CN111978279B - Method for extracting methoxy cineraria from Italian xanthium and application thereof - Google Patents

Abstract

The invention discloses a method for extracting methoxy cineraria from Italian xanthium and application thereof, the method comprises the steps of crushing Italian xanthium fruit in the mature period, soaking and extracting with ethanol, degreasing with petroleum ether, extracting with chloroform, purifying with silica gel column, purifying with Sephadex LH-20 column, and obtaining methoxy cineraria. The obtained methoxy cineraria extract is used as herbicide for trial on 3 tested plants, namely monocotyledonous plants such as bluegrass and pennisetum, and dicotyledonous plants such as amaranthus retroflexus, and the result shows that: the seedling growth of monocotyledonous bluegrass and pennisetum alopecuroides and dicotyledonous amaranthus retroflexus is obviously inhibited, and the root growth inhibition rate of the amaranthus retroflexus reaches 97%; the root length inhibition rate of pennisetum is 52%, and the bluegrass is 43%. Methoxy cineraria is a natural component of plant source, and can be rapidly decomposed in nature without polluting the environment. The herbicide can be prepared into different concentrations, can be used singly or mixed with other pesticides before or during seedling stage, is widely applied to the development of various herbicides, and has potential commercial application value.

Description

Method for extracting methoxy cineraria from Italian xanthium and application thereof

Technical Field

The invention relates to a preparation method for extracting flavonoid compounds from plants and the technical field of application of the flavonoid compounds in herbicides, in particular to a preparation method for extracting methoxy cineraria from Italian xanthium which is a harmful plant and application of the methoxy cineraria as a herbicide.

Background

With the development of modern agriculture, more than 80% of weeds in agriculture, forestry and animal husbandry are favored as chemical herbicides, and the advantages of labor saving, time saving, labor saving, low cost, simplicity, convenience, practicability and the like bring great benefits to the economic development of agriculture. However, with the mass production and wide use of chemical herbicides, serious harm is often caused to the natural environment, and the chemical herbicide is a killer for ecological environment. Meanwhile, various artificially synthesized chemical herbicides are easy to remain in nature for a long time due to the lack of corresponding microorganisms for decomposing the chemical herbicides, so that the yield and quality of agriculture, forestry, pasture and fishery are affected, and even the health of human beings is endangered. Furthermore, some chemical herbicides, although capable of decomposing in soil, are gradually resistant to weeds due to long-term massive use, so that the efficacy is reduced or even lost, see documents 1 to 3. However, the secondary metabolite extracted from the natural plants is used as herbicide, which is safe to the environment, not easy to generate drug resistance and can be rapidly degraded, thus becoming a new direction for the development of pesticide at home and abroad at present. Under such circumstances, the development of herbicides with novel targets and mechanisms of action has significant utility. The method is widely used for screening secondary metabolites with herbicidal activity in plants, separating, purifying and structurally modifying the secondary metabolites, and has become the leading edge and hot spot field of novel herbicide research. Some secondary metabolites themselves have herbicidal activity comparable to chemical herbicides, such as 1, 4-eucalyptol, artemisinin, strigol, and the like; there are also herbicides of natural origin whose efficacy is comparable to that of chemically synthesized herbicides, and thus are processed and promoted as commercial herbicides, such as phytotoxin pelargonic acid extracted from geraniaceae, and fibrinolysin isolated from cajeput, modified sulcotrione, see literature 4.

Methoxy cinerarin, named Axillarin, is flavonoid compound, and is in the form of yellow amorphous powder. Methoxy cineraria has been reported to have anti-tumor, anti-oxidant and anti-inflammatory activities, but its inhibitory effect on plant growth is not reported at home and abroad, see documents 5-8. There are reports of methods for their artificial chemical synthesis, see document 9.

The xanthium plant source for extracting methoxy cineraria is wide. The Xanthium plants are widely distributed in multiple provinces in China, and both the two exotic invasive Xanthium plants Italian Xanthium (Xanthium italicum) and Litsea palmata contain the chemical components. Because of strong ecological adaptability and large biomass, the exotic invasive xanthium sibiricum can compete with native crops for nutrients and space, and meanwhile, fruits of the exotic invasive xanthium sibiricum have hooked thorns, so that the exotic invasive xanthium sibiricum is difficult to effectively remove, and the exotic invasive xanthium sibiricum has serious influence and harm on agriculture, forestry, animal husbandry and ecosystem, and is disclosed in documents 10-11. The method for extracting the methoxy cineraria by using Italian xanthium as a raw material explores a new way for the high-valued utilization of the invasive plant, well controls the further spreading and diffusion of the invasive plant, and protects the surrounding ecological environment and the development of agricultural economy.

