Application of crocetin diester in the prevention of tobacco mosaic virus

Abstract

The invention belongs to the technical field of pesticides. The invention provides application of crocetin diester in resisting tobacco mosaic virus. The crocetin diester is any one of crocetin dimethyl ester and crocetin diethyl ester, and is used for resisting tobacco mosaic virus or for preparing pesticides resisting tobacco mosaic virus.

Description

[0001] This application is a divisional application of the invention patent with application date of December 8, 2020, application number 2020114417612, and invention name "Crocetin diester compounds, preparation methods and applications thereof". Technical Field

[0002] The invention relates to application of crocetin diester in resisting tobacco mosaic virus, and belongs to the technical field of pesticides. Background Art

[0003] With the continuous increase of global population and the change of people's diet, the pressure of food production is also increasing. Diseases, insects and weeds pose a great threat to food production and need to be controlled continuously and effectively. At the same time, the environmental, toxicological and management requirements for the application of agrochemical products are becoming increasingly stringent, and the requirements for pest control products are also increasing. The continuous development of resistance of pests, diseases and weeds to control agents further limits the application of drugs. Therefore, it is necessary to continuously discover and develop new, effective and economical pest control agents to increase crop yields and feed the growing number of humans.

[0004] Natural products are primary or secondary metabolites produced by living cells. Historically, natural products have been an important component of medicine and a major tool for controlling weeds, pathogens and pests in agriculture. Most natural products are unstable in the environment or have toxicity, insecticidal spectrum, photostability or production issues, which need to be addressed through structural transformation and modification before commercialization, especially under today's strict regulatory environment. Synthetic natural product derivatives also provide tools for studying chemical biology, which can determine the relationship between structure and biological activity and provide a deeper understanding of the mode of action of natural products and biological targets.

[0005] In order to study the structure-activity relationship of this type of compound, the present invention takes crocin as a guide and designs and synthesizes crocetin diester compounds to study their role in plant protection. Summary of the invention

[0006] The crocetin diester of the present invention has good anti-plant virus activity. The present invention provides the use of the crocetin diester in resisting tobacco mosaic virus. The crocetin diester is any one of dimethyl crocetin and diethyl crocetin, which is used for resisting tobacco mosaic virus or for preparing pesticides resisting tobacco mosaic virus.

[0007] The crocetin diester of the present invention is a compound having a structure shown in the following general formula (I):

[0008]

[0009] In the formula, R1 and R2 represent various sugar groups and substituted alkyl groups.

[0010] Another object of the present invention is to provide a method for preparing crocetin diester. The compound of general formula (I) can be prepared by method 2:

[0011]

[0012] Crocin-1 is dissolved in water, and an aqueous solution of sodium hydroxide is added, and the mixture is heated to 100° C. for three hours under stirring; after cooling, hydrochloric acid is added to adjust the pH of the reaction solution to 2. The mixture is filtered and the filter cake is washed with water to obtain crocetin.

[0013] Dissolve crocetin in DMF, then add DBU and bromohydrocarbon, stir at room temperature, add water and dichloromethane, wash the aqueous phase twice with dichloromethane, combine the organic phases, wash three times with water, wash once with saturated brine, dry, filter, desolventize, and perform silica gel column chromatography to obtain the target product, crocetin diester compound.

[0014] The compound of the general formula (I) of the present invention has excellent anti-plant virus activity, can well inhibit tobacco mosaic virus, pepper virus, rice virus, tomato virus, sweet potato virus, potato virus, melon virus and corn dwarf mosaic virus, etc., can effectively prevent and control viral diseases of various crops such as tobacco, pepper, rice, tomato, melons and vegetables, grains, vegetables, beans, etc., and is particularly suitable for preventing and controlling tobacco mosaic disease.

[0015] The compound of the general formula (I) of the present invention can be used directly as a plant virus inhibitor, or can be used with an agriculturally acceptable carrier, or can be used in combination with other anti-plant virus agents such as benzothiadiazole (BTH), thiazolamide (TDL), 4-methyl-1,2,3-thiadiazole-5-carboxylic acid (TDLA), DL-β-aminobutyric acid (BABA), ribavirin, ningnanmycin, phenanthroline indolizidine alkaloid anthophen, salicylic acid, polyhydroxybinaphthaldehyde, and amino oligosaccharides to form an interactive composition for use. Some of these compositions show synergistic effects, while others show additive effects.

