 We performed semi-preparative HPLC using the UPLC condition to obtain CBD with a 217 60:40 ratio of acetonitrile: water. Two distinct peaks emerged clearly within 90 sec. After the 218 samples were eluted using the silicon column and applied to semi-preparative HPLC, two 219 targeted peaks were entirely separated from other components and even the CBDs themselves 220 within 35 min (Fig. 3-4). The purity of the two compounds done by semi-preparative HPLC 221 was >98%, confirmed by UPLC (data not shown). The yield of compound 1 and compound 2 222 were 0.005% and 0.007%, respectively. The structures of the compounds 1 and 2 were shown 223 in Fig. 1, and the 1 H and 13 C NMR data of 1 and 2 were listed in Figures S1-S4, respectively.

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The results of qRT-PCR indicated that any distinct differences in the relative expression  Table 2). These results suggest that CBDs 271 may mainly obstruct the virus protein biosynthesis processes.  (Fig. 9). Meanwhile, no significant differences were obtained 277 between α-CBD and β-CBD treatments. These results implied that CBDs could evidently 278 enhance the generation of JA and SA in the treatment leaves.

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The transcription of JA signaling pathway (COI1 and PDF1.2) and SA signaling 280 pathway genes (PR1, NPR1 and EDS1) has made for obvious differences in performance.

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The larger elevation and longer duration of all these marker genes expression in CBDs   can effectively elicit the tobacco plant immunity against TMV.

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In conclusion, α-CBD and β-CBD with high purity (98%) were separated and purified 331 from tobacco leaves using an efficient and green extraction and purification method.