C-23 spirocholestane type steroid compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of phytochemistry and botanical pesticides, and relates to a C-23 spiro cholestane type steroid compound as well as a preparation method and application thereof. The C-23 spiro-cholestane type steroid compound disclosed by the invention has a remarkable inhibition effect on growth of grass, and can be used as an effective component of a plant source herbicide.

Description

Technical Field

[0001] The invention belongs to the technical field of plant chemistry and botanical pesticides, and in particular relates to a C-23 spirocholestane-type steroid compound and a preparation method thereof and application thereof as a herbicide. Background Art

[0002] Weeds compete with crops for water, fertilizer, light and growth space, directly affecting the photosynthesis and normal growth of crops, and are one of the important factors affecting agricultural output. According to statistics from the United Nations, the average annual reduction in agricultural production due to weeds is about 10%, and China loses nearly 4 million tons of grain due to weeds. Herbicides are the most important weed management method. For a long time, weed control has relied on chemical herbicides. Among the new active ingredients registered in conventional herbicides in the United States from 1997 to 2010, artificial synthesis accounted for 78%, and only about 8% were compounds of natural origin. The use of a large number of chemical herbicides has had a negative impact on the environment and human health. The residues of chemical herbicides can cause soil and water pollution, change the structure and function of the ecosystem, and widespread exposure to chemical herbicides may lead to many health problems, such as neurological damage, pancreatic cancer, and immune dysfunction. Natural products (NPs) are secondary metabolites extracted and separated from plants, animals, microorganisms and marine biological clocks. They are highly effective, low toxic and environmentally friendly, and are a new strategy for researching and developing new green pesticides.

[0003] In 1977, scientists isolated the triketide compound leptospermone from the plant Callistemon citrinus of the Myrtaceae family. After that, a series of herbicides developed with leptospermone as the lead compound were launched on the market, such as sulcotrione and mesotrione. In 2004, the global sales of mesotrione reached 670 million US dollars. The herbicide developed with the natural product leptospermone as the lead compound is a successful case of discovering the lead herbicide compound from plants. In addition, many herbicides such as cyproconazole, AAL toxin, Biolaphes, glufosinate and dicamba were developed from plant allelopathic substances. For example, cinmethylin is a similar compound synthesized using 1,8-cineol, a monoterpene component in plants such as Eucalyptus, as a lead compound. It can control a variety of weeds and has been developed as a rice field herbicide.

[0004] Natural products have undergone a long and protracted evolutionary process in nature. The novelty of their chemical structures and the diversity of their biological activities are one of the important resources for the research and development of drugs and pesticides. There is great potential in finding lead compounds with herbicidal activity from natural products. Summary of the invention

[0005] In view of the above, the object of the present invention is to provide six new C-23 spirocyclic cholestane steroid natural products (1-6) and their derivatives extracted from Ypsilandra plants, as well as the use of 75% alcohol eluate of Ypsilandra plants, C-23 spirocyclic cholestane steroid compounds (1-6) and their derivatives as active ingredients in the preparation of herbicides.

[0006] The purpose of the present invention can be achieved through the following technical solutions.

[0007] In a first aspect, the present invention provides a C-23 spirocyclic cholestane-type steroid compound having the following structural formula (I) or a pesticide-acceptable derivative thereof:

[0008]

[0009] wherein R1 is H, R2 is H or Ac, R3 is H or Ac, and R4 is H or Ac.

[0010] In the embodiments of the present invention, the derivative refers to the glycoside, salt or ester derivative of the C-23 spirocyclic cholestane-type steroid compound of formula (I).

[0011] In a specific embodiment of the present invention, the C-23 spirocyclic cholestane-type steroid compound is any one of the following structural formulas 1-6:

[0012]

[0013] In a second aspect, the present invention provides a method for preparing a C-23 spirocyclic cholestane-type steroid compound, the method comprising the following steps:

[0014] 1) drying and crushing the whole herb material of a Ypsilandra plant, extracting with an organic solvent, and then desolventizing to obtain an extract of the Ypsilandra plant material;

[0015] 2) dissolving the extract of the genus Asteraceae obtained in step 1) in a small amount of water, and performing macroporous resin column chromatography using water, 75% alcohol, and 95% alcohol as eluents in sequence to obtain 75% alcohol eluate and 95% alcohol eluate, wherein the alcohol is preferably methanol or ethanol, more preferably ethanol; and

[0016] 3) The 75% alcohol eluate obtained in step 2) is sequentially subjected to column chromatography and semi-preparative high performance liquid chromatography to obtain the C-23 spirocyclic cholestane-type steroid compound, especially the compound having the above structural formula 1-6.

