CN113367166A - Eugenia jambolana extract bactericide as well as preparation method and application thereof - Google Patents
Eugenia jambolana extract bactericide as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a syzygium portunuse extract bactericide and a preparation method and application thereof, the bactericide comprises an extract of stems and leaves of the syzygium portunuse, particularly a methanol crude extract and compounds 1, 2 and 3 obtained by separation and purification have the advantages of high efficiency, low toxicity, low residue, no pollution, low cost, strong bactericidal activity, wide antibacterial spectrum, simple separation process and the like, the bactericidal activity and the broad spectrum are higher than those of commercial plant-source bactericides carvacrol and eugenol, the bactericide has obvious control effects on banana anthracnose pathogen, cowpea powdery mildew and mango postharvest diseases, and can be used as a potential bactericide for plant fungal diseases.
Description
Technical Field
The invention relates to the field of agricultural sterilization, and particularly relates to a rose apple extract bactericide as well as a preparation method and application thereof.
Background
China is one of the world agricultural countries, crop diseases, insect pests and weeds have the characteristics of multiple varieties, great influence and frequent outbreak and disaster formation, and the occurrence range and the severity of the crop diseases, insect pests and weeds have serious influence on agricultural production. At present, plant diseases, insect pests and weeds are mainly controlled by chemistry, the problem of agricultural plant diseases and insect pests is relieved to a certain extent by using chemical pesticides, but due to long-term application and unreasonable use of the chemical pesticides, negative effects of ecological balance damage, serious environmental pollution, increased drug resistance of pests, over-standard pesticide residue of agricultural and sideline products, prominent food safety problem and the like are caused. Plant-derived pesticides have the characteristics of low toxicity, low residue, no pollution and the like, and people call for accelerating the development of the plant-derived pesticide industry and the application of control technology.
The plant can induce the plant to generate certain secondary metabolites with special biological activity in the long evolution process of combating pests and diseases, and the substances can resist the attack of certain pests and diseases to a certain extent and have the characteristics of safety to the environment, easy degradation, unique action mode, difficult generation of drug resistance, multiple varieties, multiple development ways, even promotion of crop growth, improvement of crop disease resistance and the like. The ancient books in Shanhai Jing, Qifol Yao Shu and Tian Gong Kai Yu have recorded the use of the whole plant product to prevent and control diseases, pests and weeds in agricultural fields. The metabolic products with biological activity are separated and screened from the plant extracts, the structures of the metabolic products are identified, the metabolic products are used as active precursors, the activity mechanism of the metabolic products is explored, and the metabolic products are important ways for creating new pesticides. However, some of these known natural products have low bactericidal activity or narrow target, do not have broad-spectrum bactericidal activity, and have complicated separation procedures and relatively high cost. Therefore, the market is urgently in need of finding and developing a series of cheap, environment-friendly, high-bactericidal-activity, wide-bactericidal-range and renewable natural products and preparation methods thereof.
The eighth major family of herbaceous plants is the Myrtaceae, and the herbaceous plants are about 140 genera and 3800-. Syzygium (Syzygium) is the 16 th largest flowering plant of the myrtaceae, with over 500 plants, mainly distributed in tropical asia, and a few in oceania and africa. About 74 kinds of Chinese plants are produced in the southern area of the Yangtze river, which are mostly seen in the two Guangdong provinces and the Yunnan province, the prunus plant contains abundant terpenoids, chalcone, flavonoid, lignanoid, alkyl phloroglucinol, hydrolyzed tannin, pigment derivatives and other secondary metabolites, and has the activities of resisting diabetes, fungi, inflammation, bacteria, oxidation, HIV, diarrhea, expelling insects, resisting viruses and the like. The most studied of the plants of the genus syzygium is syzygium jambos, which has been used as a traditional medicine for the treatment of various diseases such as dysentery, gastrointestinal diseases, diabetes, inflammation, hypertension, analgesia, antivirus, bronchitis, ulcer, etc. The syzygium jambos is also a common medicinal material in folks in the south of Ling, is recorded by the Chinese medicinal material standard of Guangdong province, is one of main components of Shaxi herbal tea, Shiqi exogenous tea and Tibetan medicine Rinqing mango in a compound prescription on the market, and has the effects of promoting blood circulation, removing blood stasis, relieving summer heat and the like. According to records in traditional Chinese medicine books such as Chinese materia medica and Chinese medicine dictionary, the syzygium jambos seeds can also check diarrhea and are mainly used for treating spleen deficiency diarrhea, chronic dysentery and diabetes. Journal literature "Alkyl phenyl pharmacological compositions from Syzygium levinei and the third Differentiation-Inducing Activity", authors J zuo, Y Mi, W Chen, Q Liu, J Wang, L Lou, W Zhao, from journal "Planta medical" disclose that the extracts of the leaves of Syzygium portentosus are useful in medical treatment, mainly as drugs in cancer cells and stem cells, but not as plant-derived bactericides.
