阅读说明：本技术 一种植物来源的苦木素及其制备方法和应用 (Plant-derived quassin and preparation method and application thereof ) 是由 陈建军 高坤 岳建民 于 2021-01-11 设计创作，主要内容包括：本发明公开了一个植物来源的苦木素及其制备方法和应用。该苦木素的制备方法如下：将常绿苦树叶子用乙醇浸泡提取,减压浓缩得粗浸膏；该提取物用大孔树脂、硅胶、凝胶及半制备的高效液相色谱柱分离,即得该化合物。该化合物对水稻纹枯病菌、西瓜尖孢镰刀菌、禾谷镰刀菌、柑桔青霉、黄瓜疫霉菌等具有明显的抑制活性。因此,该化合物可作为一种新型的植物源抗菌候选药物。(The invention discloses a plant source quassin and a preparation method and application thereof. The preparation method of the quassin comprises the following steps: soaking and extracting evergreen bitter tree leaves with ethanol, and concentrating under reduced pressure to obtain crude extract; separating the extract with macroporous resin, silica gel, gel and semi-prepared high performance liquid chromatography column to obtain the compound. The compound has obvious inhibition activity on rhizoctonia solani, fusarium oxysporum, fusarium graminearum, penicillium citrinum, phytophthora cucumis and the like. Therefore, the compound can be used as a novel botanical antibacterial candidate drug.)

1. A preparation method of plant-derived antibacterial drugs is characterized in that main components are plant-derived quassin as candidate molecules, and the structural formula of the quassin is as follows:

2. the method of claim 1, wherein the plant-derived quassin is obtained from the leaves, stems, bark, or roots of Picrasma viridis.

3. The preparation method of the botanical antibacterial drug according to claim 1, wherein the preparation method comprises the following steps: soaking the leaves of evergreen tape tree in ethanol, and concentrating under reduced pressure to obtain crude extract; separating the extract with macroporous resin, silica gel, gel and semi-prepared high performance liquid chromatography column to obtain the compound.

4. The method for preparing a botanical antibacterial drug according to claim 3, wherein the preparation method comprises the following steps:

s1, crushing the leaves of the evergreen tape tree, and soaking the leaves in ethanol (95%) for 3 times, 7 days each time, to obtain the leachate.

S2, distilling the leachate under reduced pressure to remove the solvent, thereby obtaining a crude extract.

S3, dissolving the crude extract in hot water at 50 ℃, and extracting with ethyl acetate to obtain an ethyl acetate extract.

S4, separating the ethyl acetate extract by using macroporous resin, performing gradient elution by using 30%, 50%, 80% and 95% ethanol as mobile phases respectively, and finally obtaining 4 components Fre-1-Fre-4 by TLC analysis.

S5, separating Fre-2 by normal phase silica gel column chromatography, performing gradient elution by using a mixed solvent of petroleum ether/acetone as a mobile phase, sequentially performing TLC detection on the mixed solvent in a volume ratio of 50:1, 20:1, 10:1, 5:1, 2:1 and 1:1, and combining similar samples to obtain 7 components Fre-2.1-Fre-2.7.

S6, separating Fre-2.4 by Sephadex LH-20, performing isocratic elution by using methanol/chloroform as a mobile phase at a ratio of 1:1, then performing separation by normal-phase silica gel column chromatography, performing gradient elution by using a chloroform/methanol mixed solvent as a mobile phase at a volume ratio of 20:1, 10:1, 5:1, 2:1 and 1:1 in sequence, and combining similar samples by TLC detection to obtain 4 fractions Fre-2.4.1-Fre-2.4.4.

