CN1560071A - Preparation method and application of steroid sapogenin compound - Google Patents

Abstract

The invention provides two steroidal sapogenin compounds, which belong to polyhydroxy-substituted spirostane steroid derivatives. The steroid sapogenin has obvious in vitro and living antibacterial activity on Peronophythora litchi (Peronophythora litchii Chen), and can be used as bactericide for preventing and treating Peronophythora litchi.

Description

Preparation method and application of a steroidal saponin compound

                      

The present invention relates to two steroidal saponin compounds and applications thereof. Specifically, it relates to the preparation of two steroidal saponins and their application as fungicides in the control of Peronophythoralitchii Chen.

Background technique

Litchi downy mildew is a disease caused by a lower fungus (Peronophythora litchii Chen), which can cause dry shoots, flower drop, fruit drop, and rotten fruit of litchi. It is the main disease that seriously affects the yield and quality of litchi. At present, the control of litchi downy mildew in production is still based on the chemical synthetic agent - Ridomil (Ridomil).

Contents of invention

The object of the present invention is to provide two steroidal sapogenin compounds capable of inhibiting the botanical activity of P. litchie.

Another object of the present invention is to provide a process for the preparation of this compound.

Another object of the present invention is to provide the application of this active compound in the control of litchi downy mildew.

The two compounds provided by the invention are both polyhydroxyl substituted spirostanoid steroid derivatives. The first compound (compound I for short), its name is 1β, 2β, 3β, 4β, 5β, 7α-hexahydroxyspirosta-25(27)-en-6-one (1β, 2β, 3β, 4β, 5β , 7α-hexahydroxyspirost--25(27)-en-6-one), its structure is as formula I; the second compound (referred to as compound II), its name is spiro-25(27)-en-1β, 2β , 3β, 4β, 5β, 6β, 7α-heptol (spirost-25(27)-ene-1β, 2β, 3β, 4β, 5β, 6β, 7α-heptol), the structure of which is shown in formula II. See Example 6 for the spectral data of the two compounds.

式I

Formula I

Formula II

Both compound I and compound II were obtained from the rhizome of Tupistrachinensis Bak., a plant of the family Liliaceae, and were separated by methanol immersion, organic solvent fractionation, and silica gel column chromatography. The specific separation and preparation steps are: (1) add methanol or ethanol 3-5 times the weight of the open arrow crushed product to the open arrow crushed product according to the weight volume ratio, extract under reflux conditions, the extraction temperature is 60-80 °C, extract The time is 4 to 8 hours. Or add 3-8 times the weight of methanol or ethanol to the open arrow crushed object according to the weight volume ratio, and extract for more than 24 hours to obtain methanol or ethanol extract; (2) methanol or ethanol extract, by weight volume Using 5-10 chloroform and ethyl acetate to extract more than 3 times successively, each extract is concentrated and dried to obtain the corresponding extract; (3) chloroform and ethyl acetate extract are combined, and silica gel (200-300 Purpose) column chromatography, reverse phase silica gel (RP-18) column chromatography and gel (SephadexLH-20) column chromatography separate and obtain compound I; (4) chloroform and ethyl acetate extract use silica gel (200-300 mesh ) column chromatography and reverse phase silica gel (RP-18) column chromatography to obtain compound II of the present invention.

The two compounds of the present invention have activity against P. litchi downy mildew. The spore germination method was used to carry out the in vitro biological activity tracking test of Pyrosophthora litchii to prove that: the effective inhibitory concentration (EC ₅₀ ) of compound I to sporangia germination of Pynophytophthora litchie was 100.28 μg/ml, and compound II had an effect on the sporangia germination of Pynophytophthora litchie. The effective inhibition medium concentration (EC ₅₀ ) of sporangia germination is 124.37 μ g/ml; Carry out the in vivo antibacterial test of litchi peronospora to two compounds by inoculation method and prove: concentration is the compound I of 500 μ g/mL and compound II liquid medicine, with The methods of spraying, soaking or coating can prevent and control downy mildew of litchi, and have obvious control effect on downy mildew of litchi (the variety is "Huaizhi").

The two compounds of the present invention can be used as active ingredients alone, in combination or compounded with fungicides (such as Radomil) for preventing and controlling litchi downy mildew, and are used for the prevention and treatment of litchi downy mildew. Plant extracts, fractional extracts and fractions containing the steroidal saponin compound of the present invention may also be used.

Example

The essence of the present invention will be described below in conjunction with examples, but the content of the present invention is not limited thereto.