Disclosure of Invention
Therefore, the invention provides a rose apple extract bactericide, a preparation method and application thereof to solve the problem of plant diseases.
The technical scheme of the invention is realized as follows: a rose apple extract bactericide is an extract of rose apple stems and leaves.
Preferably, the bactericide is a methanol crude extract or an organic phase extract of the stem and leaf of the syzygium portentosus.
Preferably, the extract of the stem and leaf of the syzygium portentosus contains at least one of bactericidal active ingredients of a compound 1, a compound 2 and a compound 3, wherein the chemical formula of the compound 1 is C13H19O4The chemical formula of the compound 2 is C15H21O4The chemical formula of the compound 3 is C17H23O4The structural formula is shown in the specification,
further, the preparation method of the rose apple extract bactericide comprises the steps of taking fresh rose apple stem and leaf, drying at 48-52 ℃, crushing to obtain rose apple powder, soaking the rose apple powder for 2-4 times by using a methanol solution with the volume fraction of 85-95% for 55-65 hours each time, wherein the mass-volume ratio kg/L of the rose apple powder to the methanol solution is 1.3-2.5: 25, combining soaking solutions, filtering, and concentrating under the vacuum condition of 50-60 ℃ to obtain the methanol crude extract of the rose apple stem and leaf.
Further, the preparation method of the prunus davidiana extract bactericide comprises the steps of dissolving the obtained methanol crude extract of the stem and leaf of the prunus davidiana in water, wherein the mass volume ratio kg/L of the methanol crude extract to the water is 0.8-1.2: 10 to obtain a methanol crude extract aqueous solution, extracting for 2-4 times by using an organic solvent to obtain an organic phase, wherein the volume ratio of the organic solvent to the methanol crude extract aqueous solution is 1.8-2.2: 1, and concentrating the organic phase at 40-50 ℃ under vacuum to obtain an organic phase extract, wherein the organic phase extract is at least one of a petroleum ether extract, an ethyl acetate extract or an n-butyl alcohol extract.
Further, the ketone compound bactericide comprises at least one of a compound 1, a compound 2 and a compound 3, wherein the chemical formula of the compound 1 is C13H19O4The chemical formula of the compound 2 is C15H21O4The chemical formula of the compound 3 is C17H23O4The structural formula is shown in the specification,
further, the preparation method of the bactericide comprises the following steps:
s1, preparing a methanol crude extract of the stem and leaf of the syzygium portentosus: taking fresh syzygium portunus trituberculatus stem and leaf, drying at 48-52 ℃, crushing to obtain syzygium portunus trituberculatus powder, soaking the syzygium portunus trituberculatus powder for 2-4 times by using a methanol solution with the volume fraction of 85-95%, wherein the mass-volume ratio kg/L of the syzygium portunus trituberculatus powder to the methanol solution is 1.3-2.5: 25 every time, combining soaking solutions, filtering, and concentrating under the vacuum condition of 50-60 ℃ to obtain a methanol crude extract of the stem and leaf of the syzygium portunus trituberculatus;
s2, chromatographic separation: performing column chromatography on the crude methanol extract obtained from the step S1 by using a petroleum ether or/and ethyl acetate solvent system, wherein the volume ratio of petroleum ether to ethyl acetate is 9:1, 5:1, 1:1 and 1:0, and obtaining 4 fractions, namely F1, F2, F3 and F4;
s3, elution: fraction F2 was eluted on Sephadex LH-20 gel column and detected by thin layer chromatography TLC with methanol as eluent to obtain 11 subfractions designated as Fr1, Fr2, Fr3, Fr4, Fr5, Fr6, Fr7, Fr8, Fr9, Fr10 and Fr11, and Fr3 was further purified by semi-preparative HPLC column using a volume ratio of 80: eluting with 20% methanol and ultrapure water as eluent, and separating to obtain compound 1, compound 2, and compound 3 with antibacterial activity, wherein the semi-preparative high performance liquid chromatography column has particle size of 5 μm and 10 × 200 mm.
Furthermore, the rose apple extract bactericide or the ketone compound bactericide is applied to preparation of a crop bactericide.