S7, separating Fre-2.4.4 with Sephadex LH-20, isocratically eluting with methanol/chloroform (1: 1) as mobile phase, purifying with high performance liquid chromatography (2.0 mL/min) using methanol/water (43: 57) as mobile phase to obtain target compound, and separating with Sephadex LH-20 to obtain fraction_R＝35.4min。

5. The method as claimed in claim 4, wherein the semi-preparative liquid chromatography in S7 is performed by using Waters 1525 as a liquid chromatograph, 2mL/min as a mobile phase, 2 nm/min as a detector, 200-400nm as a detection wavelength, and 10X 250mm as a chromatographic column, which is a Waters Sunfire C18 semi-preparative column.

6. Use of an antibacterial agent of plant origin, obtained by the method according to claim 1, for inhibiting the action of phytopathogenic fungi and for preparing an antibacterial agent.

7. A plant-derived quassin compound characterized by: the formula is Pj-1 in claim 1.

Technical Field

The invention belongs to the technical field of biochemical engineering, and particularly relates to plant-derived quassin, and a preparation method and application thereof.

Background

The pesticide is an indispensable production data in agricultural production and agricultural economic development, and is a major strategic substance related to global grain safety, food safety and ecological safety. The method has considerable effects on preventing and controlling crop diseases, insect pests and weeds, ensuring the yield and quality of agricultural products, ensuring food safety and the like. According to statistics, the use of the pesticide can recover 30 to 40 percent of the loss of the total crop production in the world every year. The ecological conditions of China are complex, the cultivation system is various, and the ecological environment-friendly cultivation method belongs to the countries with multiple pests, frequent pests and repeated pests. The total planting area of crops suffering from diseases, insect pests and weeds in China is up to 12190 million hectares every year. Therefore, in the past decades, chemical pesticides play an important role in controlling crop diseases, pests and weeds, guaranteeing and improving the yield and quality of grains, and guaranteeing food safety.

However, long-term unreasonable use of chemical pesticides causes a series of serious environmental and social problems, such as serious soil hardening acidification, enhanced drug resistance, serious exceeding of pesticide residue, ecological balance destruction, environmental pollution and other hidden dangers affecting human health. Particularly, the 'green technology barrier' caused by pesticide residues becomes a prominent problem for limiting the export trade of agricultural products in China at present. Therefore, the development of green pesticides with high biological activity, high selectivity, low residue, easy degradation and no pollution is urgent. The botanical pesticide has the advantages of natural active ingredients, no toxicity, no environmental pollution, difficult generation of drug resistance, safety to beneficial organisms and the like, and is an important research direction of green pesticides.

The botanical pesticides can be divided into: botanical insecticides, botanical fungicides, botanical antivirals, botanical herbicides, and the like. At present, botanical insecticides are a class of botanical pesticides which are relatively successfully researched, and mature commodities are sold on the market and applied to agricultural production. Such as azadirachtin, saponin nicotine soluble emulsion, rotenone missible oil, bisultrine aqua, fennel artemisin aqua, oleic acid nicotine, etc. However, statistics indicate that about 85% of plant diseases are caused by fungi, such as sclerotinia, fusarium, pseudosporium, brucella, phytophthora, pythium, and the like. Compared with botanical insecticides, the research on botanical fungicides is much less, and most research is limited to the antibacterial activity of plant extracts, and a botanical antibacterial agent really used in agricultural production is not available. Therefore, research and development of novel plant-derived antibacterial agents are urgently needed.

Evergreen quassia (Picrasmajavanica) is an evergreen tree of quassia of quassiaceae, and is mainly distributed in tropical regions of asia, such as indonesia, india and burma. The method is widely distributed in the places of Xishuangbanna, Jingdong and the like in China. In folk, the decoction of the bark of the Chinese medicinal herb can be used as a substitute of quinine for treating malaria. The approximate amount of the tree leaves in Java and other places is also used for treating wounds by squeezing juice. The secondary metabolite of the plant is reported in literature to be mainly a quassin compound and has the activities of resisting tumor, inflammation, virus, neuroprotection and the like. However, the activity of the quassinoid against plant pathogenic fungi is not reported.