[0016] The invention provides application of crocetin diester in resisting tobacco mosaic virus. The crocetin diester is any one of dimethyl crocetin and diethyl crocetin, and is used for resisting tobacco mosaic virus.

[0017] The invention provides application of crocetin diester in resisting tobacco mosaic virus. The crocetin diester is any one of dimethyl crocetin and diethyl crocetin, and is used for preparing pesticide resisting tobacco mosaic virus. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below by way of examples, but the present invention is not limited to these examples.

[0019] Example 1

[0020] Preparation and structural characterization of crocin-1 and crocin-2:

[0021] 150g of Gardenia fruit powder was soaked in 1200ml of methanol for 72h with constant stirring. The filtrate was filtered and dried to obtain 33.7g of Gardenia crude extract. The crude extract was separated by macroporous adsorption resin, with water and ethanol as eluents, and a total of five components (1-5) were obtained. Component 4 was separated by silica gel column chromatography to obtain six components (4.1-4.6). Component 4.6 was recrystallized to obtain crocin-1. Component 4.5 was further separated by preparative chromatography to obtain crocin-2.

[0022] Compound 1: Crocin-1: mp: 229–231°C; 1 H NMR(400MHz,DMSO)δ7.36(d,J=10.8Hz,2H),6.90–6.79(m,4H),6.72–6.63(m,2H),6.54(d,J=10.2Hz,2H ),5.42(d,J=7.6Hz,2H),5.33(d,J=4.2Hz,2H),5.20(s,2H),5.10(d,J=3.1Hz,2H),4.98–4.83(m,6H),4. 46(t,J=5.7Hz,2H),4.17(d,J=7.8Hz,2H),3.99(d,J=10.4Hz,2H),3.70–3.54(m,4H),3.46–3.39(m,4H), 3.28–3.19(m,6H),3.14–3.09(m,2H),3.05(s,4H),2.95(td,J=8.3,4.4Hz,2H),1.99(d,J=10.0Hz,12H); 13 C NMR (100MHz, DMSO) δ166.16,144.59,139.88,136.91,135.95,131.98,125.27,123.90,103.07,94.52,7 6.86,76.75,76.27,76.25,73.44,72.44,69.96,69.22,67.92,60.97,12.67,12.55; HRMS(MALDI)calcd for C 44 H 64 NaO 24 [M+Na] + 999.3680, found 999.3677.

[0023] Compound 2: Crocin-2: mp: 201–203°C; 1H NMR (400MHz, DMSO) δ7.36(d,J=11.2Hz,1H),6.90–6.84(m,2H),6.81(d,J=2.9Hz,1H),6.72–6.63(m,2H),6.54(d,J=8.3Hz,1H),5.42(d,J=7.7Hz,2H) ,5.31(dd,J=11.3,4.8Hz,2H),5.19(d,J=3.4Hz,1H),5.14–5.08(m,2H),5. 03(d,J=5.1Hz,1H),4.94(d,J=4.5Hz,1H),4.90(d,J=1.8Hz,1H),4.86(d,J =4.7Hz,1H),4.59(t,J=5.9Hz,1H),4.46(t,J=5.9Hz,1H),4.18(d,J=7.8Hz ,1H),3.99(d,J=10.4Hz,1H),3.79(d,J=11.3Hz,1H),3.68–3.62(m,3H),3. 59(dd,J=11.2,5.2Hz,1H),3.47–3.41(m,3H),3.27–3.21(m,6H),3.19–3.1 0(m,3H),3.06(s,2H),2.96(td,J=8.3,4.8Hz,1H),1.99(d,J=9.5Hz,12H); 13 C NMR (100MHz, DMSO) δ166.15,144.56,144.49,139.84,139.74,136.87,135.91,131.95,125.34,125.27,123.87,103.06,94.57,94.52,77 .82,76.84,76.74,76.44,76.26,73.44,72.51,72.44,72.42,69.96,69.50,69.23,67.92,60.96,60.54,12.64,12.53; HRMS(MALDI)calcd for C 38 H 54 NaO 19 [M+Na] + 837.3152, found 837.3149.