[0017] In the embodiments of the present invention, the pesticide-acceptable derivative refers to the pesticide-acceptable glycoside, salt, ester and other derivatives of C-23 spirocyclic cholestane-type steroid compounds.

[0018] In a third aspect, the present invention provides a 75% alcohol eluate of a plant of the genus Asteraceae, which is obtained by step 2) of the above method.

[0019] In a fourth aspect, the present invention provides a herbicidal composition comprising a C-23 spirocholestane-type steroid compound or a pesticidally acceptable derivative thereof or a 75% alcohol eluate of a plant of the genus Asteraceae.

[0020] In an embodiment of the present invention, grasses include grasses in the Poaceae family, such as barnyard grass, crabgrass, ryegrass, goosegrass and foxtail grass, etc.; grasses in the Cyperus family, such as sedge and Cyperus rotundus, etc.; grasses in the Cruciferae family, such as Arabidopsis thaliana and Artemisia selengensis, etc.; and grasses in the Amaranthaceae family, such as Amaranthus retroflexus and Amaranthus sphaerocephalus, etc.

[0021] In a fifth aspect, the present invention provides a C-23 spirocholestane steroid compound or its pesticide-acceptable derivative or a 75% alcohol eluate of a plant of the genus Asteraceae for use in the preparation of a herbicide. The C-23 spirocholestane steroid compound provided by the present invention is a natural product, has significant herbicidal activity, can be used to remove weeds in agriculture, forestry and gardening, and is environmentally friendly and does not pollute the natural environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the chemical structural formula of the C-23 spirocyclic cholestane type steroid compound 1-6 in the present invention.

[0023] Figure 2 The results of the inhibitory effect of the compound C-23 spirocholestane steroid 1-6 of the present invention on Arabidopsis roots, wherein A is a phenotypic diagram of the inhibitory effect of compound 1-6 (50 μM) and glyphosate (3.13 μM) on Arabidopsis root growth, and B is a statistical diagram of root length after the action of compound 1-6 and glyphosate on Arabidopsis.

[0024] Figure 3 The results of the inhibitory effects of the compounds C-23 spirocholestane-type steroid compounds 3 / 4, 5 / 6 of the present invention on barnyard grass roots, wherein A is a phenotypic diagram of the inhibitory effects of compounds 1-6 (50 μM) and glyphosate (50 μM) on barnyard grass root growth, and B is a statistical diagram of the root length of barnyard grass after the action of compounds 1-6 and glyphosate.

[0025] Figure 4 The results of the inhibitory effect of the compound C-23 spirocholestane steroid compound 3 / 4 of the present invention on ryegrass roots, wherein A is a phenotypic diagram of the inhibitory effect of compound 1-6 (50 μM) and glyphosate (50 μM) on ryegrass root growth, and B is a statistical diagram of the root length of ryegrass after the action of compound 1-6 and glyphosate.

[0026] Figure 5 The 75% ethanol eluate (YRH-1) of the YRH-1 plant in the present invention inhibits the growth of Arabidopsis thaliana, wherein A is a phenotypic diagram of the root inhibition of YRH-1 extract (200 μg / ml), YRH-1 (200 μg / ml), and glyphosate (3.13 μM) on Arabidopsis thaliana, and B is a statistical diagram of the root length of Arabidopsis thaliana after the YRH-1 extract, YRH-1 and glyphosate acted on Arabidopsis thaliana. DETAILED DESCRIPTION

[0027] In order to facilitate the understanding of the present invention, the present invention will be described more fully below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0029] According to the first aspect, the present invention provides a C-23 spirocyclic cholestane steroid compound and its pesticide acceptable derivatives, wherein the C-23 spirocyclic cholestane steroid compound has the following structural formula (I):

[0030]

[0031] wherein R1 is H, R2 is H or Ac, R3 is H or Ac, and R4 is H or Ac.

[0032] In the embodiments of the first aspect, the pesticidally acceptable derivative refers to a derivative such as a glycoside, a salt, or an ester of a C-23 spirocyclic cholestane steroid compound.

[0033] In a specific embodiment of the first aspect, the C-23 spirocyclic cholestane-type steroid compound may be any one of the following structural formulas 1-6:

[0034]

[0035] It is well known to those skilled in the art that the pesticide-acceptable glycoside can be, for example, a glycoside formed by dehydration condensation of a hydroxyl group in a pesticide-acceptable compound with a hemiacetal hydroxyl group of a sugar or a derivative of a sugar. Pesticide-acceptable sugars include monosaccharides or sugar chains composed of monosaccharides. Monosaccharides include any one of monosaccharides such as glucose, rhamnose, xylose, fucose, celery sugar or arabinose.