Disclosure of Invention

One of the purposes of the present invention is to provide a novel plant-derived quassin compound Pj-1, the structural formula of which is shown as the following formula:

the second purpose of the invention is to provide a method for preparing a novel quassin compound Pj-1, which is characterized in that the compound is obtained from leaves of evergreen quassia, and specifically, the leaves of evergreen quassia are crushed and soaked by ethanol (95%) to obtain leachate, the leachate is decompressed and distilled to remove a solvent to obtain a crude extract, the crude extract is dissolved in hot water at 50 ℃, then ethyl acetate is used for extraction to obtain an ethyl acetate extract, and then macroporous resin, silica gel, gel and semi-prepared high performance liquid chromatography columns are used for separation to obtain the compound. The method comprises the following steps:

s1, crushing the leaves of the evergreen tape tree, and soaking the leaves in ethanol (95%) for 3 times, 7 days each time, to obtain the leachate.

S2, distilling the leachate under reduced pressure to remove the solvent, thereby obtaining a crude extract.

S3, dissolving the crude extract in hot water at 50 ℃, and extracting with ethyl acetate to obtain an ethyl acetate extract.

S4, separating the ethyl acetate extract by using macroporous resin, performing gradient elution by using 30%, 50%, 80% and 95% ethanol as mobile phases respectively, and finally obtaining 4 components Fre-1-Fre-4 by TLC analysis.

S5, separating Fre-2 by normal phase silica gel column chromatography, performing gradient elution by using a mixed solvent of petroleum ether/acetone as a mobile phase, sequentially performing TLC detection on the mixed solvent in a volume ratio of 50:1, 20:1, 10:1, 5:1, 2:1 and 1:1, and combining similar samples to obtain 7 components Fre-2.1-Fre-2.7.

S6, separating Fre-2.4 by Sephadex LH-20, performing isocratic elution by using methanol/chloroform as a mobile phase at a ratio of 1:1, then performing separation by normal-phase silica gel column chromatography, performing gradient elution by using a chloroform/methanol mixed solvent as a mobile phase at a volume ratio of 20:1, 10:1, 5:1, 2:1 and 1:1 in sequence, and combining similar samples by TLC detection to obtain 4 fractions Fre-2.4.1-Fre-2.4.4.

S7, Fre-2.4.4 using Sephadex LH-20 separation, methanol/chloroform 1:1 as mobile phase for isocratic elution, then using high performance liquid chromatography for purification, methanol/water 43:57 as mobile phase for isocratic elution, the flow rate is 2.0 mL/min, get the target compound Pj-1, t_R＝35.4min。

The liquid chromatograph of the semi-preparative liquid chromatogram in the S7 is Waters 1525, the mobile phase is methanol-water, the flow rate is 2mL/min, the detector is a Waters 2998 photodiode array detector, the detection wavelength is 200-400nm, and the chromatographic column is a Waters Sunfire C18 semi-preparative column with the specification of 10 multiplied by 250 mm.

The invention also aims to provide the compound Pj-1 with good effect of inhibiting plant pathogenic fungi and application in preparing antibacterial drugs.

The invention has the beneficial effects that: a quassinoid is isolated from the leaves of the evergreen quassia (Picrasmajavanica). The compound has obvious inhibition activity on Rhizoctonia solani (Rhizoctonia solani), Fusarium oxysporum (Fusarium oxysporum f.sp.niveum), Fusarium graminearum (Fusarium graminearum), Penicillium citrinum (Penicillium citrinum), Phytophthora cucumerinum (Phytophthora melonis) and the like. Therefore, the compound can be used as a novel plant source antibacterial candidate drug and provides scientific basis for reasonable development and utilization of evergreen bitter tree resources.

