Gynostemma pentaphyllum extract and preparation method and application thereof, dammarane type triterpenoid saponin and preparation method and application thereof, and gynostemma pentaphyllum botanical fungicide

Abstract

The invention provides a gynostemma pentaphyllum extract and a preparation method and application thereof, dammarane type triterpenoid saponin and a preparation method and application thereof, and a gynostemma pentaphyllum botanical fungicide, and relates to the technical field of fungicides. Active substances in the gynostemma pentaphylla extract provided by the invention comprise dammarane type triterpenoid saponin. The fiveleaf gynostemma herb extract provided by the invention shows an obvious effect of resisting plant anthracnose pathogenic bacteria, the EC50 value is below 97 mu g / mL, the antibacterial effect is superior to that of sophocarpidine, d-limonene and chlorothalonil, and the fiveleaf gynostemma herb extract has the advantages of high safety, low toxicity, low residue and difficulty in generating drug resistance, can be used as a lead compound of a botanical fungicide, and has a wide application prospect. The compound is used for preparing pesticides meeting green prevention and control requirements, can improve the pesticide effect and reduce the pollution of the pesticides to the ecological environment, and has important significance for improving the quality safety of agricultural products and guaranteeing human health.

Description

Technical Field

[0001] The invention relates to the technical field of fungicides, and in particular to a gynostemma pentaphyllum extract and a preparation method and application thereof, a dammarane-type triterpenoid saponin and a preparation method and application thereof, and a gynostemma pentaphyllum plant-derived fungicide. Background Art

[0002] Plant fungal diseases seriously affect the yield and quality of crops. Chemical fungicides are still the main means of preventing and controlling plant fungal diseases. Pesticide residues are still a common problem in international "green trade barriers". Therefore, it is imperative to find and develop new pesticides that are highly efficient, low-toxic and low-residue.

[0003] Plants are a natural treasure trove of bioactive compounds. At present, more than 4,000 plants with pesticide active ingredients have been discovered, of which more than 1,400 have fungicide activity. Compared with chemical pesticides, botanical fungicides developed using these active substances have the advantages of strong selectivity, relative safety to non-target organisms, biodegradability, and environmental friendliness. Although there are many plant species with pesticide activity, the research and development of the chemical composition and mechanism of action of these plant antibacterial active substances is still relatively lacking. At present, the active ingredients of botanical pesticides registered in my country for the prevention and control of plant anthracnose are only the alkaloid compound matrine, the coumarin compound osthole, and the terpene compound d-limonene. Therefore, the development of new botanical fungicides with high efficiency, low toxicity, and low residue from plant resources is of great significance for promoting the green and sustainable development of agriculture.

[0004] Gynostemmapentaphyllum (Thunb.) Makino is a perennial herbaceous climbing plant of the genus Gynostemma of the Cucurbitaceae family, also known as the second ginseng, immortal grass, etc. Gynostemma is a plant with both medicinal and edible properties. The whole plant can be used as medicine, and has multiple functions such as delaying aging, anti-cancer, improving brain function, enhancing human immunity, and regulating endocrine. Modern research shows that gypenosides are the main active ingredients in Gynostemma. Gynostemma saponins belong to dammarane-type triterpenoid saponins. At present, more than 200 gypenosides have been isolated from Gynostemma, and many active substances with neuroprotective, anti-tumor, hypoglycemic, and antibacterial effects have been found. At present, there are relatively few studies on the anti-plant pathogenic activity of dammarane triterpenoid saponins, and the research objects are mainly concentrated on plant extracts rich in dammarane triterpenoid saponins, protopanaxadiol ginsenoside compounds and protopanaxatriol ginsenoside compounds, but there is no relevant research on the anti-anthrax pathogenic bacteria activity of dammarane triterpenoid saponins. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a Gynostemma pentaphyllum extract and a preparation method and application thereof, a dammarane-type triterpenoid saponin and a preparation method and application thereof, and a Gynostemma pentaphyllum plant-derived fungicide. The Gynostemma pentaphyllum extract and dammarane-type triterpenoid saponin provided by the present invention have high anti-anthrax pathogenic bacteria activity.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a Gynostemma pentaphyllum extract, comprising an active substance, wherein the active substance comprises a dammarane-type triterpene saponin, and the dammarane-type triterpene saponin has one or more structures shown in Formula I, Formula II, Formula III and Formula IV:

[0008]

[0009] The present invention also provides a method for preparing the Gynostemma pentaphyllum extract of the above technical solution, comprising the following steps: mixing Gynostemma pentaphyllum with a lower alcohol extractant, performing alcohol extraction, and obtaining the Gynostemma pentaphyllum extract; the lower alcohol extractant is a lower alcohol or a mixed solvent of a lower alcohol and water; the lower alcohol includes methanol and / or ethanol; the volume fraction of the lower alcohol in the lower alcohol extractant is 70-100%;

[0010] Preferably, the ratio of the dry weight of Gynostemma pentaphyllum to the volume of the lower alcohol extractant is 1 g: 5 to 50 mL.

[0011] Preferably, the alcohol extraction includes one or more of reflux extraction, ultrasound-assisted extraction and room temperature cold soaking extraction; the number of alcohol extractions is 2 to 5 times; the time for a single reflux extraction is 0.5 to 5 hours; the time for a single ultrasound-assisted extraction is 0.5 to 5 hours; and the time for a single room temperature cold soaking extraction is 5 to 7 days.