Further, the application of the syzygium fusceolatum extract bactericide comprises bacteriostasis of crop pathogenic fungi and prevention and treatment of crop diseases, wherein the pathogenic fungi comprise dragon fruit ulcer fungi, apple ulcer fungi, rice sheath blight fungi, rice blast fungi, tomato botrytis cinerea, rape sclerotinia or mango pedicel rot fungi, and the diseases comprise cucumber powdery mildew, cowpea powdery mildew, rice sheath blight, rice blast, banana anthracnose, mango anthracnose, tomato gray mold, rape sclerotinia, dragon fruit ulcer diseases or dragon fruit ulcer fungi.
Further, the pathogenic fungi comprise rhizoctonia solani, rice blast, banana colletotrichum, tomato botrytis cinerea, dragon fruit canker, sclerotinia sclerotiorum or phytophthora capsici, and the diseases comprise cowpea powdery mildew, banana colletotrichum and mango stem rot.
The invention provides an extraction method of a syzygium portentosus stem and leaf crude extract and an active ingredient separation method, wherein two separated active compounds are identified, the crude extract and the active compounds are used for carrying out in-vitro activity test on 22 common plant pathogenic fungi by a hypha growth rate method, and the living body protection effect of the crude extract and the active compounds is measured on cowpea powdery mildew, banana anthracnose and mango postharvest diseases.
The 22 common plant pathogenic fungi specifically comprise:
dragon fruit ulcer (neospora dimyritum);
apple ring lines (Botryosphaeria dothidea);
banana wilt (Fusarium oxysporum Schl);
wheat grain withering (Rhizoctonia cerealis);
banana anthrax (Colletotrichum musae);
colletotrichum gloeosporioides (Colletotrichum gloeosporioides);
gibberella zeae (Gibberella zeae);
pestalotiopsis guilinii (Pestalotiopsis guilinii);
wax gourd anthrax (Colletotrichum orbicular);
fusarium moniliforme (Fusarium moniliforme);
phytophthora nicotianae (phytophthora nicotiana);
alternaria solani (Alternaria solani);
botrytis cinerea (Botrytis cinerea);
pestalotiopsis longirostatica (Pestalotiopsis longirosta)
Sclerotium of colza (sclerotiotinia sclerotiorum)
Mango base rot (Botryodiploid ia the obroma)
Gibberella zeae (Gibberella zeae)
Rice sheath blight disease (Rhizoctonia solani)
Phytophthora capsici (Phytophthora capsicii)
Rice blast (Pyricularia oryzae)
Tomato gray mold (Botrytis cinerea)
Powdery mildew of cowpea (Podosphaera xanthontii).
Compared with the prior art, the invention has the beneficial effects that:
the syzygium portentosus stem extract, particularly the methanol crude extract, the petroleum ether extract phase extract and the compounds 1, 2 and 3 obtained by separation and purification have the advantages of high efficiency, low toxicity, low residue, no pollution, low cost, strong bactericidal activity, wide antibacterial spectrum, simple separation process and the like, the bactericidal activity and the broad spectrum of the syzygium portentosus stem extract are far higher than those of commercial plant-source bactericides carvacrol and eugenol, and the syzygium portentosus stem extract has special effects on banana anthracnose pathogen, cowpea powdery mildew and mango postharvest diseases, and can be used as a potential bactericide for plant fungal diseases.
The EC50 values of the compounds 1, 2 and 3 on banana colletotrichum are respectively 4.89 mu g/mL, 8.53 mu g/mL and 14.90 mu g/mL, wherein the inhibiting effect of the 3 compounds on banana colletotrichum is higher than that of carvacrol and eugenol.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Example 1 preparation of crude extract of leaf and leaf of Syzygium portulacea
Cutting fresh stem and leaf of Syzygium portulacea L.var.portulaca Maxim into pieces, drying at 48 ℃ by using an air blower drying box, crushing to obtain Syzygium portulaca Maxim powder, weighing 1.3kg of Syzygium portulaca Maxim powder, soaking for 2 times with 25L of 85% methanol solution with volume fraction, each time for 55h, combining soaking solutions, filtering, and concentrating by using a rotary evaporator under the vacuum condition of 50 ℃ to obtain the methanol crude extract of the stem and leaf of Syzygium portulaca Fortunei L.var.Maxim.
Example 2 preparation of crude extract of leaf and Stem of Syzygium portulacea
Cutting fresh stem and leaf of Syzygium portulacea L.var.portulaca Maxim into pieces, drying at 52 ℃ by using an air blower drying box, crushing to obtain Syzygium portulaca Maxim powder, weighing 2.5kg of Syzygium portulaca Maxim powder, soaking for 4 times with 25L of 95% methanol solution with volume fraction, 65 hours each time, combining soaking solutions, filtering, and concentrating by using a rotary evaporator under the vacuum condition of 60 ℃ to obtain the methanol crude extract of the stem and leaf of Syzygium portulaca Fortunei L.var.Maxim.