Drawings

FIG. 1 is a drawing of compound Pj-1¹HNMR spectra;

FIG. 2 is a drawing of compound Pj-1¹³A CNMR spectrum;

FIG. 3 is an HMBC spectrum of compound Pj-1;

FIG. 4 is an IR spectrum of the compound Pj-1;

FIG. 5 is an HRESIMS spectrum of compound Pj-1;

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples.

In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.

Example 1:

the compound Pj-1 is separated and extracted from the leaves of evergreen picrasma quassioides (Picrasmajavanica), and the extraction process is as follows:

crushing 10kg of evergreen picrasma quassioides leaves collected from Xishuangbanna, soaking and extracting the crushed leaves with 95% ethanol at room temperature for 3 times, each time for 7 days, concentrating the crushed leaves under reduced pressure to obtain 380g of total extract, dissolving the total extract in hot water of 500 ℃, and extracting the total extract with ethyl acetate to obtain an ethyl acetate extract. Separating the ethyl acetate extract with macroporous resin, gradient eluting with 30%, 50%, 80%, and 95% ethanol as mobile phase, and analyzing by TLC to obtain 4 fractions Fre-1-Fre-4. Separating Fre-2 by normal phase silica gel column chromatography, gradient eluting with petroleum ether/acetone mixed solvent as mobile phase at volume ratio of 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, detecting by TLC, and combining similar samples to obtain 7 components Fre-2.1-Fre-2.7. Separating Fre-2.4 with Sephadex LH-20, isocratically eluting with methanol/chloroform at a ratio of 1:1 as mobile phase, separating with normal phase silica gel column chromatography, and gradient eluting with chloroform/methanol mixed solvent as mobile phaseThe volume ratio is 20:1, 10:1, 5:1, 2:1 and 1:1 in sequence, similar samples are combined by TLC detection, and 4 components Fre-2.4.1-Fre-2.4.4 are obtained. Separating Fre-2.4.4 with Sephadex LH-20, isocratically eluting with methanol/chloroform (1: 1) as mobile phase, purifying with high performance liquid chromatography, isocratically eluting with methanol/water (43: 57) as mobile phase at flow rate of 2.0 mL/min to obtain target compound, and separating with Sephadex LH-20 to obtain fraction_R35.4 min. The compound was identified as Pj-1 by integrated spectroscopic analysis.

Example 2

The plant-derived quassin compound can also be obtained from leaf, stem, bark, root, etc. of evergreen quassia. Meanwhile, other lignin molecules with antibacterial effect, namely the compounds Pj-2-Pj-5 can be provided, but the experimental comparison shows that Pj-1 is the optimized compound, and the structural formula of the compounds Pj-2-Pj-5 is as follows:

other compounds were prepared by the following method:

crushing 10kg of evergreen bitter tree stems (or bark, root and leaves are the most preferred) collected from Xishuangbanna, soaking and extracting with 95% ethanol at room temperature for 3 times, each time for 7 days, concentrating under reduced pressure to obtain 380g of total extract, dissolving in hot water of 50 ℃, and extracting with ethyl acetate to obtain an ethyl acetate extract. Separating the ethyl acetate extract with macroporous resin, gradient eluting with 30%, 50%, 80%, and 95% ethanol as mobile phase, and analyzing by TLC to obtain 4 fractions Fre-1-Fre-4. Separating Fre-2 by normal phase silica gel column chromatography, gradient eluting with petroleum ether/acetone mixed solvent as mobile phase at volume ratio of 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, detecting by TLC, and combining similar samples to obtain 7 components Fre-2.1-Fre-2.7. Separating Fre-2.4 with Sephadex LH-20, isocratically eluting with methanol/chloroform 1:1 as mobile phase, separating with normal phase silica gel column chromatography, and separating with chloroform/methanol mixed solventGradient elution is carried out on the mobile phase, the volume ratio is 20:1, 10:1, 5:1, 2:1 and 1:1 in sequence, similar samples are combined through TLC detection, and 4 components Fre-2.4.1-Fre-2.4.4 are obtained. Separating Fre-2.4.4 with Sephadex LH-20, isocratically eluting with methanol/chloroform (1: 1) as mobile phase, purifying with high performance liquid chromatography, isocratically eluting with methanol/water (43: 57) as mobile phase at flow rate of 2.0 mL/min to obtain target compound Pj-1, t_R35.4 min. Fre-2.4.2 was purified by high performance liquid chromatography, and the product was isocratically eluted with methanol/water 35:65 as a mobile phase at a flow rate of 2.0 mL/min to give Pj-3 (t: -3) as a target compound_R42.1min) and Pj-4 (t)_R41.2 min). Fre-2.4.3 was purified by high performance liquid chromatography, and was isocratically eluted with methanol/water 40:60 as a mobile phase at a flow rate of 2.0 mL/min to give Pj-2 (t/min) as the target compound_R35.4min) and Pj-5 (t)_R37.32 min). The structure of the compound is determined by comprehensive spectrum analysis.