[0036] The acceptable salt in pesticides can be, for example, an acceptable base addition salt in pesticides. The acceptable base addition salt in pesticides includes salts derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc. The salts derived from the acceptable organic base in pesticides include salts of primary amines, secondary amines and tertiary amines, including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine and ethanolamine and triethanolamine.

[0037] The pesticidally acceptable esters may be esters of the C-23 spirocyclic cholestane-type steroid compounds of the present invention and pesticidally acceptable non-toxic organic acids, such as acetic acid, propionic acid, malic acid, citric acid, oxalic acid and palmitic acid.

[0038] According to a second aspect, the present invention provides a method for preparing a C-23 spirocyclic cholestane-type steroid compound, comprising the following steps:

[0039] 1) drying and crushing the whole herb material of a Ypsilandra plant, extracting with an organic solvent, and then desolventizing to obtain an extract of the Ypsilandra plant material;

[0040] 2) dissolving the Yerba spp. plant material extract obtained in step 1) with a small amount of water, and then performing macroporous resin column chromatography using water, 75% alcohol, and 95% alcohol as eluents in sequence to obtain a 75% alcohol eluate (referred to as YRH) and a 95% alcohol eluate; and

[0041] 3) The 75% alcohol eluate (YRH) obtained in step 2) is sequentially subjected to column chromatography and semi-preparative high performance liquid chromatography to obtain a C-23 spirocholestane-type steroid compound, especially a compound having structural formula 1-6.

[0042] In the embodiments of the present invention, the Y. genus plant is a plant in the genus Y. thibetica, Y. cavaleriei, Y. yunnanensis, and Y. alpina. In a preferred embodiment, the Y. genus plant is Y. thibetica and Y. cavaleriei.

[0043] In an embodiment of the present invention, in step 1), the organic solvent may be at least one of acetone, ethanol, methanol, n-butanol and acetonitrile; preferably, the organic solvent is 75% methanol or 75% ethanol.

[0044] In an embodiment of the present invention, in step 1), the extraction method may be organic solvent cold extraction, heating reflux extraction, organic solvent ultrasonic extraction, etc. Preferably, heating reflux extraction.

[0045] In an embodiment of the present invention, the plant material of the genus Asteraceae is extracted with an organic solvent at least once, for example, 2, 3, 4 times, preferably 3 times.

[0046] In an embodiment of the present invention, the volume ratio of the organic solvent to the dried genus Aconitum plant material can be 1:1 to 5:1, for example 1:1, 2:1, 3:1, 4:1, 5:1, and ratios between any two of the above ratios, for example 1.5:1, 2.5:1, 3.5:1, 4.5:1, etc., preferably 3:1.

[0047] In an embodiment of the present invention, in step 2), the alcohol may be methanol or ethanol; preferably, ethanol.

[0048] In a specific embodiment of the present invention, in step 3), column chromatography includes normal phase silica gel column chromatography, reverse phase silica gel column chromatography (e.g. RP-18 column chromatography), resin column chromatography (e.g. PRP resin), gel column chromatography (e.g. Sephadex LH-20), medium pressure chromatography separation gel (e.g. MCI gel) and preparative or semi-preparative high performance liquid chromatography (hereinafter referred to as semi-preparative HPLC).

[0049] In a specific embodiment of the present invention, in step 3), the eluent used for column chromatography is at least one of petroleum ether, dichloromethane, chloroform, ethyl acetate, acetone, ethanol, methanol, n-butanol, acetonitrile, water and formic acid, for example, a combination of any two, any three, any four of the above organic solvents. In a more specific embodiment of the present invention, for normal phase silica gel column chromatography, chloroform and methanol can be used for gradient elution in a volume ratio of 1:0 to 0:1, for example, 1:0, 50:1, 25:1, 10:1, 8:1, 4:1, 0:1. In a more specific embodiment of the present invention, for reverse phase silica gel column chromatography, such as reverse phase RP-18 column chromatography, methanol and water can be used for gradient elution in a volume ratio of 3:7 to 7:3, for example, the volume ratio of methanol to water can be 3:7, 5:5, 7:3. In a more specific embodiment of the present invention, for resin column chromatography, such as PRP resin column chromatography, methanol and water can be used for gradient elution in a volume ratio of 3:7 to 7:3, for example, the volume ratio of methanol to water can be 3:7, 5:5, 7:3. In a more specific embodiment of the present invention, for gel column chromatography, such as Sephadex LH-20 gel column chromatography, methanol or methanol-water (9:1, v:v) can be used for elution. In a more specific embodiment of the present invention, for medium pressure chromatography separation gel, such as MCI gel, ethanol and water can be used for gradient elution in a volume ratio of 3:7 to 7:3, for example, the volume ratio of ethanol to water can be 3:7, 5:5, 7:3. In a more specific embodiment of the present invention, for semi-preparative HPLC, acetonitrile and water can be used for isocratic elution, for example, elution is performed with 50% acetonitrile-water as the mobile phase. In a more specific embodiment of the present invention, for semi-preparative HPLC, gradient elution with acetonitrile and water can also be used, for example, gradient elution with acetonitrile and water in a volume ratio of 2:8 to 8:2.