Example 3 preparation of crude extract of leaf and Stem of Syzygium portulacea
Cutting fresh stem and leaf of Syzygium portulacea L.var.portulaca Maxim into pieces, drying at 50 ℃ by using an air blower drying box, crushing to obtain Syzygium portulaca Maxim powder, weighing 1.9kg of Syzygium portulaca Maxim powder, soaking for 3 times by using 25L of 90% methanol solution with volume fraction for 60 hours each time, combining soaking solutions, filtering, and concentrating by using a rotary evaporator under the vacuum condition of 55 ℃ to obtain the methanol crude extract of the stem and leaf of Syzygium portulaca Fortunei L.var.Maxim.
(1) In vitro antifungal Activity test
And (3) detecting the bactericidal activity of the methanol crude extract by adopting a hypha growth rate method.
The crude methanol extracts of examples 1-3 were dissolved in DMF (0.1mL) respectively, and then mixed with potato dextrose agar (PDA; 9.9mL) to give the desired concentration. The crude extract was tested for activity at a concentration of 1000. mu.g/mL. All strains are cultured on PDA at 27 + -1 deg.C for 4-7 days to ensure the mycelium has strong activity, and can be used for identifying antifungal activity. The test was carried out on a sterile bench, the cake of about 5 mm in diameter was cut from the medium full of hyphae, inoculated with a sterile inoculating needle in the middle of a drug-treated PDA plate, and cultured at 27. + -. 1 ℃ for 4-7 days. An equal volume of DMF was added to sterile distilled water as a negative control, and three replicates were used for each treatment condition in examples 1-3. Fungal colony diameter length was measured. The inhibition rate was calculated using the following formula:
I(％)＝〔(C－T)/(C－0.5)〕×100
c: diameter of fungal growth on control PDA;
t: diameter of fungi on treated PDA;
i: the inhibition rate.
The results are shown in table 1 below.
TABLE 1 bacteriostatic activity of Syzygium cumini crude extract against 16 plant pathogenic fungi (1000. mu.g/mL)
As can be seen from the above table 1, the crude extract of Syzygium fuscata has bacteriostatic activity against 16 plant pathogenic fungi, while the bacteriostatic activity in example 3 is strong, which indicates that reasonable adjustment of the process in the extraction process improves the extraction rate to a certain extent, thereby improving the bacteriostatic activity.
(2) Field test
In the town of Fushan of Chengmei county, Hainan province, 10 months in 2019, the field test was divided into 3 treatments, each treatment comprising 3 test areas with an area of 100m2And/plot. The crude methanol extract obtained in example 3 is prepared into 5 mass percent of syzygium cumini methanol extract microemulsion, the experimental dosage per hectare is 375g/ha, a cell sprayed with clear water is used as a blank control, and registered bactericides Lunacsen (21.5 percent of trifloxystrobin and 21.5 percent of fluopyram SC) are used as positive controls.
The experiment adopts a spraying mode, and the spraying amount is 700 liters per hectare. The spraying is carried out for the first time at the early disease stage of the powdery mildew of the cucumbers, and the spraying is carried out for 2 times at intervals of 7 days. The disease condition and the control effect after 2 times of spraying are respectively observed. Implementing the grading standard of experimental diseases, calculating disease indexes and preventing and treating effects. 7 leaves were randomly selected for each treatment, 6 leaves per plant. The control effect of each treatment was investigated 6d and 7d after the first and second spraying, respectively. The incidence of all inoculated leaves was investigated and the percentage of lesion area per leaf to total leaf area was classified.
The classification criteria are as follows:
no disease spot at level 0;
grade 1, the lesion area is less than 5 percent of the whole leaf area;
grade 3, the lesion area accounts for 6 to 10 percent of the whole leaf area;
grade 5, the lesion area accounts for 11 to 20 percent of the whole leaf area;
7, the lesion area accounts for 21 to 40 percent of the whole leaf area;
the area of the 9-grade lesion spots accounts for more than 40% of the whole leaf area.
The results of the experiment are shown in Table 2 below
TABLE 25% field control effect of Syzygium cumini methanol extract microemulsion on cucumber powdery mildew
The detection shows that the 5% syzygium cumini methanol extract microemulsion has high bactericidal activity and high control effect on cucumber powdery mildew.