[0024] Example 2

[0025] Synthesis and structural characterization of crocetin:

[0026] Crocin-1 (2.00 g, 2.05 mmol) was dissolved in 100 mL of water, and an aqueous solution of sodium hydroxide (2.00 g, 50 mmol) was added. The mixture was heated to 100 °C and reacted for three hours under stirring. After cooling, 2 M hydrochloric acid was added to adjust the pH of the reaction solution to 2. The filter cake was filtered and washed with water to obtain crude crocetin (0.60 g, 1.83 mmol). mp>320 °C; 1 H NMR (400MHz, DMSO) δ7.20 (d, J = 10.5 Hz, 1H), 6.83 (d, J = 6.6 Hz, 1H), 6.72 (d, J = 14. 7Hz,1H),6.63(d,J=11.5Hz,1H),6.49(d,J=5.5Hz,1H),1.97(s,3H),1.92(s,3H); 13 C NMR (100MHz, DMSO) δ169.65,143.64,138.31,137.08,135.68,132.04,127.68,124.58,13.28,13.00.

[0027] Example 3

[0028] Synthesis and structural characterization of crocetin diester:

[0029] The crude crocetin (0.20 g, 0.61 mmol) was dissolved in DMF (50 mL), and then DBU (0.37 g, 2.44 mmol) and bromohydrocarbon (2.44 mmol) were added. After stirring at room temperature for 24–96 h, water and dichloromethane were added. The aqueous phase was washed twice with dichloromethane, and the organic phases were combined, washed three times with water, washed once with saturated brine, dried over anhydrous magnesium sulfate, filtered, desolventized, and purified by silica gel column chromatography to obtain the target product (3–19).

[0030] Compound 3:

[0031] Yield: 65.3%; mp 214–216℃; 1 H NMR (400MHz, CDCl3) δ7.29 (d, J = 10.6Hz, 2H),

[0032] 6.76–6.66(m,2H),6.66–6.50(m,4H),6.42–6.31(m,2H),3.77(s,6H),2.00(d,J=4.2Hz,12H); 1313C NMR (100 MHz, CDCl3) δ 168.90, 143.73, 138.86, 136.70, 135.32, 131.31, 126.44, 123.80, 51.79, 12.88, 12.77; HRMS (MALDI) calcd for C 22 H 28 O4 [M]+ 356.1988, found 356.1980.

[0033] Compound 4:

[0034] Yield: 58.5%; mp 205–207 °C; 1 1H NMR (400 MHz, CDCl3) δ 7.29 (d, J = 10.9 Hz, 2H),

[0035] 6.73–6.51 (m, 6H), 6.37 (d, J = 7.6 Hz, 2H), 4.22 (dd, J = 14.0, 7.0 Hz, 4H), 2.00 (s, 12H), 1.32 (t, J = 7.0 Hz, 6H); 13 13C NMR (100 MHz, CDCl3) δ 168.60, 143.71, 138.70, 136.85, 135.37, 131.41, 126.93, 124.00, 60.68, 14.49, 13.01, 12.93; HRMS (MALDI) calcd for C 24 H 32 O4 [M] + 384.2301, found 384.2293.

[0036] Compound 5:

[0037] Yield: 42.4%; mp 152–153 °C; 1 1H NMR (400 MHz, CDCl3) δ 7.28 (d, J = 11.7 Hz, 2H), 6.70

[0038] (dd, J = 7.8, 2.2 Hz, 2H), 6.65–6.50 (m, 4H), 6.37 (d, J = 7.6 Hz, 2H), 4.17 (t, J = 6.5 Hz, 4H), 2.00 (s, 12H), 1.67 (dd, J = 14.5, 6.9 Hz, 4H), 1.43 (dd, J = 14.7, 7.5 Hz, 4H), 0.96 (t, J = 7.3 Hz, 6H); 13C NMR (100MHz, CDCl3) δ168.52,143.56,138.53,136.69,135.21,131.26,126.86,123.86,64.47,30.84,19.28,13.76,12.87,12.78; HRMS(MALDI)calcd forC 28 H 40 O4[M] + 440.2927, found 440.2919.