[0012] The present invention also provides a dammarane-type triterpene saponin having a structure shown in Formula I, Formula II, Formula III or Formula IV:

[0013]

[0014] The present invention also provides a method for preparing the dammarane-type triterpenoid saponins described in the above technical solution, including method 1 and / or method 2;

[0015] The method 1 comprises the following steps:

[0016] The Gynostemma pentaphyllum extract is subjected to macroporous adsorption resin chromatography separation to obtain component A4; the Gynostemma pentaphyllum extract is the Gynostemma pentaphyllum extract described in the above technical solution or the Gynostemma pentaphyllum extract prepared by the preparation method described in the above technical solution, wherein the lower alcohol in the lower alcohol extractant is methanol; the eluent used in the macroporous adsorption resin chromatography separation includes methanol -Water, the volume fraction of methanol in the methanol-water is 0 to 100%;

[0017] The component A4 is subjected to silica gel column chromatography to obtain component FrA.6; the eluent used in the silica gel column chromatography separation includes chloroalkane-methanol, and the volume ratio of chloroalkane to methanol in the chloroalkane-methanol is 50:1 to 1:1;

[0018] The component FrA.6 is subjected to high performance liquid chromatography separation to obtain the dammarane-type triterpenoid saponin; the mobile phase used in the high performance liquid chromatography separation includes organic solvent-water, the organic solvent in the organic solvent-water includes methanol and / or acetonitrile, and the volume fraction of the organic solvent in the organic solvent-water is 60-90%.

[0019] The method 2 comprises the following steps:

[0020] The Gynostemma pentaphyllum extract is subjected to macroporous adsorption resin chromatography separation to obtain component B6; the Gynostemma pentaphyllum extract is the Gynostemma pentaphyllum extract described in the above technical solution or the Gynostemma pentaphyllum extract prepared by the preparation method described in the above technical solution, wherein the lower alcohol in the lower alcohol extractant is ethanol; the eluent used in the macroporous adsorption resin chromatography separation includes methanol - Water, the volume fraction of methanol in the methanol-water is 0 to 100%;

[0021] The component B4 is subjected to silica gel column chromatography to obtain component FrB.4; the eluent used in the silica gel column chromatography separation includes chloroalkane-methanol, and the volume ratio of chloroalkane to methanol in the chloroalkane-methanol is 50:1 to 1:1;

[0022] The component FrB.4 is separated by high performance liquid chromatography to obtain the dammarane-type triterpenoid saponin; the mobile phase used in the high performance liquid chromatography separation includes organic solvent-water, the organic solvent in the organic solvent-water includes methanol and / or acetonitrile, and the volume fraction of the organic solvent in the organic solvent-water is 60-90%

[0023] The present invention also provides the use of the Gynostemma pentaphyllum extract described in the above technical solution, the Gynostemma pentaphyllum extract prepared by the preparation method described in the above technical solution, or the dammarane-type triterpenoid saponin described in the above technical solution in the preparation of medicines.

[0024] Preferably, the medicine comprises a pesticide or a human medicine.

[0025] Preferably, the drug comprises a bactericide.

[0026] The present invention also provides a Gynostemma pentaphyllum plant-derived fungicide, comprising an active component and a pharmaceutically acceptable excipient; the active component comprises a Gynostemma pentaphyllum extract and / or a dammarane-type triterpenoid saponin; the Gynostemma pentaphyllum extract is the Gynostemma pentaphyllum extract described in the above technical solution or the Gynostemma pentaphyllum extract obtained by the preparation method described in the above technical solution; the dammarane-type triterpenoid saponin is the dammarane-type triterpenoid saponin described in the above technical solution.

[0027] The active substance in the Gynostemma pentaphyllum extract provided by the present invention includes one or more dammarane-type triterpenoid saponins having the structures shown in formula I, formula II, formula III and formula IV. The Gynostemma pentaphyllum extract provided by the present invention exhibits a significant anti-plant anthracnose pathogenic bacteria (Colletotrichum gloeosporioides (Penz.) Penz. & Sacc.) effect, and can be used as a potential botanical fungicide for preventing and controlling plant fungal diseases.

[0028] The dammarane-type triterpenoid saponins (Formula I, Formula II, Formula III and Formula IV) provided by the present invention exhibit significant anti-anthrax pathogenic bacteria activity, EC 50 The value is below 97 μg / mL, with significant anti-anthracnose pathogenic bacteria activity, and the antibacterial effect is better than the registered botanical pesticides matrine, d-limonene and spectral chemical fungicide chlorothalonil for the prevention and treatment of anthrax, and has the advantages of high safety, low toxicity and low residue, and not easy to produce drug resistance. The dammarane-type triterpenoid saponin provided by the present invention can be used as a botanical fungicide lead compound for the preparation of pesticides that meet the requirements of green prevention and control, which can improve the effect of pesticides and reduce the pollution of pesticides to the ecological environment. It is of great significance to improve the quality and safety of agricultural products and protect human health, and it is helpful to provide material guarantee and technical support for the research of botanical fungicides.

[0029] The preparation method of the gynostemma pentaphyllum extract provided by the invention has the advantages of simple operation, simple process, green environmental protection, low production cost and suitability for industrial production.

[0030] The preparation method of dammarane-type triterpenoid saponins provided by the present invention has the advantages of simple operation, simple process, green environmental protection, low production cost and suitability for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The effects of methanol extract of Gynostemma pentaphyllum (a) and ethanol extract of Gynostemma pentaphyllum (b) on the mycelial growth of anthrax pathogenic bacteria, wherein ① the concentration is 100 mg / mL, ② the concentration is 50 mg / mL, ③ the concentration is 25 mg / mL, and ④ is a negative control;

[0032] Figure 2The antibacterial effect diagram of compounds DT-12, DT-13, DT-14 and DT-16 on anthrax pathogenic bacteria, wherein A is the effect of the compound on the growth of anthrax pathogenic mycelium, B is the effect of the compound on the colony diameter, and C is the inhibition rate of the compound on mycelium growth;