Example 4 preparation of the Petroleum Ether extract phase from the stems and leaves of Syzygium portulacea
Dissolving 100g of the crude methanol extract of the stem and leaf of syzygium portentosus obtained in example 3 in 500ml of ultrapure water to obtain an aqueous solution of the crude methanol extract, extracting the aqueous solution of the crude methanol extract with petroleum ether for 3 times, wherein the volume ratio of the aqueous solution of the petroleum ether to the aqueous solution of the crude methanol extract is 2:1 to obtain a petroleum ether phase, and concentrating the petroleum ether phase under a vacuum condition at 45 ℃ to obtain a petroleum ether extract.
Example 5 preparation of the Ethyl acetate extract phase of Syzygium portulacea Stem and leaf
100g of the crude methanol extract of the stem and leaf of Syzygium portulacea obtained in example 3 was dissolved in 500ml of ultrapure water to obtain an aqueous methanol extract, which was then extracted with ethyl acetate 3 times at a volume ratio of 2:1 to obtain an ethyl acetate phase, and then concentrated under vacuum at 45 ℃ to obtain an ethyl acetate extract.
Example 6 preparation of n-butanol extract phase of Syzygium portulacea Stem and leaf
Dissolving 100g of crude methanol extract of the stem and leaf of Syzygium portulacea obtained in example 3 in 500ml of ultrapure water to obtain an aqueous solution of the crude methanol extract, extracting the aqueous solution with n-butanol at a volume ratio of 2:1 for 3 times to obtain an n-butanol phase, and concentrating the n-butanol phase under vacuum at 45 ℃ to obtain an n-butanol extract.
(3) Bacteriostatic activity of each of the crude extracts of the syzygium cumini against 7 plant pathogenic fungi
And (3) carrying out the in-vitro antifungal activity test on each phase of the syzygium portentosus crude extract obtained in the example 3-6, wherein the treatment conditions of each example are repeated three times, and the diameter and the length of the fungal colony are measured. The inhibition rate was calculated using the following formula:
I(％)＝〔(C－T)/(C－0.5)〕×100
c: diameter of fungal growth on control PDA;
t: diameter of fungi on treated PDA;
i: the inhibition rate.
The results are shown in table 3 below.
TABLE 3 crude extracts of Syzygium fusceolatum each having 7 relative phytopathogenic fungi bacteriostatic activity (500. mu.g/mL)
aR.S. rice sheath blight disease; P.O blast; C.M banana anthracnose; B.C, tomato gray mold; N.D. dragon fruit canker; S.S. sclerotinia rot of colza; P.C Phytophthora capsici Leonian;beach treatment was 3 times and data are shown as mean ± SD.
As can be seen from Table 2, the methanol extract and each organic extract phase extract of the stem and leaf of Eugenia jambolana have inhibitory effects on hyphae, and the petroleum ether extract phase extract showed the highest bactericidal activity.
(4) Protective test
The petroleum ether extract obtained in example 4 was dissolved in 0.1ml of dmf, and mixed with tween 80 water (0.1% v/v) to prepare solutions of different concentrations for measuring the inhibitory effects on the symptoms of wilting of cowpea caused by powdery mildew of cowpea, banana rot caused by banana anthracnose, and mango postharvest disease. And (3) uniformly spraying 10mL of petroleum ether extract solutions with different concentrations on cowpea leaves, banana fruits and mango fruits, airing, and inoculating after treating for 24 hours. All spores were cultured on PDA at 27 + -1 deg.C for 4-7 days to ensure strong activity of spores for protective tests. Uniformly spraying the prepared spore suspension on the treated leaves or fruits, wherein the mangoes do not need to be treated, and the leaves and fruits are cultured under the conditions of 25 ℃,16 h illumination and 80% relative humidity. 14. After 8 and 20 days, the disease conditions of the cowpea leaves, the banana fruits and the mango fruits are respectively observed and graded.
The classification criteria are as follows:
grade 0, no disease;
grade 1, the lesion area is less than 5 percent of the whole area;
grade 3, the lesion area accounts for 6 to 10 percent of the whole area;
grade 5, the lesion area accounts for 11 to 20 percent of the whole area;
7, the lesion area accounts for 21 to 40 percent of the whole area;
grade 9, the lesion area accounts for more than 40% of the whole area.
Protective effect calculation method:
equal volumes of DMF were dissolved in sterile distilled water as negative Control (CR), propiconazole and azoxystrobin positive controls, repeated three times.
The results of the experiments are shown in tables 4,5 and 6 below.
TABLE 4 potted plant control of cowpea powdery mildew by petroleum ether phase extract
Each treatment was 3 times and data are shown as mean ± SD.