[0050] According to a third aspect, the present invention provides a 75% alcohol eluate (YRH) of a plant of the genus Yerba rapa, which is obtained by step 2) of the above method.

[0051] According to a fourth aspect, the present invention provides a herbicidal composition comprising at least one of the C-23 spirocyclic cholestane-type steroid compounds of formula (I) or derivatives thereof or the 75% alcohol eluate (YRH) of the Yerba rapa plant of the third aspect.

[0052] When the C-23 spirocyclic cholestane type steroid compound of the present invention is used as a herbicidal active ingredient, it can be applied directly or in the form of a herbicidal composition. The composition comprises at least one of the compounds of formula (I) or its derivatives. In the herbicidal composition of the present invention, the herbicidal composition may contain 0.05-99% C-23 spirocyclic cholestane type steroid compound, preferably 0.1-50%, based on the total weight of the herbicidal composition.

[0053] When the 75% alcohol eluate (YRH) of the Yerba spp. plant of the present invention is used as a herbicidal active ingredient, it can be applied directly or in the form of a pharmaceutical composition. In the herbicidal composition, the herbicidal composition can contain 0.1-99% of the 75% alcohol eluate (YRH) of the Yerba spp. plant, preferably 0.5-90%, based on the total weight of the herbicidal composition.

[0054] In an embodiment of the present invention, grasses include but are not limited to grasses in the Poaceae family, such as barnyard grass, crabgrass, ryegrass, goosegrass and foxtail grass, etc.; grasses in the Cyperus family, such as sedge and Cyperus rotundus, etc.; grasses in the Cruciferae family, such as Arabidopsis thaliana and Artemisia selengensis, etc.; and grasses in the Amaranthaceae family, such as Amaranthus retroflexus and Amaranthus sphaerocephalus, etc.

[0055] Those skilled in the art will appreciate that, in addition to the C-23 spirocyclic cholestane steroid compound or its derivative or YRH as an active ingredient, the composition of the present invention may also contain carriers, adjuvants or excipients that are compatible with the compound of the present invention or other ingredients or other herbicidal ingredients that contribute to its activity.

[0056] Pesticide-acceptable carriers, adjuvants or excipients are well known to those skilled in the art. Pharmaceutical carriers or excipients are one or more solid, semisolid and liquid diluents, fillers and adjuvants for pharmaceutical products, including but not limited to fillers (diluents), lubricants (glidants or anti-adhesives), dispersants, wetting agents, adhesives, solubilizers, antioxidants, antibacterial agents, emulsifiers, disintegrants, etc. Binders include syrup, gum arabic, gelatin, sorbitol, tragacanth, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose or hydroxypropyl methylcellulose, etc.), gelatin slurry, syrup, starch slurry or polyvinyl pyrrolidone, etc.; fillers include lactose, powdered sugar, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salts (such as calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol or glycine, etc.; lubricants include micropowdered silica gel, magnesium stearate, talc, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, etc. Disintegrants include starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinyl pyrrolidone or microcrystalline cellulose, etc.; wetting agents include sodium lauryl sulfate, water or alcohol, etc.; antioxidants include sodium sulfite, sodium bisulfite, sodium metabisulfite, dibutyl benzoic acid, etc.; antibacterial agents include 0.5% phenol, 0.3% cresol, 0.5% trichlorobutanol, etc.; emulsifiers include polysorbate-80, sorbitan monohydrate, lecithin, soybean lecithin, etc.; solubilizers include Tween-80, bile, glycerol, etc.

[0057] In a fifth aspect, the present invention provides the use of a C-23 spirocholestane steroid compound or its pesticide acceptable derivative or a 75% alcohol eluate (YRH) of a plant of the genus Yerba rapa in the preparation of a herbicide.

[0058] The preferred embodiments of the present invention are described in detail below in conjunction with the examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention, and all of these modifications and substitutions fall within the scope of the claims of the present invention.

[0059] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained through commercial channels.