[0039] Compound 6:

[0040] Yield:29.9%; mp 190–192℃; 1 H NMR (400MHz, CDCl3) δ7.35 (d, J = 11.1Hz, 2H),

[0041] 6.75–6.51(m,6H),6.38(d,J=8.1Hz,2H),4.52(dd,J=12.5,2.4Hz,2H),4.01(dd,J=12.3,6.3H z,2H),3.31–3.26(m,2H),2.88(t,J=4.4Hz,2H),2.71–2.67(m,2H),2.02(s,6H),2.00(s,6H); 13 C NMR(100MHz, CDCl3)δ168.14,144.29,139.68,136.87,135.71,131.56,125.99,123.83,65.25,49.73,44.82,12.98,12.90; HRMS(MALDI)calcdfor C 26 H 32 O6[M] + 440.2199,found440.2191.

[0042] Compound 7:

[0043] Yield:45.3%; mp 231–232℃; 1 H NMR (400MHz, CDCl3) δ7.37 (d, J = 11.2Hz, 2H),

[0044] 6.77–6.66(m,4H),6.58(d,J=11.6Hz,2H),6.42(d,J=6.6Hz,2H),4.82(s,4H),2.02(s,6H),2.01(s,6H); 13 C NMR(100MHz,CDCl3)δ166.70,145.59,141.62,137.04,136.50,131.96,124.21,123.58,114.92,48.71,12.94,12.88;HRMS(MALDI)calcd forC 24 H 26 N2O4[M] + 406.1893,found 406.1885。

[0045] Compound 8:

[0046] Yield:49.8%;mp 158–160℃; 1 H NMR(400MHz,CDCl3)δ7.24(d,J=4.4Hz,2H),6.68(d,

[0047] J=8.0Hz,2H),6.63–6.48(m,6H),6.36(d,J=8.1Hz,2H),3.84(s,4H),1.98(s,6H),1.98(s,6H),0.09(s,18H); 13 C NMR(100MHz,CDCl3)δ169.40,143.65,138.53,136.82,135.32,131.39,127.08,123.97,57.91,13.14,12.93,-2.81;HRMS(MALDI)calcd forC 28 H 44 O4Si2[M] + 500.2778,found 500.2770。

[0048] Compound 9:

[0049] Yield:42.5%;mp 162–163℃; 1 H NMR(400MHz,CDCl3)δ7.39(d,J=10.8Hz,2H),

[0050] 6.75–6.52(m,6H),6.38(d,J=6.6Hz,2H),4.72(s,4H),3.78(s,6H),2.04(s,6H),2.00(s,6H); 13 C NMR(100MHz,CDCl3)δ168.62,167.62,144.46,140.20,136.79,135.70,131.50,125.38,123.70,60.84,52.16,12.82,12.77;HRMS(MALDI)calcd for C 26 H 32 O8[M] + 472.2097,found472.2089。

[0051] Compound 10:

[0052] Yield:24.04%; mp 175–177 °C; 1 H NMR(400MHz,CDCl3)δ7.37(d,J=10.4Hz,2H),

[0053] 6.79–6.50(m,6H),6.40(s,2H),5.85(s,4H),2.12(s,6H),2.01(s,12H); 13 C NMR(100MHz,CDCl3)δ169.79,166.87,144.82,140.65,136.83,135.94,131.61,125.18,123.65,79.51,20.81,12.77,12.70;HRMS(MALDI)calcd for C 26 H 32 O8[M] + 472.2097,found472.2089。

[0054] Compound 11:

[0055] Yield:45.4%; mp 189–191 °C; 1 H NMR(400MHz,CDCl3)δ7.33(d,J=10.7Hz,2H),

[0056] 6.74–6.50(m,6H),6.37(d,J=7.7Hz,2H),5.99(ddt,J=16.2,10.7,5.5Hz,2H),5.35(d,J=17.2Hz,2H),5.25(d,J=10.4Hz,2H),4.68(d,J=5.2Hz,4H),2.02(s,6H),1.99(s,6H); 13 C NMR(100MHz,CDCl3)δ168.18,143.98,139.16,136.87,135.52,132.79,131.49,126.59,123.95,117.92,65.37,13.02,12.93;HRMS(MALDI)calcd for C 26 H 32 O4[M] + 408.2301,found 408.2293。