[0033] Figure 3 The positive controls were the effects of matrine, d-limonene and thiophanate-methyl on the mycelial growth of anthracnose pathogenic bacteria;

[0034] Figure 4 This is a graph showing the efficacy of compound DT-14 in preventing and controlling postharvest anthracnose of apples (A1-A3), grapes (B1-B3) and tomatoes (C1-C3). DETAILED DESCRIPTION

[0035] The present invention provides a Gynostemma pentaphyllum extract, comprising an active substance, wherein the active substance comprises a dammarane-type triterpene saponin, and the dammarane-type triterpene saponin has one or more structures shown in Formula I, Formula II, Formula III and Formula IV:

[0036]

[0037] The present invention also provides a method for preparing the Gynostemma pentaphyllum extract described in the above technical solution, comprising the following steps: mixing Gynostemma pentaphyllum with a low-grade alcohol extractant, performing alcohol extraction, and obtaining the Gynostemma pentaphyllum extract.

[0038] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0039] In the present invention, the lower alcohol extractant is a lower alcohol or a mixed solvent of a lower alcohol and water; the lower alcohol includes methanol and / or ethanol; the volume fraction of the lower alcohol in the lower alcohol extractant is 70-100%, preferably 75-95%, more preferably 80-95%, further preferably 85-95%, and most preferably 90-95%.

[0040] In the present invention, the ratio of the dry weight of Gynostemma pentaphyllum to the volume of the lower alcohol extractant is preferably 1g:5-50mL, more preferably 1g:10-40mL, further preferably 1g:10-30mL, and further preferably 1g:10-20mL.

[0041] In the present invention, the Gynostemma pentaphyllum is preferably the dried aerial part of Gynostemma pentaphyllum, and the Gynostemma pentaphyllum is preferably cut into segments before use to obtain Gynostemma pentaphyllum segments, and the length of the Gynostemma pentaphyllum segments is preferably 2 to 4 cm.

[0042] In the present invention, the alcohol extraction preferably includes one or more of reflux extraction, ultrasound-assisted extraction and room temperature cold soaking extraction; the ultrasonic power of the ultrasound-assisted extraction is preferably 10-60KHz, more preferably 30-50KHz; the number of alcohol extractions is preferably 2-5 times, more preferably 3-4 times; the time for a single reflux extraction is preferably 0.5-5h, more preferably 1-4h, and more preferably 2-3h; the time for a single ultrasound-assisted extraction is preferably 0.5-5h, more preferably 1-4h, and more preferably 2-3h; the time for a single room temperature cold soaking extraction is preferably 5-7 days, more preferably 5.5-6.5 days, and more preferably 6 days. In the present invention, when the alcohol extraction is two or more of reflux extraction, ultrasound-assisted extraction and room temperature cold soaking extraction, the present invention has no special limitation on the order of different alcohol extraction methods, and any order is acceptable.

[0043] After completing the alcohol extraction, the present invention preferably further comprises subjecting the obtained alcohol extraction system to solid-liquid separation, concentrating the obtained liquid component to constant weight, and obtaining the Gynostemma pentaphyllum extract. The present invention has no special limitation on the solid-liquid separation, and may adopt a solid-liquid separation method well known to those skilled in the art, such as filtration, suction filtration or centrifugal separation. In the present invention, the concentration preferably comprises reduced pressure concentration, and the temperature of the reduced pressure concentration is preferably 40 to 50°C; the vacuum degree of the reduced pressure concentration is preferably -0.05 to -0.10MPa, more preferably -0.07MPa.

[0044] The present invention also provides a dammarane-type triterpene saponin having a structure shown in Formula I, Formula II, Formula III or Formula IV:

[0045]

[0046] The present invention also provides a method for preparing the dammarane-type triterpenoid saponins described in the above technical solution, comprising method 1 and / or method 2. In the present invention, the method 1 comprises the following steps:

[0047] The Gynostemma pentaphyllum extract (i.e., Gynostemma pentaphyllum methanol extract) is subjected to macroporous adsorption resin chromatography separation to obtain component A4; the Gynostemma pentaphyllum extract is the Gynostemma pentaphyllum extract described in the above technical solution or the Gynostemma pentaphyllum extract prepared by the preparation method described in the above technical solution, wherein the lower alcohol in the lower alcohol extractant is methanol; the eluent used in the macroporous adsorption resin chromatography separation includes methanol - Water, the volume fraction of methanol in the methanol-water is 0 to 100%;

[0048] The component A4 is subjected to silica gel column chromatography to obtain component FrA.6; the eluent used in the silica gel column chromatography separation includes chloroalkane-methanol, and the volume ratio of chloroalkane to methanol in the chloroalkane-methanol is 50:1 to 1:1;

[0049] The component FrA.6 is subjected to high performance liquid chromatography separation to obtain the dammarane-type triterpenoid saponin; the mobile phase used in the high performance liquid chromatography separation includes organic solvent-water, the organic solvent in the organic solvent-water includes methanol and / or acetonitrile, and the volume fraction of the organic solvent in the organic solvent-water is 60-90%.

[0050] The present invention performs macroporous adsorption resin chromatography separation on a Gynostemma pentaphyllum extract (i.e., a Gynostemma pentaphyllum methanol extract) to obtain component A4; the Gynostemma pentaphyllum extract is the Gynostemma pentaphyllum extract described in the above technical solution or the Gynostemma pentaphyllum extract obtained by the preparation method described in the above technical solution, wherein the lower alcohol in the lower alcohol extractant is methanol. In the present invention, the eluent used in the macroporous adsorption resin chromatography separation includes methanol-water; the volume fraction of methanol in the methanol-water is 0-100%. In the present invention, the elution mode of the macroporous adsorption resin chromatography separation is preferably gradient elution, and the eluent used in the gradient elution is preferably water, 40 vol% methanol-water, 80 vol% methanol-water and methanol in sequence. In the present invention, the macroporous adsorption resin used in the macroporous adsorption resin chromatography separation preferably includes D101 macroporous adsorption resin. In the present invention, the macroporous adsorption resin chromatography separation obtains 4 components, which are sequentially recorded as component A1, component A2, component A3 and component A4.