Embodiment one: crude extraction and separation of compound

Open arrow rhizome dry powder 3kg, soaked repeatedly with 95% and 9L methanol until the leachate was almost colorless, concentrated the methanol extract under reduced pressure to obtain 283g of brown paste, and washed the concentrate with 1500mL petroleum ether, chloroform and ethyl acetate in sequence Fractional extraction, concentrated under reduced pressure to obtain the extracts of each fraction, combined 15.5 g of ethyl acetate extract and chloroform extract, put it on a silica gel (100-200 mesh, 300 g) column, and carried out gradient gradient washing with a mixture of chloroform and methanol De, chloroform and methanol mixing ratio, the volume ratio is 9: 1 and 8: 1, receive as a fraction by 400mL, and carry out the in vitro biological activity tracking test to Phytophthora litchie by spore germination method, get 22-26 fraction 1.78g And 27-37 fraction 1.32g two active parts.

Embodiment two: the extraction and separation of compound I

The 22nd-26th fraction described in Example 1 was put on a silica gel (200-300 mesh, 60g) column, and eluted with chloroform and methanol. The volume ratio of chloroform and methanol was 8:1.5, and 50 mL was accepted as a fraction. In addition, the spore germination method was used to carry out the in vitro biological activity tracking test on Pythophthora litchie, and 0.37g of the active part of the 9-12 fraction was obtained. Put this active part on the reversed-phase silica gel (RP-18) column, elute with methanol and water mixed solution, methanol and water volume ratio are 8: 2, receive by 10mL as a cut, and use spore germination method to the peronospora of litchi The in vitro biological activity tracking test was carried out, and 0.11 g of the active part of the 11th-15th fraction was obtained. Put the active part on a gel (Sephadex LH-20) column, elute with tetrahydrofuran, receive 2 mL as a fraction, and obtain 32 mg of the 5th-7th fraction, which is compound I.

Embodiment three: the extraction and separation of compound II

Put the 27th-37th fraction described in Example 1 on a reverse-phase silica gel column (RP-18), elute with methanol and water mixture, methanol and water volume ratio 8:2, receive 10 mL as a fraction, and use spores The in vitro biological activity tracking test was carried out on Pythophthora litchie by germination method, and 852 mg of the active part of the 17th-30th fraction was obtained, which was compound II.

Embodiment four: compound I and compound II are to the in vitro activity assay of the sporangium germination inhibitory action of peronoplasty mildew of litchi

Compound I and compound were first prepared as a stock solution with a concentration of 1000 μg/mL. Then, cultured under the conditions of 25°C and 98% RH, the fungal strains of Phytophthora litchie with a bacterial age of 5 days were washed with sterile water to prepare sporangia suspensions with different concentrations. Sporangia suspensions of different concentrations were mixed with the stock solutions of Compound I and Compound II to form drug-containing bacterial solutions with concentrations of 50, 100, 150, 200, and 250 μg/ml, and the concentration of drug-containing bacterial solutions was 20 per field of view. For the sporangia, shake the drug-containing bacteria liquid, and culture it at 25°C and 98% RH for 6 hours, and set a blank control at the same time, then take the cultured drug-containing bacteria and count the germination rate or inhibition rate under a 10×40 times microscope. Rate. Statistical analysis of the data shows that the effective inhibitory concentration (EC ₅₀ ) of compound I to the sporangia germination of P. litchi downy mildew is 100.28 μg/ml, and the effective inhibitory medium concentration (EC ₅₀ ) of compound II to the sporangial germination of P. litchi downy mildew is 124.37 μg/ml. The results are shown in Figure 1.

Figure 1 is the inhibition of compound I and compound II on the sporangia germination of P. litchi peronospora

In vivo antibacterial assay of compound I and compound II against Peronophytophthora litchii

The selected lychees (variety is Huaizhi) were first washed with a surface disinfectant, then rinsed with tap water, dried, and then dipped in a brush with a concentration of 500 μg/mL compound I and compound II liquid (prepared The method is the same as in Example 4), evenly spread on the tip of litchi fruit, dry it in air, then dip the sporangia suspension (2× ¹⁰⁵ /mL) of downy mildew with a brush, and spread evenly on the place where the medicine is applied. After packaging and culturing for 3 days under the conditions of 25° C. and 98% RH, the diseased and healthy fruits were counted, and the infection rate was calculated. The test results are shown in Table 1:

Table 1: In vivo antibacterial results of Compound I and Compound II against Peronophytophthora litchii ^* deal with Concentration (μg/mL) diseased fruit Nuts (pieces) Infection rate (%) control 79 11 ^87.8a Compound I 500 15 75 ^16.7b Compound II 500 17 73 ^18.9b

^* Following the same letter means no significant difference at the 5% level (DMRT method).