[0057] Compound 12:

[0058] Yield:62.3%;mp 157–159℃; 1 H NMR(400MHz,CDCl3)δ7.35(d,J=10.7Hz,2H),

[0059] 6.75–6.51(m,6H),6.38(d,J=7.1Hz,2H),4.78(s,4H),2.48(s,2H),2.02(s,6H),2.00(s,6H); 13 C NMR(100MHz,CDCl3)δ167.54,144.35,139.88,136.80,135.67,131.50,125.67,123.72,78.19,74.58,52.11,12.84,12.80;HRMS(MALDI)calcd for C 26 H 28 O4[M] + 404.1988,found404.1980。

[0060] Compound 13:

[0061] Yield:41.9%;mp 183–185℃; 1 H NMR(400MHz,CDCl3)δ7.37–7.27(m,4H),6.98(d,J=

[0062] 7.4 Hz, 2H), 6.94 (s, 2H), 6.87 (d, J = 8.2 Hz, 2H), 6.70 (d, J = 10.4 Hz, 2H), 6.59 (dd, J = 21.2, 12.8 Hz, 4H), 6.36 (d, J = 7.7 Hz, 2H), 5.19 (s, 4H), 3.82 (s, 6H), 2.03 (s, 6H), 1.99 (s, 6H); 13 C NMR (100 MHz, CDCl3) δ 168.32, 159.87, 144.08, 139.34, 138.15, 136.87, 135.57, 131.50, 129.72, 126.49, 123.92, 120.34, 113.69, 113.60, 66.32, 55.39, 13.08, 12.92; HRMS (MALDI) calcd for C 36 H 40 O6 [M] + 568.2825, found 568.2816.

[0063] Compound 14:

[0064] Yield: 41.5%; mp 167–169 °C; 1 H NMR (400 MHz, CDCl3) δ 7.24–7.17 (m, 6H), 7.00 (t, J =

[0065] 8.6 Hz, 4H), 6.71 (dd, J = 7.9, 2.4 Hz, 2H), 6.63–6.49 (m, 4H), 6.38 (d, J = 7.8 Hz, 2H), 4.34 (t, J = 6.8 Hz, 4H), 2.97 (t, J = 6.8 Hz, 4H), 1.99 (s, 6H), 1.97 (s, 6H); 13 C NMR (100 MHz, CDCl3) δ 168.28, 162.93, 160.51, 143.85, 138.97, 136.73, 135.43, 133.82, 133.79, 131.38, 130.42, 130.34, 126.46, 123.79, 115.38, 115.17, 65.03, 34.50, 12.85, 12.79; HRMS (MALDI) calcd for C 36 H 38 F2O4 [M] + 572.2738, found 572.2729.

[0066] Compound 15:

[0067] Yield: 30.3%; mp 164–165 °C; 1 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 7.8 Hz, 4H), 7.37 (d,

[0068] J = 7.8 Hz, 4H), 7.24 (d, J = 10.5 Hz, 2H), 6.72 (dd, J = 7.9, 2.3 Hz, 2H), 6.63–6.48 (m, 4H), 6.38 (d, J = 7.4 Hz, 2H), 4.40 (t, J = 6.7 Hz, 4H), 3.06 (t, J = 6.6 Hz, 4H), 1.99 (s, 6H), 1.97 (s, 6H); 13 13C NMR (100 MHz, CDCl3) δ 168.21, 143.96, 142.35, 139.12, 136.72, 135.49, 131.41, 129.29, 126.28, 125.45, 125.42, 125.38, 125.34, 123.73, 64.46, 35.10, 29.70, 12.83, 12.78; HRMS (MALDI) calcd for C 38 H 38 F6O4 [M] + 672.2674, found 672.2665.