[0060] In the following examples, the optical rotation was measured using an AutopolVI polarimeter. The ultraviolet spectrum (UV) was measured using a Shimadzu UV2700 ultraviolet spectrometer. 1 HNMR, 13 C NMR and 2D NMR spectra were recorded using a Bruker AM-500 or AM-600 nuclear magnetic resonance instrument. High-resolution mass spectrometry (HR-ESI-MS) was measured using an Agilent 1290UPLC / 6540Q-TOF mass spectrometer. Semi-preparative HPLC used an Agilent 1260 liquid phase, and the chromatographic column was a Zorbax SB-C18 preparative column with a flow rate of 3.0 mL / min. Column chromatography involved the use of silica gel (200-300 mesh, Qingdao Ocean Chemical Company, China), RP-18 (40-60 μm, Merk), D101 macroporous resin (Shanghai McLean Biochemical Technology Co., Ltd., China), HPD-100 macroporous resin (Tianjin Yunkai Resin Technology Co., Ltd.), PRP macroporous resin (Beijing Jufu Resin Factory), MCI gel (75-150 μm, Mitsubishi Chemical Corporation, Japan) and Sephadex LH-20 (GE Healthcare, USA). Thin layer chromatography (TLC) was performed on silica gel GF254 plates (Qingdao Ocean Chemical Co., Ltd., China) and the spots were observed by 10% sulfuric acid-ethanol solution. Plant growth climate chamber (Ningbo Kesheng Experimental Instrument Co., Ltd.) Root length was measured by Image J measurement.

[0061] Example 1: Preparation method of C-23 spirocyclic cholestane steroid compound 1-6

[0062] 10 kg of dried Y. thibetica whole plant was crushed, and 30 L of 75% ethanol (the volume ratio of organic solvent to plant material was 3:1) was added, and reflux extraction was performed at 90°C for 3 times, with extraction times of 3, 3, and 2 hours respectively. The filtrates were combined, and the solvent was recovered under reduced pressure to obtain an extract (i.e., Y. thibetica extract). The extract was dispersed in water, passed through HPD-100 macroporous resin, and eluted with water, 75% ethanol, and 95% ethanol in sequence; the 75% ethanol eluate (YRH-1) was recovered, mixed with 80-100 mesh silica gel, and chromatographed on a 300-400 mesh silica gel column. The eluent was chloroform-methanol gradient elution (20:1, 10:1, 8:2, 7:3, v / v), and each gradient was 4 to 6 column volumes. According to thin layer chromatography, 10% sulfuric acid-ethanol solution was used for color development and then combined to obtain 4 components, namely Fr.1-Fr.7. Fr.1 (200.0 g) was chromatographed on a 200-300 mesh silica gel column and eluted with a chloroform-methanol gradient (20:1, 15:1, 10:1, 8:1, 4:1, 1:1, v / v), with 4 to 6 column volumes for each gradient, to obtain 6 components, namely Fr.1.1-Fr.1.6. Fr.1.2 (26.0 g) was chromatographed on an RP-18 reverse phase silica gel column and eluted with a methanol-water gradient (2:8, 3:7, 1:1, 7:3, 8:2, v / v), with 4 to 6 column volumes for each gradient, to obtain 5 components, namely Fr.1.2.1-Fr.1.2.5. Fr.1.2.2 (200.0 mg) was prepared by semi-preparative HPLC using isocratic elution and 50% acetonitrile-water as mobile phase to obtain compound 1 (20.3 mg) (yield: 0.00020%) and compound 2 (14.6 mg) (yield: 0.00015%). Fr.1.2.3 (270.0 mg) was prepared by semi-preparative HPLC using acetonitrile-water (3:7-7:3, v / v) gradient elution to obtain compound 3 (27.5 mg) (yield: 0.00028%) and compound 4 (26.2 mg) (yield: 0.00026%). Fr.1.2.4 (130.0 mg) was purified by semi-preparative HPLC using acetonitrile-water (3:7 to 7:3, v / v) gradient elution to give compound 5 (17.5 mg) (yield: 0.00018%) and compound 6 (21.5 mg) (yield: 0.00022%).

[0063] Physical constants and spectral data of compound 1: white amorphous powder; IR(KBr)ν max 3441,2936,2873,1708,1633,1450,1287,1137,1068cm -1 ; 1H NMR (C5D5N, 500 MHz) and 13 C NMR (125 MHz, C5D5N) data are shown in Tables 1 and 2; HRESIMS m / z 615.3509 [M+Na] + (calcd.for C 33 H 52 oeLh + ,615.3504).

[0064] Physical constants and spectral data of compound 2: white amorphous powder; IR(KBr)ν max 3441,2935,2874,1743,1712,1633,1450,1383,1236,1138,1069cm-1; 1 H NMR (500 MHz, C5D5N) and 13 C NMR (125 MHz, C5D5N) data are shown in Tables 1 and 2; HRESIMS m / z: 657.3617 [M+Na] + (calcd.for C 35 H 54 O 10 Na + ,657.3609).