[0051] After obtaining component A4, the present invention performs silica gel column chromatography separation on the component A4 to obtain component FrA.6. In the present invention, the eluent used in the silica gel column chromatography separation includes chloroalkane-methanol; the chloroalkane in the chloroalkane-methanol preferably includes dichloromethane and / or chloroform, more preferably dichloromethane; the volume ratio of chloroalkane to methanol in the chloroalkane-methanol is 50:1 to 1:1, preferably 40:1 to 1:1, more preferably 30:1 to 1:1, and further preferably 20:1 to 1:1; the elution mode of the silica gel column chromatography separation is preferably gradient elution, and the eluent used in the gradient elution process is preferably chloroalkane-methanol with a volume ratio of 20:1, 10:1, 10:1, 8:1, 6:1, 4:1, 2:1 and 1:1, respectively. In the present invention, the particle size of the silica gel used in the silica gel column chromatography separation is preferably 200 to 300 meshes. In the present invention, the silica gel column chromatography separation obtains 9 components, which are respectively recorded as component FrA.1, component FrA.2, component FrA.3, component FrA.4, component FrA.5, component FrA.6, component FrA.7, component FrA.8 and component FrA.9.

[0052] After obtaining component FrA.6, the present invention performs high performance liquid chromatography separation on the component FrA.6 to obtain the dammarane-type triterpenoid saponin. In the present invention, the mobile phase used in the high performance liquid chromatography separation includes organic solvent-water, and the organic solvent in the organic solvent-water includes methanol and / or acetonitrile; the volume fraction of the organic solvent in the organic solvent-water is 60-90%, preferably 65-85%, more preferably 70-80%, and further preferably 70-75%; the high performance liquid chromatography separation is preferably preparative high performance liquid chromatography separation, and the chromatographic column used is preferably a reverse phase chromatography column, more preferably a Megres C18 reverse phase chromatography column or a PRC-ODS reverse phase chromatography column; the high performance liquid chromatography separation sequentially obtains dammarane-type triterpenoid saponins of structures shown in formula IV, formula II, formula III and formula I.

[0053] In the present invention, the method 2 comprises the following steps:

[0054] The Gynostemma pentaphyllum extract (i.e., the Gynostemma pentaphyllum ethanol extract) is subjected to macroporous adsorption resin chromatography separation to obtain component B6; the Gynostemma pentaphyllum extract is the Gynostemma pentaphyllum extract described in the above technical solution or the Gynostemma pentaphyllum extract prepared by the preparation method described in the above technical solution, wherein the lower alcohol in the lower alcohol extractant is ethanol; the eluent used in the macroporous adsorption resin chromatography separation includes methanol - Water, the volume fraction of methanol in the methanol-water is 0 to 100%;

[0055] The component B6 is subjected to silica gel column chromatography to obtain component FrB.4; the eluent used in the silica gel column chromatography separation includes chloroalkane-methanol, and the volume ratio of chloroalkane to methanol in the chloroalkane-methanol is 50:1 to 1:1;

[0056] The component FrB.4 is subjected to high performance liquid chromatography separation to obtain the dammarane-type triterpenoid saponin; the mobile phase used in the high performance liquid chromatography separation includes organic solvent-water, the organic solvent in the organic solvent-water includes methanol and / or acetonitrile, and the volume fraction of the organic solvent in the organic solvent-water is 60 to 90%.

[0057] The present invention performs macroporous adsorption resin chromatography separation on a Gynostemma pentaphyllum extract (i.e., a Gynostemma pentaphyllum ethanol extract) to obtain component B6; the Gynostemma pentaphyllum extract is the Gynostemma pentaphyllum extract described in the above technical solution or the Gynostemma pentaphyllum extract prepared by the preparation method described in the above technical solution, wherein the lower alcohol in the lower alcohol extractant is ethanol. In the present invention, the eluent used in the macroporous adsorption resin chromatography separation includes methanol. -Water, the volume fraction of methanol in the methanol-water is 0 to 100%. In the present invention, the elution mode of the macroporous adsorption resin chromatography separation is preferably gradient elution, and the eluents used in the gradient elution are preferably water, 40vol% methanol-water, 80vol% methanol-water and methanol in sequence. In the present invention, the macroporous adsorption resin used in the macroporous adsorption resin chromatography separation preferably includes D101 macroporous adsorption resin. In the present invention, the macroporous adsorption resin chromatography separation obtains 6 components, which are sequentially recorded as component B1, component B2, component B3, component B4, component B5 and component B6.

[0058] After obtaining component B6, the present invention performs silica gel column chromatography separation on the component B6 to obtain component FrB.4. In the present invention, the eluent used in the silica gel column chromatography separation includes chloroalkane-methanol, and the volume ratio of chloroalkane to methanol in the chloroalkane-methanol is 50:1 to 1:1. In the present invention, the eluent used in the silica gel column chromatography separation includes chloroalkane-methanol; the chloroalkane in the chloroalkane-methanol preferably includes dichloromethane and / or chloroform, more preferably dichloromethane; the volume ratio of chloroalkane to methanol in the chloroalkane-methanol is 50:1-1:1, preferably 40:1-1:1, more preferably 30:1-1:1, and further preferably 20:1-1:1; the elution mode of the silica gel column chromatography separation is preferably gradient elution, and the eluent used in the gradient elution process is preferably chloroalkane-methanol with a volume ratio of 20:1, 10:1, 10:1, 8:1, 6:1, 4:1, 2:1 and 1:1, respectively. In the present invention, the particle size of the silica gel used in the silica gel column chromatography separation is preferably 200-300 meshes. In the present invention, the silica gel column chromatography separation obtains 10 components, which are respectively recorded as component FrB.1, component FrB.2, component FrB.3, component FrB.4, component FrB.5, component FrB.6, component FrB.7, component FrB.8, component FrB.9 and component FrB.10.