Example 3 Activity test

And (3) carrying out in-vitro antifungal activity detection on the target compound by adopting a mycelium growth inhibition method. The specific method comprises the following steps: the 5 plant pathogenic fungi used for the test were inoculated on Potato Dextrose Agar (PDA) and cultured in an incubator at 28 ℃ for 4 days to obtain a vigorously growing hypha. Respectively dissolving the compound Pj-1-Pj-5 and carbendazim (positive control) in DMSO-H₂O (v: v ═ 1:1), and this was mixed with Potato Dextrose Agar (PDA) medium to obtain a mixture of 50 μ g/mL. The above mixture was poured into 6cm sterile petri dishes, and 6mL of each dish was poured to prepare a PDA plate containing the test compound Pj-1-Pj-5 or carbendazim. The cultured hyphae of each pathogen were cut into small pieces of 5mm in diameter and placed in the center of each PDA plate containing the test compound Pj-1-Pj-5 or carbendazim, while 5mm different hyphae small pieces were also placed in the center of the PDA plate without any drug as blank controls, with three replicates per experiment. All the hypha-containing PDA plates were incubated in an incubator at 28 ℃ for 48 hours. Adding test compound Pj-1-Pj-5 or more bacteriaIn the respective plates of the treated (T) and blank control (C), the diameter (mm) of each colony was measured by the cross method, and the inhibition ratio (I) was calculated using the following formula: i (%) - (C-T)/C]×100。

TABLE 1 inhibitory Activity of the Compound Pj-1-Pj-5 against 5 phytopathogenic fungi at 50. mu.g/mL

TABLE 2 NMR data (600M, CDCl) for Compound Pj-1₃)

Other physicochemical data for Compound Pj-1: a colorless oil.IR(KBr)ν_max3376, 2924,1721,1602,1383,1095,1044,792cm^-1；HRESIMS m/z 417.1878[M+Na]⁺(calcd for C₂₁H₃₀O₇Na,417.1884)。

The experimental result shows that the compound Pj-1-Pj-5 has certain inhibition effect on 5 tested plant pathogenic fungi. Wherein, the compound Pj-1 has the most obvious inhibition effect on 5 tested plant pathogenic fungi, and the inhibition rate ranges from 70.6 percent to 82.9 percent. In particular, Pj-1 has higher inhibitory activity against Fusarium oxysporum (sp. niveum) and Fusarium graminearum (Fusarium graminearum) than that of the positive control drug carbendazim. Meanwhile, the inhibitory activity of Pj-1 on Rhizoctonia solani (Rhizoctonia solani), Penicillium citrinum (Penicillium citrinum) and Phytophthora cucumerinum (Phytophtora melonis) is equivalent to that of a positive control medicament, carbendazim. These results indicate that Pj-1 has the potential to be deeply developed into botanical antibacterial drugs.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, improvement and the like made within the content and principle of the present invention shall be included in the protection scope of the present invention.