[0065] Physical constants and spectral data of compound 3: white amorphous powder; IR(KBr)ν max 3440,2939,2873,1737,1713,1633,1450,1370,1252,1135,1069cm -1 ; 1 H NMR (600 MHz, C5D5N) and 13 C NMR (150 MHz, C5D5N) data are shown in Tables 1 and 2; HRESIMS m / z: 657.3619 [M+Na] + (calcd.for C 35 H 54 O 10 Na + ,657.3609).

[0066] Physical constants and spectral data of compound 4: white amorphous powder; IR(KBr)ν max3442,2939,2873,1735,1711,1633,1450,1385,1250,1136,1068cm -1 ; 1 H NMR (600 MHz, C5D5N) and 13 C NMR (150 MHz, C5D5N) data are shown in Tables 1 and 2; HRESIMS m / z: 657.3615 [M+Na] + (calcd.for C 35 H 54 O 10 Na + ,657.3609).

[0067] Physical constants and spectral data of compound 5: white amorphous powder; IR(KBr)ν max 3440,2937,2875,1741,1713,1633,1542,1450,1383,1237,1138,1069cm -1 ; 1 HNMR (500MHz, C5D5N) and 13 C NMR (125 MHz, C5D5N) data are shown in Tables 1 and 2; HRESIMS m / z: 699.3717 [M+Na] + (calcd.for C 37 H 56 O 11 Na + ,699.3715).

[0068] Physical constants and spectral data of compound 6: white amorphous powder; IR(KBr)ν max 3442,2936,2874,1743,1714,1634,1556,1449,1383,1237,1139,1068cm -1 ; 1 HNMR (500MHz, C5D5N) and 13 C NMR (125 MHz, C5D5N) data are shown in Tables 1 and 2; HRESIMS m / z: 699.3719 [M+Na] + (calcd.for C 37 H 56 O 11 Na + ,699.3715).

[0069] Table 1. Compounds 1-6 1 HNMR data (solvent is C5D5N)

[0070]

[0071] Note: s, d, t, m, br, and o refer to the fragmentation peaks of the signals, respectively (s: singlet; d: doublet; t: triplet; br: broad; m: multiplet; o: overlapping peak). Compounds 3 and 4 were tested at 600 MHz, and the remaining compounds were tested at 500 MHz.

[0072] Table 2. Compounds 1-6 13 C NMR data (solvent is C5D5N)

[0073]

[0074] Note: Compounds 3 and 4 were tested at 150 MHz, and the remaining compounds were tested at 125 MHz.

[0075] Example 2: Preparation Method 2 of C-23 Spirocyclic Cholestane Steroid Compound 1-6

[0076] 15 kg of dried whole plant of Y. cavaleriei was crushed, and 75 L of 75% methanol (the volume ratio of organic solvent to plant material was 5:1) was added. The extraction was refluxed at 80°C for 3 times, with extraction time of 3, 3 and 2 hours respectively. The filtrates were combined, and the solvent was recovered under reduced pressure to obtain an extract. The extract was dispersed in water, passed through D101 macroporous resin, and eluted with water, 75% methanol and 95% methanol in sequence; the 75% methanol eluate (YRH-2) was recovered, mixed with 80-100 mesh silica gel, and chromatographed on a 300-400 mesh silica gel column. The eluent was chloroform-methanol gradient elution (20:1, 10:1, 8:2, 7:3, v / v), and each gradient was 4 to 6 column volumes. According to thin layer chromatography, 10% sulfuric acid-ethanol solution was used for color development and then combined to obtain 4 components, namely Fr.1-Fr.7. Fr.1 (258.9 g) was chromatographed on a 200-300 mesh silica gel column and eluted with a chloroform-methanol gradient (20:1, 15:1, 10:1, 8:1, 4:1, 1:1, v / v), with 4 to 6 column volumes for each gradient, to obtain 6 components, namely Fr.1.1-Fr.1.6. Fr.2.2 (34.8 g) was chromatographed on a 200-300 mesh silica gel column and eluted with a chloroform-methanol gradient (20:1, 15:1, 10:1, 8:1, 4:1, 1:1, v / v), with 4 to 6 column volumes for each gradient, to obtain 6 components, namely Fr.1.2.1-Fr.1.2.6. Fr.1.2.3 (427.6 mg) was subjected to semi-preparative HPLC using acetonitrile-water (3:7-7:3, v / v) gradient elution to obtain compound 1 (16.8 mg) (yield: 0.000168%), compound 2 (12.4 mg) (yield: 0.000124%), compound 3 (7.3 mg) (yield: 0.000073%), and compound 4 (16.2 mg) (yield: 0.000162%). Fr.1.2.1 (128.3 mg) was subjected to Sephadex LH-20 gel column chromatography using chloroform-methanol (1:1, v / v) as eluent to obtain four components, namely Fr.1.2.1.1-Fr.1.2.1.4. Fr.1.2.1.1 (60.7 mg) was purified by semi-preparative HPLC using acetonitrile-water (3:7 to 7:3, v / v) gradient elution to give compound 5 (5.4 mg) (yield: 0.000054%) and compound 6 (4.6 mg) (yield: 0.000046%).