[0059] After obtaining component FrB.4, the present invention performs high performance liquid chromatography separation on the component FrB.4 to obtain the dammarane-type triterpenoid saponin. In the present invention, the mobile phase used in the high performance liquid chromatography separation includes organic solvent-water, and the organic solvent in the organic solvent-water includes methanol and / or acetonitrile; the volume fraction of the organic solvent in the organic solvent-water is 60-90%, preferably 65-85%, more preferably 70-80%, and further preferably 70-75%; the high performance liquid chromatography separation is preferably preparative high performance liquid chromatography separation, and the chromatographic column used is preferably a reverse phase chromatography column, more preferably a Megres C18 reverse phase chromatography column or a PRC-ODS reverse phase chromatography column; the high performance liquid chromatography separation sequentially obtains dammarane-type triterpenoid saponins of structures shown in formula IV, formula II, formula III and formula I.

[0060] The present invention also provides the use of the Gynostemma pentaphyllum extract described in the above technical solution, the Gynostemma pentaphyllum extract prepared by the preparation method described in the above technical solution, or the dammarane-type triterpenoid saponin described in the above technical solution in the preparation of medicines. In the present invention, the medicine preferably includes pesticides or human medicines, and the medicine more preferably includes bactericides, and the bactericides preferably include bactericides for anthrax pathogens.

[0061] The present invention studies the anti-plant pathogenic activity of gypenosides for the first time, and finds dammarane-type triterpenoid saponins (Formulas I to IV) with antibacterial activity superior to that of existing registered botanical pesticides. The triterpenoid saponins can be used as lead compounds of botanical fungicides and can be used to prepare pesticides that meet green prevention and control requirements, which can improve the effect of pesticides and reduce pollution to the ecological environment. They are of great significance for improving the quality and safety of agricultural products and protecting human health, and at the same time, they play a good role in promoting the development of related industries.

[0062] The present invention provides a Gynostemma pentaphyllum botanical fungicide, comprising an active component and a pharmaceutically acceptable excipient; the active component comprises a Gynostemma pentaphyllum extract and / or a dammarane-type triterpenoid saponin; the Gynostemma pentaphyllum extract is the Gynostemma pentaphyllum extract described in the above technical solution and the Gynostemma pentaphyllum extract prepared by the preparation method described in the above technical solution; the dammarane-type triterpenoid saponin is the dammarane-type triterpenoid saponin described in the above technical solution; the dammarane-type triterpenoid saponin has one or more of the structures shown in formula I, formula II, formula III and formula IV. The present invention has no special restrictions on the pharmaceutically acceptable excipients, and pharmaceutically acceptable excipients well known to those skilled in the art can be used. The present invention has no special restrictions on the dosage form and preparation method of the Gynostemma pentaphyllum botanical fungicide, and dosage forms and preparation methods well known to those skilled in the art can be used.

[0063] To further illustrate the present invention, the Gynostemma pentaphyllum extract, preparation method and application thereof, dammarane-type triterpenoid saponins, preparation method and application thereof, and Gynostemma pentaphyllum plant-derived fungicide provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1

[0065] Preparation of methanol extract of Gynostemma pentaphyllum

[0066] The dried aerial part of Gynostemma pentaphyllum was cut into small pieces of 2 to 4 cm, and 95 vol% methanol-water was added, and the extraction was heated and refluxed for 3 times, and the extracts were combined and filtered, and the obtained filtrate was concentrated to constant weight under reduced pressure at 40-50°C and -0.07MPa vacuum conditions to obtain the methanol extract of Gynostemma pentaphyllum. The solid-liquid ratio of dry weight of Gynostemma pentaphyllum to methanol-water was 1g:10mL.

[0067] Example 2

[0068] Preparation of Gynostemma pentaphyllum ethanol extract

[0069] The dried aerial part of Gynostemma pentaphyllum was cut into small pieces of 2 to 4 cm, and 95 vol% ethanol-water was added, and the extraction was heated and refluxed for 3 times, and the extracts were combined and filtered, and the obtained filtrate was concentrated to constant weight under reduced pressure at 40 to 50°C and -0.07 MPa vacuum conditions to obtain a methanol extract of Gynostemma pentaphyllum. The solid-liquid ratio of Gynostemma pentaphyllum dry weight to ethanol-water was 1 g:10 mL.

[0070] Example 3

[0071] Isolation and structural identification of active substances from methanol extract of Gynostemma pentaphyllum

[0072] The methanol extract of Gynostemma pentaphyllum prepared in Example 1 was separated by chromatography using D101 macroporous adsorption resin, and gradient elution was performed using water, 40 vol% methanol-water, 80 vol% methanol-water and methanol as eluents, respectively, to obtain four components, which were recorded as A1 to A4.

[0073] Component A4 was separated by silica gel column chromatography (200-300 mesh silica gel) using gradient elution with dichloromethane-methanol in volume ratios of 20:1, 10:1, 10:1, 8:1, 6:1, 4:1, 2:1 and 1:1 to obtain 9 components, which were designated as FrA.1-FrA.9.