TABLE 5 Petroleum ether phase extract for in vivo prevention of mango pedicel rot
Each treatment was 3 times and data are shown as mean ± SD.
TABLE 6 in vivo control of banana anthracnose by petroleum ether phase extract
Each treatment was 3 times and data are shown as mean ± SD.
The test results show that the syzygium cumini petroleum ether extract has obvious control effect on banana anthracnose and mango stalk rot.
Example 7 preparation of Compounds 1, 2, 3 and measurement of fungicidal Activity
(1) Preparation of
S1, chromatographic separation: subjecting the crude methanol extract obtained in example 3 to column chromatography with a solvent system of petroleum ether and/or ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate is 9:1, 5:1, 1:1, 1:0, obtaining 4 fractions, which are marked as F1, F2, F3 and F4;
s2, elution: fraction F2 was eluted on Sephadex LH-20 gel column and detected by thin layer chromatography TLC with methanol as eluent to obtain 11 subfractions designated as Fr1, Fr2, Fr3, Fr4, Fr5, Fr6, Fr7, Fr8, Fr9, Fr10 and Fr11, and Fr3 was further purified by semi-preparative HPLC column using a volume ratio of 80: eluting with methanol and ultrapure water 20 as eluent, and separating to obtain compound 1, compound 2, and compound 3 with bactericidal activity, which are white powders.
(2) Structural identification
The structures of the prepared compounds 1, 2 and 3 were detected by hydrogen and carbon nuclear magnetic resonance spectroscopy, and the results are as follows.
Compound 1:
white powder, mp111.0 ℃. -111.0 ℃. (2,6-dihydroxy-4-methoxyphenyl) hexan-1-one (1).
1H NMR(400MHz,Chloroform-d)δ5.93(s,2H),3.78(s,3H),3.06(t,J＝7.5Hz,2H),1.75–1.63(m,2H),1.40–1.30(m,4H),0.95–0.83(m,3H).13C NMR(100MHz,Chloroform-d)δ206.5(d,J＝7.6Hz),165.5(d,J＝2.9Hz),163.4,104.9,94.4,55.5,44.0,31.7,24.5(d,J＝1.8Hz),22.6,14.0.HRMS(ESI):m/z calcd for C13H19O4[M+H]+239.1278,found:239.1223.
Compound 2:
white powder, mp 83.0 ℃ -84.0 ℃. (2,6-dihydroxy-4-methoxyphenyl) oct-5-en-1-one (2).1H NMR (400MHz, Chloroform-d) δ 5.94(s,2H), 5.44-5.29 (m,2H),3.77(s,3H),3.08(t, J ═ 7.5Hz,2H),2.12(q, J ═ 6.9Hz,2H), 2.07-1.98 (m,2H), 1.81-1.71 (m,2H),0.94(t, J ═ 7.5Hz,3H), 13C NMR (100MHz, Chloroform-d) δ 206.3,165.6,163.3,132.4,128.4,104.9,94.4,55.5,43.5,26.7,24.7,20.5, 20.4, 13C NMR (hresi/lcc) (C/C), c.5: (m,2H), (C, C5, 26.7,24.7,20.5, 20.4, esi, 14.5, C /) (C/C)15H21O4[M+H]+265.1435,found:265.1368.
Compound 3:
white powder, mp 73.0 deg.C
-74.0℃.(3E,7E)-1-(2,6-dihydroxy-4-methoxyphenyl)deca-3,7-dien-1-one(3).
1H NMR(400MHz,Chloroform-d)δ5.94(s,2H),5.77–5.67(m,1H),5.65–5.54(m,1H),5.43–5.26(m,2H),3.80(dd,J＝6.5,1.1Hz,2H),3.78(s,3H),2.11(t,J＝7.5Hz,4H),2.06–1.97(m,2H),0.94(t,J＝7.5Hz,3H).13C NMR(101MHz,Chloroform-d)δ204.1,165.8,163.4,134.1,132.1,128.3,123.1,104.7,94.4,55.5,47.5,32.8,26.9,20.6,14.3.HRMS(ESI):m/z calcd for C17H23O4[M+H]+291.1591,found:291.1517.
Analysis of the nuclear magnetic data confirmed Compound 1 to be of formula C13H19O4The chemical formula of the compound 2 is C15H21O4The chemical formula of the compound 3 is C17H23O4The structural formula III is shown as ketone compounds.
(3) In vitro antifungal Activity test
And (3) detecting the bactericidal activity of the compounds by adopting a hypha growth rate method.