[0077] Example 3: Inhibition experiment of C-23 spirocyclic cholestane steroids 1-6 on Arabidopsis

[0078] Arabidopsis root inhibition experiment: Arabidopsis seeds were first disinfected with an aqueous solution of sodium dichloroisocyanurate for 10 minutes. After rinsing three times with sterile water, the seeds were placed in a 7 cm culture dish containing 1 / 2 MS medium and cultured at 4 ° C for one day, and then transferred to a plant growth climate box with a constant temperature of 22 ° C, 16 hours of light, and 8 hours of dark cycle for 3 days. After that, the Arabidopsis seedlings were transferred to a culture dish of 1 / 2 MS medium with compound 1-6, negative control DMSO (control) and positive control drug glyphosate (Glyphosate) added respectively. After 8 days of culture under the above temperature and light conditions, the length of Arabidopsis roots was measured by ImageJ.

[0079] Experimental results: When the concentration of compounds 1-6 was 50 μM (Note: in the solvent, compounds 3 and 4 were a pair of tautomeric isomers, and compounds 5 and 6 were a pair of tautomeric isomers), the inhibition rates on Arabidopsis root growth were 64.87%, 68.89%, 75.56% and 87.44%, respectively ( Figure 2 ), and the positive control drug was glyphosate (3.13 μM). Further experiments calculated the EC values ​​of compounds 1, 2, 3 / 4 and 5 / 6 for Arabidopsis root inhibition. 50 The values ​​were 18.01±5.72, 26.83±5.41, 9.13±1.79 and 6.89±2.11 μM, respectively.

[0080] Example 4: Inhibition experiment of C-23 spirocholestane steroids 3 / 4 and 5 / 6 on barnyard grass

[0081] Compounds 3 / 4 and 5 / 6, which have significant inhibitory effects on Arabidopsis, were used as test samples, glyphosate as a positive control, and DMSO as a negative control (control). The filter paper method was used to determine their inhibitory activity against barnyard grass. Two layers of filter paper were laid in a 7 cm diameter culture dish, 3 ml of 50 μM test solution was added to each dish, 10 barnyard grass seeds that had germinated for 2 days were placed in each dish, the dish was covered with a sealing film, and three replicates were set for each test sample. Finally, the culture dish was placed in a light incubator, the temperature was set to 25 ° C, the light intensity was 5000 lux, and the light cycle was 16:8. After culturing for 5 days, the length of the barnyard grass roots was measured. The results showed that when the concentrations of compounds 3 / 4 and 5 / 6 had significant inhibitory activity on the root growth of barnyard grass ( Figure 3 ), the inhibition rates were 91.3% and 94.0% respectively, and the inhibition rate of the positive control drug glyphosate on barnyard grass growth was 57.3%. Further experiments determined the EC values ​​of 3 / 4 and 5 / 6 for the inhibition of barnyard grass growth. 50 The values ​​were 11.83±0.63 and 14.35±0.44 μM, respectively, both better than the positive control drug glyphosate (EC 50 =47.34±7.69 μM).

[0082] Example 5: Inhibition experiment of C-23 spirocholestane steroid 3 / 4 on Lolium multiflorum

[0083] The activity of compound 3 / 4 on Lolium multiflorum was determined by the method in Example 3. When the concentration was 50 μM, the inhibition rate of compound 3 / 4 on Lolium multiflorum was 58.2% ( Figure 4 Further experiments determined that compounds 3 / 4 inhibited the EC 50 was 34.7±1.58μM, and the positive control drug glyphosate (EC 50 =21.89±5.92 μM).

[0084] Example 6: Inhibition experiment of 75% ethanol eluate of Yerba spp. (YRH-1) on Arabidopsis thaliana

[0085] The Y. rueba extract obtained in Example 1 and the 75% ethanol eluate (YRH-1) were used to perform an Arabidopsis thaliana growth inhibition experiment.