[0074] The component FrA.6 was separated by preparative high performance liquid chromatography using a Megres C18 reverse phase column and 72 vol% methanol-water as the mobile phase for isocratic elution. The retention time was 30 min to obtain compound DT-16 (Formula IV), the retention time was 40 min to obtain compound DT-13 (Formula II), the retention time was 45 min to obtain compound DT-14 (Formula III), and the retention time was 59 min to obtain compound DT-12 (Formula I).

[0075] The structures of compounds DT-16, DT-13, DT-14 and DT-12 were determined by nuclear magnetic resonance spectroscopy (NMR spectroscopy).

[0076] Compound DT-12: 1 H NMR(400MHz,MeOH-d4)δH:5.41(brs,H-1′),5.37(m,H-23),5.21(d,J=8.8Hz,H-24),4.46(d,J=7.5Hz,H-1′),4.45(d,J=7.5Hz,H -1″′),4.38(dd,J=12.0,2.0Hz,H-6′),4.23(dd,J=11.7,6.5Hz,H-6′),3.71(dd,J=9.6,3.1Hz,Hb-6′),3.14(dd,J=11.4,4.0Hz, H-3), 2.21 (dd, J = 13.3, 5.6 Hz, Ha-22), 2.06 (s, H3-Ac), 1.80 (s, H3-26), 1.78 (s, H3-27), 1.21 (d, J = 6.2 Hz, H3-6″′), 1.03 (s, H3-28), 1.00 (s, H3-18), 0.95 (s, H3-30), 0.89 (s, H3-19), 0.85 (s, H3-29), 0.78 (d, J = 11.1 Hz, H-5); The carbon spectrum data are shown in Table 1, which confirms that the compound DT-12 has the structure shown in Formula I.

[0077] Compound DT-13: 1H NMR(400MHz,MeOH-d4)δH:5.42(brs,H-1″),5.37(m,H-23),5.20(m,H-24),4.46(d,J=7.5Hz,H-1′),4.46( d, J=7.5Hz, H-1″′), 3.70 (dd, J=9.5, 3.3Hz, Hb-6′), 3.20 (dd, J=11.5, 3.9Hz, H-3), 2.21 (dd, J=13.3, 5.6H z, Ha-22), 1.80 (s, H3-26), 1.78 (s, H3-27), 1.21 (d, J = 6.1 Hz, H3-6″), 1.03 (s, H3-28), 1.00 (s, H3-18), 0.94 (s, H3-30), 0.89 (s, H3-19), 0.86 (s, H3-29), 0.79 (d, J = 10.3 Hz, H-5); The carbon spectrum data are shown in Table 1, which confirms that the compound DT-13 has the structure shown in Formula II.

[0078] Compound DT-14: 1 H NMR(400MHz,MeOH-d4)δH:5.41(brs,H-1″),5.31(d,J=8.8Hz,H-24),5.21(m,H-23),4.46(d,J=7.5Hz,H-1′),4.45(d,J=7.5Hz,H -1″′),4.38(dd,J=12.0,2.0Hz,Ha-6′),4.23(dd,J=11.7,6.5Hz,H-6′),3.72(dd,J=9.5,2.9Hz,Hb-6′),3.14(dd,J=11.4,4.1Hz, H-3), 2.52 (dd, J = 13.4, 7.0 Hz, Ha-22), 2.06 (s, H3-Ac), 1.78 (s, H3-26), 1.76 (s, H3-27), 1.21 (d, J = 6.1 Hz, H3-6″), 1.03 (s, H3-28), 1.03 (s, H3-18), 0.93 (s, H3-30), 0.89 (s, H3-19), 0.85 (s, H3-29), 0.78 (d, J = 10.9 Hz, H-5); The carbon spectrum data are shown in Table 1, which confirms that the compound DT-14 has the structure shown in Formula III.

[0079] Compound DT-16: 1H NMR(400MHz,MeOH-d4)δH:5.42(brs,H-1″),5.31(d,J=8.9Hz,H-24),5.21(m,H-23),4.46(d,J=7.4,H-1′), 4.45(d,J=7.4Hz,H-1″′),3.70(dd,J=9.5,3.0Hz,Hb-6′),3.20(dd,J=11.5,3.9Hz,H-3),2.52(dd,J=13.3,7 .0Hz, Ha-22), 1.78(s, H3-26), 1.76(s, H3-27), 1.21(d, J=6.2Hz, H3-6″), 1.03(s, H3-28), 1.02(s, H3-18), 0.93(s, H3-30), 0.89(s, H3-19), 0.86(s, H3-29), 0.79(d, J=9.6Hz, H-5); The carbon spectrum data are shown in Table 1, which confirms that the compound DT-16 has the structure shown in Formula IV.

[0080] Table 1 Compounds DT-12, DT-13, DT-14 and DT-16 13 C NMR data (100 MHz, MeOH-d 4)

[0081]

[0082]

[0083] Example 4

[0084] Separation of Active Substances from Gynostemma Pentaphyllum Ethanol Extract

[0085] The ethanol extract of Gynostemma pentaphyllum prepared in Example 2 was separated by chromatography using D101 macroporous adsorption resin, and gradient elution was performed using water, 20 vol% methanol-water, 40 vol% methanol-water, 60 vol% methanol-water, 80 vol% methanol-water and methanol as eluents, respectively, to obtain 6 components, which were recorded as B1 to B6.

[0086] Component B6 was separated by silica gel column chromatography (200-300 mesh silica gel) using gradient elution with dichloromethane-methanol in volume ratios of 20:1, 10:1, 10:1, 8:1, 6:1, 4:1, 2:1 and 1:1 to obtain 10 components, which were designated as FrB.1-FrB.10.