[0069] Compound 16:

[0070] Yield: 47.9%; mp 186–188 °C; 1 1H NMR (400 MHz, CDCl3) δ 8.60 (d, J = 4.3 Hz, 2H), 7.71

[0071] (td, J = 7.7, 1.3 Hz, 2H), 7.42–7.38 (m, 4H), 7.26–7.21 (m, 2H), 6.71 (dd, J = 7.9, 2.7 Hz, 2H), 6.59 (dt, J = 15.0, 12.9 Hz, 4H), 6.38 (d, J = 8.7 Hz, 2H), 5.34 (s, 4H), 2.06 (s, 6H), 2.00 (s, 6H); 13CNMR(100MHz,CDCl3)δ167.98,156.42,149.40,144.15,139.55,136.76,136.74,135.55,131.42,126.08,123.76,122.72,121.64,66.94,12.95,12.79; HRMS(MALDI) calcd for C 32 H 34 N2O4[M + H] + 511.2591, found 511.2588。

[0072] Compound 17:

[0073] Yield: 47.3%; mp 183–184 °C; 1 H NMR(400MHz,CDCl3)δ7.27(d,J = 9.1Hz,2H),7.08(s,

[0074] 2H),6.98(d,J = 7.8Hz,2H),6.76–6.67(m,4H),6.64–6.50(m,4H),6.42–6.32(m,2H),4.55(t,J = 8.6Hz,4H),4.31(t,J = 6.9Hz,4H),3.19(t,J = 8.6Hz,4H),2.92(t,J = 6.9Hz,4H),1.99(s,12H); 13 C NMR(100MHz,CDCl3)δ168.35,158.82,143.70,138.82,136.72,135.32,131.33,129.95,128.51,127.18,126.66,125.46,123.85,109.08,71.19,65.59,34.73,29.78,12.87,12.79; HRMS(MALDI) calcd for C 40 H 44 O6[M] + 620.3138, found 620.3128。

[0075] Compound 18:

[0076] Yield: 46.2%; mp 143–145 °C; 1 H NMR(400MHz,CDCl3)δ7.32–7.26(m,6H),6.95(t,J =

[0077] 7.6 Hz, 2H), 6.91 (d, J = 8.1 Hz, 4H), 6.70 (dd, J = 8.0, 2.5 Hz, 2H), 6.64–6.50 (m, 4H), 6.37 (d, J = 8.4 Hz, 2H), 4.37 (t, J = 6.2 Hz, 4H), 4.09 (t, J = 6.1 Hz, 4H), 2.19 (p, J = 6.2 Hz, 4H), 2.02–1.96 (m, 12H); 13 C NMR (100 MHz, CDCl3) δ 168.48, 158.94, 143.95, 139.02, 136.86, 135.50, 131.47, 129.60, 126.63, 123.92, 120.92, 114.65, 64.58, 61.64, 28.97, 13.03, 12.93; HRMS (MALDI) calcd for C 38 H 44 O6 [M] + 596.3138, found 596.3129。

[0078] Compound 19:

[0079] Yield: 48.6%; mp 205–206 °C; 1 H NMR (400 MHz, CDCl3) δ 7.84 (dd, J = 4.9, 3.2 Hz, 4H),

[0080] 7.72–7.68 (m, 4H), 7.24 (d, J = 10.2 Hz, 2H), 6.72 (d, J = 10.0 Hz, 2H), 6.58–6.46 (m, 4H), 6.37 (d, J = 8.5 Hz, 2H), 4.23 (t, J = 5.9 Hz, 4H), 3.85 (t, J = 6.7 Hz, 4H), 2.16–2.08 (m, 4H), 1.99 (s, 6H), 1.97 (s, 6H); 13 C NMR (100 MHz, CDCl3) δ 168.24, 168.19, 143.77, 138.91, 136.70, 135.30, 133.93, 132.15, 131.31, 126.39, 123.77, 123.25, 62.10, 35.34, 27.77, 12.78; HRMS (MALDI) calcd for C 42 H 42 N2O8 [M] + 702.2941, found 702.2931。

[0081] Example 4

[0082] Taking the anti-tobacco mosaic virus (TMV) activity as an example,

[0083] 1. Virus purification and concentration determination:

[0084] Virus purification and concentration determination were carried out in accordance with the SOP specification for tobacco mosaic virus compiled by the Laboratory of the Institute of Element Analysis of Nankai University. The crude virus extract was centrifuged twice with polyethylene glycol, the concentration was determined, and the extract was refrigerated at 4°C for later use.