The three compounds were dissolved in DMF (0.1mL) respectively, then mixed with potato dextrose agar (PDA; 9.9mL) to finally make the desired concentration, and activity test was performed. All strains are cultured on PDA at 27 + -1 deg.C for 4-7 days to ensure the mycelium has strong activity, and can be used for identifying antifungal activity. The test was carried out on a sterile bench, the cake of about 5 mm in diameter was cut from the medium full of hyphae, inoculated with a sterile inoculating needle in the middle of a drug-treated PDA plate, and cultured at 27. + -. 1 ℃ for 4-7 days. Equal volumes of DMF were added to sterile distilled water as negative controls and carvacrol and eugenol as positive controls, with three replicates per treatment condition. Fungal colony diameter length was measured. The inhibition rate was calculated using the following formula:
I(％)＝〔(C－T)/(C－0.5)〕×100
c: diameter of fungal growth on control PDA;
t: diameter of fungi on treated PDA;
i: the inhibition rate.
The results are shown in Table 7 below.
TABLE 7 bacteriostatic activity of Compounds 1-3 against 7 phytopathogenic fungi
aR.S. rice sheath blight disease; P.O blast; C.M banana anthracnose; B.C, tomato gray mold; N.D. dragon fruit canker; S.S. sclerotinia rot of colza; P.C Phytophthora capsici Leonian;beach treatment was 3 times and data are shown as mean ± SD.
Compared with 2 commercial natural products (carvacrol and eugenol), the compounds 1, 2 and 3 have higher bactericidal effect than commercial products and have wider spectrum.
(4) Protective test
The compounds 1-3 are respectively dissolved in 0.1ml DMF, and mixed with Tween 80 water (0.1% v/v) to prepare solutions with different concentrations, which are used for measuring the inhibition effect on cowpea wilt symptoms caused by cowpea powdery mildew, banana rot caused by banana anthracnose and mango postharvest diseases. And (3) uniformly spraying 10mL of the compound 1 solution on cowpea leaves, banana fruits and mango fruits, airing, and inoculating after treating for 24 h. All spores were cultured on PDA at 27 + -1 deg.C for 4-7 days to ensure strong activity of spores for protective tests. Uniformly spraying the prepared spore suspension on the treated leaves or fruits, wherein the mangoes do not need to be treated, and the leaves and fruits are cultured under the conditions of 25 ℃,16 h illumination and 80% relative humidity. 14. After 8 and 20 days, the infection degree is determined by measuring the diameter of the scab on the leaf, and the diameter of the scab used for calculation is the average value of the length of the longest diameter and the shortest diameter.
Protective effect calculation method: protective effect (%) - (negative control-treated leaf lesion diameter-drug-treated leaf lesion diameter)/negative control-treated leaf lesion diameter × 100.
Equal volumes of DMF dissolved in equal volumes of sterile water were used as negative Controls (CR), propiconazole and azoxystrobin positive controls, repeated three times.
The results of the experiments are shown in tables 8, 9 and 10 below.
TABLE 8 potted control of cowpea powdery mildew by compounds
Each treatment was 3 times and data are shown as mean ± SD.
TABLE 9 in vivo control of mango pedicel rot by compounds
Each treatment was 3 times and data are shown as mean ± SD.
TABLE 10 in vivo control of banana anthracnose with the compounds
Each treatment was 3 times and data are shown as mean ± SD.
From the test results, it can be seen that compound 1 has significant control effects on cowpea powdery mildew (table 8), banana anthracnose (table 10) and mango stem rot (table 9).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The rose apple extract bactericide is characterized in that: the bactericide is an extract of the stem and leaf of syzygium portentosus.
2. The Eugenia jambolana extract fungicide of claim 1, wherein: the bactericide is a methanol crude extract or an organic phase extract of the stem and leaf of the syzygium portentosus.
3. The Eugenia jambolana extract fungicide of claim 1, wherein: the extract of the stem and leaf of Eugenia jambolana contains at least one of compound 1, compound 2 and compound 3 as bactericidal active ingredient, which are ketone compounds, and the chemical formula of compound 1 is C13H19O4The chemical formula of the compound 2 is C15H21O4The chemical formula of the compound 3 is C17H23O4The structural formula is shown in the specification,
4. a preparation method of a rose apple extract bactericide is characterized by comprising the following steps: taking fresh syzygium portunus trituberculatus stem and leaf, drying at 48-52 ℃, crushing to obtain syzygium portunus trituberculatus powder, soaking the syzygium portunus trituberculatus powder for 2-4 times by using a methanol solution with the volume fraction of 85-95%, wherein the mass-volume ratio kg/L of the syzygium portunus trituberculatus powder to the methanol solution is 1.3-2.5: 25, combining soaking solutions, filtering, and concentrating under the vacuum condition of 50-60 ℃ to obtain a methanol crude extract of the stem and leaf of the syzygium portunus trituberculatus.