[0087] The component FrB.4 was separated by preparative high performance liquid chromatography, using a Megres C18 reverse phase column, and the mobile phase was 72 vol% methanol-water for isocratic elution, with a retention time of 30 min to obtain compound DT-16 (Formula IV), a retention time of 40 min to obtain compound DT-13 (Formula II), a retention time of 45 min to obtain compound DT-14 (Formula III), and a retention time of 59 min to obtain compound DT-12 (Formula I). ​​The hydrogen and carbon spectrum data of the compounds DT-16, DT-13, DT-14 and DT-12 prepared in this example are consistent with those in Example 3.

[0088] Test Example 1

[0089] The in vitro anti-plant pathogenic bacteria activity test of the Gynostemma pentaphyllum extract prepared in Examples 1-2.

[0090] Test strains: Anthrax pathogenic bacteria (C. gloeosporioides) were used as the test strains.

[0091] Activity test method: The Oxford cup method was used to study the anti-plant pathogenic activity of Gynostemma pentaphyllum methanol extract and Gynostemma pentaphyllum ethanol extract. Take 100 μL of the experimental fungal strain cultured to the logarithmic growth phase and spread it on potato dextrose agar medium (PDA medium). Use sterile tweezers to grab the sterile Oxford cup and place it in the agar medium coated with the bacterial solution. Pipette 200 μL of Gynostemma pentaphyllum extract solution (solvent is methanol) with concentrations of 100 mg / mL, 50 mg / mL and 25 mg / mL into the above Oxford cup, place the culture dish in a constant temperature incubator and culture at a suitable temperature for 5 days, and observe the antibacterial situation. Set up three parallels for each group, and use PDB medium without samples as the negative control.

[0092] Figure 1 The effect of Gynostemma pentaphyllum methanol extract (a) and Gynostemma pentaphyllum ethanol extract (b) on the mycelial growth of anthracnose pathogenic bacteria, wherein ① concentration is 100 mg / mL, ② concentration is 50 mg / mL, ③ concentration is 25 mg / mL, and ④ is a negative control. It can be seen that Gynostemma pentaphyllum methanol extract and Gynostemma pentaphyllum ethanol extract can significantly inhibit the mycelial growth of anthracnose pathogenic bacteria at a concentration of 25 mg / mL, showing anti-plant pathogenic bacteria activity.

[0093] Test Example 2

[0094] In vitro anti-plant pathogen activity test of compounds DT-16, DT-13, DT-14 and DT-12.

[0095] The mycelium growth rate method was used to determine the anti-anthracnose pathogenic bacteria activity of compounds DT-16, DT-13, DT-14 and DT-12. Compounds DT-16, DT-13, DT-14 and DT-12 were dissolved in methanol, and the resulting compound solutions were mixed with sterile molten PDA culture medium, respectively, so that the compound concentrations in the resulting mixtures were 12.5 mg / L, 50 mg / L, 100 mg / L and 200 mg / L, respectively. PDA containing the compound was poured into a 60 mm disposable culture dish (10 mL), and after solidification, a 6 mm hole was formed by a puncher, and a 6 mm bacterial cake of the experimental strain was inoculated. Each treatment group was paralleled three times, and PDA culture medium containing methanol at the corresponding concentration was used as a blank control. In addition, the active substances of plant-derived pesticides, matrine, d-limonene and chemical pesticide thiophanate-methyl were used as positive controls at concentrations of 50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L and 800 mg / L. The culture dish was placed in a constant temperature incubator at 28°C for 6 days and then the mycelial growth diameter was measured. The inhibition rate (I) was calculated according to formula (1). The half effective concentration (EC) of the compound was obtained by Graphpad-Prism software analysis. 50 ), the results are shown in Table 2 and Figures 2-3 . Figure 2 A is the effect of compounds DT-12, DT-13, DT-14 and DT-16 on the growth of anthrax pathogenic hyphae, B is the colony diameter, and C is the hyphae growth inhibition rate. Figure 3 The positive controls were matrine, d-limonene and thiophanate-methyl on the mycelial growth of anthracnose pathogenic bacteria.

[0096] I(%)=[(Cd)-(Td)] / (Cd)×100% Formula (1);

[0097] In formula (1), I is the inhibition rate (%); C is the average colony diameter of the control mycelium (mm); T is the average colony diameter of the treated mycelium (mm); d is the diameter of the initial bacterial cake (mm), d = 6 mm.

[0098] Table 2 EC values ​​of compounds DT-16, DT-13, DT-14 and DT-12 against anthrax pathogens 50 (μg / mL) value

[0099] Compound DT-12 DT-13 DT-14 DT-16 <![CDATA[EC 50 (μg / mL)]]> 38.48 61.59 27.50 96.98

[0100] It can be seen that the monomer compounds DT-12, DT-13, DT-14 and DT-16 isolated from Gynostemma pentaphyllum showed significant antibacterial activity against anthrax pathogens, EC 50 The values ​​were 38.48 μg / mL, 61.59 μg / mL, 27.50 μg / mL and 96.98 μg / mL respectively.

[0101] Compared with the positive control, the antibacterial activities of compounds DT-16, DT-13, DT-14 and DT-12 were superior to those of the active components of botanical pesticides, matrine, d-limonene and the chemical pesticide thiophanate-methyl.

[0102] Test Example 3

[0103] In vivo anti-plant pathogenic activity of compound DT-14

[0104] Tomatoes (Solanum lycopersicum Mill.), grapes (Vitis labruscaxvinifera'Shine Muscat'), and apples (Malus domestica Borkh.cv.RedFuji) of similar maturity and size were selected and soaked in 0.5wt% sodium hypochlorite aqueous solution for 1 min and then dried. Sterile stainless steel nails were used to punch holes along the equator of the fruit to form holes with a diameter of 3 mm and a depth of 3 mm. 10 μL of anthrax pathogenic bacteria spore suspension (bacterial concentration was 10 6 cfu / mL) were injected into the wound surface formed above, and naturally air-dried at room temperature to allow the spore suspension to be completely absorbed. Then 10 μL of antimicrobial active substance solution (compound DT-14, concentration of 0, 50 mg / L, 500 mg / L or 1000 mg / L, solvent is methanol) was injected and naturally air-dried at room temperature. The treated fruits were placed in a plastic basket, sealed with polyethylene cling film, and placed in a fresh-keeping cabinet (temperature 28°C, humidity 90%) for constant temperature and humidity culture for 6 days, 9 days and 12 days.