[0086] Arabidopsis root inhibition experiment: Arabidopsis seeds were first disinfected with an aqueous solution of sodium dichloroisocyanurate for 10 minutes. After rinsing three times with sterile water, the seeds were placed in a 7 cm culture dish containing 1 / 2 MS medium and cultured at 4 ° C for one day, and then transferred to a plant growth climate box with a constant temperature of 22 ° C, 16 hours of light, and 8 hours of dark cycle for 3 days. After that, the Arabidopsis seedlings were transferred to a culture dish of 1 / 2 MS medium supplemented with Yarrowia extract (200 μg / mL), 75% alcohol eluate (YRH-1) (200 μg / mL), negative control DMSO (control) and positive control drug glyphosate (3.13 μM). After culturing for 8 days under the above temperature and light conditions, the length of Arabidopsis roots was measured by Image J.

[0087] Experimental results: The inhibition rates of the extract of Y. rueba, 75% alcohol eluate (YRH-1), and the positive control drug glyphosate on the root growth of Arabidopsis thaliana were 71.25%, 89.44%, and 79.53%, respectively. Figure 5 shown.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the concept, spirit and scope of the present invention; and these modifications or replacements fall within the scope of protection claimed in the claims of the present invention.

Claims

1. A C-23 spirocholestane steroid compound or a pesticide acceptable derivative thereof, characterized in that: The compound has the following structural formula (I): wherein R1 is H, R2 is H or Ac, R3 is H or Ac, and R4 is H or Ac, And wherein, the derivative refers to glycoside, salt, ester derivative.

2. The C-23 spirocyclic cholestane steroid compound or its pesticide acceptable derivative according to claim 1, characterized in that: The C-23 spirocyclic cholestane-type steroid compound has any one of the following structural formulas 1-6:

3. A method for preparing the C-23 spirocyclic cholestane-type steroid compound according to claim 1, characterized in that: The following steps are involved: 1) drying and crushing the whole herb material of a Ypsilandra plant, extracting with an organic solvent and then desolventizing to obtain an extract of the Ypsilandra plant material, wherein the volume ratio of the organic solvent to the Ypsilandra plant material is preferably 1:1 to 5:1, preferably 3:1; 2) dissolving the extract of the genus Asteraceae obtained in step 1) in a small amount of water, and performing macroporous resin column chromatography using water, 75% alcohol, and 95% alcohol as eluents in sequence to obtain 75% alcohol eluate and 95% alcohol eluate, wherein the alcohol is preferably methanol or ethanol, more preferably ethanol; and 3) The 75% alcohol eluate obtained in step 2) is sequentially subjected to column chromatography and semi-preparative high performance liquid chromatography to obtain the C-23 spirocyclic cholestane-type steroid compound.

4. The method according to claim 3, characterized in that The plants of the genus Y. are Y. thibetica, Y. cavaleriei, Y. yunnanensis, and Y. alpina.

5. The method according to claim 3, characterized in that: In step 1), the organic solvent comprises at least one of acetone, ethanol, methanol, n-butanol and acetonitrile. Preferably, the organic solvent comprises 75% methanol or 75% ethanol.

6. The method according to claim 3, characterized in that In step 3), the column chromatography includes normal phase silica gel column chromatography, reverse phase silica gel column chromatography, resin column chromatography, gel column chromatography, medium pressure chromatography separation gel (MCI Gel) and preparative or semi-preparative high performance liquid chromatography (HPLC).

7. The method according to claim 3, characterized in that In step 3), the eluent used for column chromatography includes one or a combination of two or more of petroleum ether, chloroform, dichloromethane, ethyl acetate, acetone, ethanol, methanol, n-butanol, acetonitrile, water and formic acid; preferably, normal phase silica gel column chromatography uses chloroform and methanol in a volume ratio of 20:1 to 7:3 for gradient elution; reverse phase RP-18 column chromatography uses alcohol and water in a volume ratio of 3:7 to 1:0 for gradient elution; gel column chromatography uses methanol, chloroform and methanol (1:1, v / v), methanol and water (7:3, v / v) for elution; medium pressure chromatography separation gel (MCI Gel) uses methanol and water in a volume ratio of 2:8 to 8:2 for gradient elution; preparative or semi-preparative HPLC uses acetonitrile and water in a volume ratio of 2:8 to 7:3 for gradient elution.

8. A 75% alcohol eluate of a plant of the genus Asteraceae, obtained by the step 2) of claim 3.

9. A herbicidal composition, characterized in that The composition comprises the C-23 spirocyclic cholestane steroid compound or its pesticide acceptable derivative as described in claim 1 or the 75% alcohol eluate of the genus Asteraceae as described in claim 8.

10. Use of the C-23 spirocholestane steroid compound or its pesticide acceptable derivative according to claim 1 or the 75% alcohol eluate of the genus Asteraceae according to claim 8 in the preparation of a herbicide.

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