Structural Identification of Steroidal Saponins

Compound I: It is a white powder, and its molecular formula is C ₂₇ H ₄₀ O ₉ . Proton NMR spectrum (400MHz, tritiated pyridine): δ4.32 (1H, br s, H-1), 4.40 (1H, br s, H-2), 4.74 (1H, br s, H-3), 5.81 (1H, d, J=3.2Hz, H-4), 4.54 (1H, br s, H-7), 4.56 (1H, m, H-16), 0.87 (3H, s, H-18), 1.45 (3H, s, H-19), 1.06 (3H, d, J=6.8Hz, H-21), 4.01 (1H, d, J=12.0Hz, H-26eq), 4.41 (1H, d, J =12.0Hz, H-26ax), 4.76 (1H, br s, H-27a), 4.79 (1H, brs, H-27b): Carbon NMR spectrum (100MHz, tritiated pyridine): See Table 2.

Compound II: It is a white powder with a molecular formula of C ₂₇ H ₄₂ O ₉ . Proton NMR spectrum (400MHz, tritiated pyridine): δ4.34(1H, br s, H-1), 4.31(1H, br s, H-2), 4.74(1H, d, J=3.0Hz, H -3), 5.31(1H, d, J=3.0Hz, H-4), 5.00(1H, d, J=2.8Hz, H-6), 4.48(1H, br s, H-7), 2.61( 1H, br t, J=11.6Hz, H-8), 4.56 (1H, dd, J=14.4, 7.6Hz, H-16), 0.93 (3H, s, H-18), 1.98 (3H, s, H-19), 1.06 (3H, d, J=6.8Hz, H-21), 3.98 (1H, d, J=12.0Hz, H-26eq), 4.40 (1H, d, J=12.0Hz, H- 26ax), 4.75 (1H, br s, H-27a), 4.78 (1H, br s, H-27b): Carbon NMR spectrum (100MHz, tritiated pyridine): see Table 2.

Table 2: Carbon NMR spectrum data of compound I and compound II Carbon Compound I Compound II Carbon Compound I Compound II 1 76.5 79.72 67.23 75.5 75.64 71.2 69.85 86.3 78.26 211.1 73.67 75.2 71.88 40.9 34.0 37.610 50.2 40.411 22.0.41.4 39.813 40.40.414 49.3 50.1 50.1 15 31.5 32.016 81.31.317 62.9 63.118 16.3 16.419 13.0 15.620 42.1 42.121 15.022 109.5 109.523 33.224 29.025 144.526 65.027 108.8.7.7.7

Claims (5)

1. A steroidal saponin compound whose name is 1β, 2β, 3β, 4β, 5β, 7α-hexahydroxyspirosta-25(27)-en-6-one (1β, 2β, 3β, 4β, 5β , 7α-hexahydroxyspirost--25(27)-en-6-one), its chemical structure is as follows:

2. A preparation method of a steroidal saponin compound, which is characterized in that the open arrow is used as a raw material. After the raw material is crushed, methanol or 3-5 times the weight of the open arrow crushed object is added to the open arrow crushed object according to the weight volume ratio. Ethanol is extracted under reflux conditions, the extraction temperature is 60-80° C., and the extraction time is 4-8 hours. Or add 3-8 times the weight of methanol or ethanol to the open arrow crushed product according to the weight volume ratio, and extract for more than 24 hours to obtain methanol or ethanol extract; methanol or ethanol extract, according to the weight volume ratio Use 5-10 times of chloroform and ethyl acetate to extract sequentially for more than 3 times, and each extract is concentrated to obtain the corresponding extract; the chloroform and ethyl acetate extracts are combined, and 200-300 mesh silica gel column chromatography, RP-18 reverse-phase silica gel column chromatography and SephadexLH-20 gel column chromatography to obtain the compound, chloroform and ethyl acetate extracts were obtained by 200-300 mesh silica gel column chromatography and RP-18 reverse-phase silica gel column chromatography This compound. 3. The compound according to claim 1, characterized in that it is used as a fungicide with a concentration ≥ 100 mg/L, and can prevent and treat downy mildew of litchi by spraying, soaking or coating. 4. The compound according to claim 1, characterized in that it is used for the control of downy mildew of litchi alone or in combination or compounded with Ridomil, a fungicide for preventing and controlling downy mildew of litchi. 5. The use of the compound according to claim 1, characterized in that it is used as an active ingredient or additive for preventing and treating litchi downy mildew with other single compounds or two or more compounds mixed in any proportion, and also includes Plant extracts, fractional extracts and isolated parts of Compound I and Compound II of the present invention.