[0105] Figure 4 The control effect of compound DT-14 on postharvest anthracnose of apples (A1-A3), grapes (B1-B3) and tomatoes (C1-C3) is shown. It can be seen that compound DT-14 can effectively slow down or inhibit the development of anthracnose of apples and tomatoes at a concentration of 500 mg / L, and can effectively slow down or inhibit the development of anthracnose of tomatoes at a concentration of 1000 mg / L.

[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Gynostemma pentaphyllum extract, comprising an active substance, wherein the active substance comprises a dammarane-type triterpene saponin, wherein the dammarane-type triterpene saponin has one or more of the structures shown in Formula I, Formula II, Formula III and Formula IV:

2. The method for preparing the Gynostemma pentaphyllum extract according to claim 1, comprising the following steps: The gynostemma pentaphyllum is mixed with a lower alcohol extractant and subjected to alcohol extraction to obtain the gynostemma pentaphyllum extract; the lower alcohol extractant is a lower alcohol or a mixed solvent of lower alcohol and water; the lower alcohol includes methanol and / or ethanol; the volume fraction of the lower alcohol in the lower alcohol extractant is 70-100%.

3. The preparation method according to claim 2, characterized in that: The ratio of the dry weight of Gynostemma pentaphyllum to the volume of the lower alcohol extractant is 1g:5-50mL.

4. The preparation method according to claim 2 or 3, characterized in that: The alcohol extraction includes one or more of reflux extraction, ultrasound-assisted extraction and room temperature cold soaking extraction; the number of alcohol extractions is 2 to 5 times; the time for a single reflux extraction is 0.5 to 5 hours; the time for a single ultrasound-assisted extraction is 0.5 to 5 hours; and the time for a single room temperature cold soaking extraction is 5 to 7 days.

5. A dammarane-type triterpene saponin having a structure shown in Formula I, Formula II, Formula III or Formula IV:

6. The method for preparing the dammarane-type triterpenoid saponins according to claim 5, comprising method 1 and / or method 2; The method 1 comprises the following steps: The Gynostemma pentaphyllum extract is subjected to macroporous adsorption resin chromatography separation to obtain component A4; the Gynostemma pentaphyllum extract is the Gynostemma pentaphyllum extract according to claim 1 or the Gynostemma pentaphyllum extract prepared by the preparation method according to any one of claims 2 to 4, wherein the lower alcohol in the lower alcohol extractant is methanol; the eluent used in the macroporous adsorption resin chromatography separation includes methanol - Water, the volume fraction of methanol in the methanol-water is 0 to 100%; The component A4 is subjected to silica gel column chromatography to obtain component FrA.6; the eluent used in the silica gel column chromatography separation includes chloroalkane-methanol, and the volume ratio of chloroalkane to methanol in the chloroalkane-methanol is 50:1 to 1:1; The component FrA.6 is subjected to high performance liquid chromatography separation to obtain the dammarane-type triterpenoid saponin; the mobile phase used in the high performance liquid chromatography separation includes organic solvent-water, the organic solvent in the organic solvent-water includes methanol and / or acetonitrile, and the volume fraction of the organic solvent in the organic solvent-water is 60-90%. The method 2 comprises the following steps: The Gynostemma pentaphyllum extract is subjected to macroporous adsorption resin chromatography separation to obtain component B6; the Gynostemma pentaphyllum extract is the Gynostemma pentaphyllum extract according to claim 1 or the Gynostemma pentaphyllum extract prepared by the preparation method according to any one of claims 2 to 4, wherein the lower alcohol in the lower alcohol extractant is ethanol; the eluent used in the macroporous adsorption resin chromatography separation includes methanol - Water, the volume fraction of methanol in the methanol-water is 0 to 100%; The component B6 is subjected to silica gel column chromatography to obtain component FrB.4; the eluent used in the silica gel column chromatography separation includes chloroalkane-methanol, and the volume ratio of chloroalkane to methanol in the chloroalkane-methanol is 50:1 to 1:1; The component FrB.4 is subjected to high performance liquid chromatography separation to obtain the dammarane-type triterpenoid saponin; the mobile phase used in the high performance liquid chromatography separation includes organic solvent-water, the organic solvent in the organic solvent-water includes methanol and / or acetonitrile, and the volume fraction of the organic solvent in the organic solvent-water is 60 to 90%.

7. Use of the Gynostemma pentaphyllum extract according to claim 1, the Gynostemma pentaphyllum extract prepared by the preparation method according to any one of claims 2 to 4, or the dammarane-type triterpenoid saponin according to claim 5 in the preparation of medicines.

8. The use according to claim 8, characterized in that: The medicine includes pesticides or human medicines.

9. The use according to claim 8 or 9, characterized in that: The medicaments include antiseptics.

10. A Gynostemma pentaphyllum plant-derived fungicide, comprising an active component and a pharmaceutically acceptable excipient; the active component comprises a Gynostemma pentaphyllum extract and / or a dammarane-type triterpenoid saponin; the Gynostemma pentaphyllum extract is the Gynostemma pentaphyllum extract according to claim 1 or the Gynostemma pentaphyllum extract obtained by the preparation method according to any one of claims 2 to 4; the dammarane-type triterpenoid saponin is the dammarane-type triterpenoid saponin according to claim 5.

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