5. The method for preparing the rose apple extract bactericide as claimed in claim 4, wherein the rose apple extract bactericide is prepared by the following steps: dissolving the obtained crude methanol extract of the stem and leaf of the syzygium portentosus in water, wherein the mass volume ratio kg/L of the crude methanol extract to the water is 0.8-1.2: 10, obtaining an aqueous solution of the crude methanol extract, extracting for 2-4 times by using an organic solvent to obtain an organic phase, wherein the volume ratio of the organic solvent to the aqueous solution of the crude methanol extract is 1.8-2.2: 1, and concentrating the organic phase at 40-50 ℃ under a vacuum condition to obtain an organic phase extract, wherein the organic phase extract is at least one of a petroleum ether extract, an ethyl acetate extract or an n-butyl alcohol extract.
6. A ketone compound bactericide is characterized in that: what is needed isThe bactericide contains at least one of compound 1, compound 2 and compound 3, wherein the chemical formula of the compound 1 is C13H19O4The chemical formula of the compound 2 is C15H21O4The chemical formula of the compound 3 is C17H23O4The structural formula is shown in the specification,
7. the method for producing a bactericide as claimed in claim 6, wherein: the method comprises the following steps:
s1, preparing a methanol crude extract of the stem and leaf of the syzygium portentosus: taking fresh syzygium portunus trituberculatus stem and leaf, drying at 48-52 ℃, crushing to obtain syzygium portunus trituberculatus powder, soaking the syzygium portunus trituberculatus powder for 2-4 times by using a methanol solution with the volume fraction of 85-95%, wherein the mass-volume ratio kg/L of the syzygium portunus trituberculatus powder to the methanol solution is 1.3-2.5: 25 every time, combining soaking solutions, filtering, and concentrating under the vacuum condition of 50-60 ℃ to obtain a methanol crude extract of the stem and leaf of the syzygium portunus trituberculatus;
s2, chromatographic separation: performing column chromatography on the crude methanol extract obtained from the step S1 by using a petroleum ether or/and ethyl acetate solvent system, wherein the volume ratio of petroleum ether to ethyl acetate is 9:1, 5:1, 1:1 and 1:0, and obtaining 4 fractions, namely F1, F2, F3 and F4;
s3, elution: and (2) eluting fraction F2 by using a Sephadex LH-20 gel column and using methanol as an eluent to obtain 11 sub-fractions which are marked as Fr1, Fr2, Fr3, Fr4, Fr5, Fr6, Fr7, Fr8, Fr9, Fr10 and Fr11, and then passing Fr3 through a semi-preparative high performance liquid chromatography column at a volume ratio of 70-90: eluting with methanol and ultrapure water 20 as eluent, and separating and purifying to obtain compound 1, compound 2 and compound 3 with bactericidal activity.
8. The rose apple extract bactericide as claimed in any one of claims 1 to 5 or the ketone compound bactericide as claimed in any one of claims 6 to 7 is used for preparing a crop bactericide.
9. The use of claim 8, wherein: the application of the syzygium fusceolatum extract bactericide comprises bacteriostasis of crop pathogenic fungi and prevention and control of crop diseases, wherein the pathogenic fungi comprise dragon fruit canker, apple ring rot fungi, rice sheath blight fungi, banana anthracnose, rape sclerotium, mango stem rot fungi, rice blast fungi or tomato botrytis cinerea, and the diseases comprise cucumber powdery mildew, cowpea powdery mildew, banana anthracnose and mango stem rot.
10. The use of claim 8, wherein: the ketone compound bactericide comprises bacteriostasis of crop pathogenic fungi and prevention and control of crop diseases, wherein the pathogenic fungi comprise rhizoctonia solani, magnaporthe oryzae, banana colletotrichum, botrytis cinerea, dragon fruit ulcer fungi, sclerotinia sclerotiorum or phytophthora capsici, and the diseases comprise cowpea powdery mildew, banana colletotrichum and mango base rot.
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| CN1210469A (en) * | 1996-02-06 | 1999-03-10 | 马达加斯加应用研究所 | Mixtures derived from eugenia jambolana lamarck, preparation and use of said mixtures and some of their constituents as medicaments |
| WO2010143061A1 (en) * | 2009-06-12 | 2010-12-16 | Generex Pharmaceuticals, Inc. | Composition used to prevent and treat red blood cell coagulation |
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