[0085] 2. Preparation of compound solution:

[0086] After weighing, the original drug was dissolved in DMF to obtain 1×10 5 μg / mL stock solution, and then diluted to the required concentration with an aqueous solution containing 1‰ Tween 80; the virazole preparation is directly diluted with water.

[0087] 3. In vivo protection:

[0088] Select 3-5 leaf-stage Sansi tobacco plants with uniform growth, spray the whole plant with 3 replicates per treatment, and set a 1‰ Tween 80 aqueous solution as a control. After 24 hours, sprinkle emery (500 mesh) on the leaves, dip a brush in the virus solution, and gently rub the entire leaf surface along the branch veins twice, support the lower part of the leaf with the palm of your hand, and rinse with running water after inoculation. Record the number of lesions after 3 days and calculate the prevention effect.

[0089] 4. In vivo therapeutic effect:

[0090] Select Sansi tobacco plants with uniform growth at the 3-5 leaf stage, inoculate the whole leaf with the virus using a brush, with a virus concentration of 10μg / mL, and rinse with running water after inoculation. After the leaf surface is dry, spray the whole plant with the pesticide, with 3 replicates per treatment, and set a 1‰ Tween 80 aqueous solution as a control. Record the number of lesions after 3 days and calculate the prevention effect.

[0091] 5. In vivo passivation effect:

[0092] Select Sansi tobacco plants with uniform growth at the 3-5 leaf stage, mix the agent with an equal volume of virus juice and inactivate for 30 minutes, then inoculate by friction, with a virus concentration of 20 μg / mL. Rinse with running water immediately after inoculation, repeat 3 times, and set a 1‰ Tween 80 aqueous solution as a control. Count the number of lesions after 3 days and calculate the results.

[0093] Inhibition rate (%) = [(number of control necrosis spots - number of treated necrosis spots) / number of control necrosis spots] × 100%

[0094] Table 1 Results of the anti-tobacco mosaic virus in vivo test of crocetin diester (0.5 mg / mL) a

[0095]

[0096] When the in vivo inactivation activity of the tested compound is higher than 40%, the in vivo protection and in vivo therapeutic effects of the compound are tested.

[0097] It can be seen from Table 1 that all compounds showed activity against tobacco mosaic virus at a concentration of 0.5 mg / mL. The natural products crocin-1 and crocin-2 showed activity levels significantly higher than the commercial variety ribavirin. Most of the synthetic crocetin diester derivatives showed good in vivo anti-TMV activity, and compounds 5, 9, 13, 14 and 15 showed higher activity levels than ribavirin, especially compound 14. Compounds 3-5 containing linear alkyl chains had moderate anti-TMV activity, but their passivation effects were all less than 40%. The presence of different functional groups on the alkyl chain (compounds 6-12) had a greater effect on the anti-TMV activity. Compound 9 with a methoxy functional group showed an activity level higher than ribavirin. The anti-TMV activity can be improved when the substituted benzene ring is introduced into the alkyl chain through the CC bond (inhibition rate: compound 13>compound 3; compound 14>compound 15>compound 4). Crocetin di(4-fluorophenylethyl) ester (compound 14) showed the highest anti-TMV activity among the synthesized crocetin diesters, and was higher than the lead compounds crocin-1 and crocin-2. However, the introduction of heteroaromatic rings did not improve the anti-TMV activity.

Claims

1. The application of crocetin diester in resisting tobacco mosaic virus, characterized in that: Crocetin diester is any one of dimethyl crocetin and diethyl crocetin, and is used to resist tobacco mosaic virus.

2. The application of crocetin diester in resisting tobacco mosaic virus, characterized in that: The crocetin diester is any one of crocetin dimethyl ester and crocetin diethyl ester, and is used for preparing pesticides against tobacco mosaic virus.

3. The use of crocetin diester in resisting tobacco mosaic virus, characterized in that: The crocetin diester is any one of crocetin dimethyl ester and crocetin diethyl ester, and the crocetin diester and any one of benzothiadiazole, thiazolamide, 4-methyl-1,2,3-thiadiazole-5-carboxylic acid, DL-β-aminobutyric acid, virazole, ningnanmycin, phenanthroindolizidine alkaloid anthophen, salicylic acid, polyhydroxybinaphthaldehyde, and amino oligosaccharide form an interactive composition for use.

Images