Reference is made to:

1. zhang Linlong. 2012. Chemical herbicides: killers of ecological environment. Ecological economy, 11:14-17.

2. Wang Chunhua 2017. The current use situation and countermeasures of chemical herbicides in China. Shandong pesticide information, 01:19-20.

3. Dan Xiaoxu, chen Yi, huang Rong, 2016. The current state of use of chemical herbicides in China and countermeasures, modern agriculture technology, 19:133-134.

4. Mo Wenyan, he Gongwu, 2005. Research on herbicidally active substances of plant origin. Chemical and biological engineering, 08:7-9.

5、Park,E.J.；Kim,Y.；Kim,J.Acylated Flavonol Glycosides from the Flower of Inula britannica.JournaL of Natural Products.2000,63(1):34-36.

6、Forgo,P.；István Zupkó.；Judit MoLnár.Bioactivity-guided isolation of antiproliferative compounds from Centaurea jacea L.Fitoterapia.2012,83(5).

7、Juan,B.M.；Habtemariam,S.；Jackson,C.Antioxidant Principles of Tanacetum vulgare L.Aerial Parts.Natural Product Communications.2009,4(11):1561-1564.

8、MoscateLLi,V.；Hnatyszyn,O.；Acevedo,C.Flavonoids from Artemisia copa with anti-inflammatory activity.Planta Medica.2006,72(01):72-74.

9、Fukui,K.；Nakayama,M.；Horie,T.The syntheses of axillarin and its related compounds.Experientia.1968,24(8):769-770.

10. Wang Rubing, deng Shidong, qu Jing, shishan.2017. Study of chemical composition of Siberian cocklebur fruit. Study and development of natural products, (05): 69-72.

11. Du Zhenzhu, xu Wen, yan Ping, wang Shaoshan, guo Yimin.2012. 3 exotic invasive new plants of Xanthium genus, 49 (05) of Xinjiang agricultural science 879-886.

Disclosure of Invention

The invention aims to provide a method for extracting methoxy cineraria from Italian xanthium and application thereof, the method comprises the steps of crushing Italian xanthium fruit in the mature period, soaking in ethanol, filtering, steaming until no alcohol smell exists by a rotary evaporator, degreasing by petroleum ether, extracting by chloroform, carrying out chromatography by a 100-200 mesh silica gel column, eluting, carrying out Sephadex LH-20 column chromatography, eluting, and obtaining the methoxy cineraria with the purity of more than 95%. The methoxy cineraria extract obtained by the method of the invention is used as herbicide, can be degraded in nature, does not pollute the environment, simultaneously utilizes waste of harmful plants, is suitable for preventing and killing farmland weeds, and is shown by experimental results for 3 tested plants, namely monocotyledonous plants such as bluegrass and pennisetum and dicotyledonous plants such as amaranthus retroflexus: the seedling growth of monocotyledonous bluegrass and pennisetum alopecuroides and dicotyledonous amaranthus retroflexus is obviously inhibited, and the root growth inhibition rate of the monocotyledonous amaranthus retroflexus reaches 97%; the root length inhibition rate of the pennisetum is 52 percent, and the bluegrass is 43 percent. In addition, the roots of the tested plants were distorted and brown in color, suggesting that they had been severely poisoned. High inhibition rate to seedlings: the amaranth on the back branch reaches 93 percent, the pennisetum 40 percent and the bluegrass 15 percent. In addition, the seedlings also undergo a distortion phenomenon.

The invention discloses a method for extracting methoxy cineraria from Italian xanthium, which comprises the following steps:

a. collecting Italian xanthium plants in the mature period, separating fruits from other parts, airing and crushing for later use;

b. soaking the crushed Italian xanthium fruits in the step a with 5 times of ethanol with the concentration of 95% for 7 days at room temperature, repeating the operation for 3 times, filtering the soaking solution to remove residues, collecting filtrate, concentrating and drying the filtrate by a rotary evaporator to obtain an extract;

c. stirring the extract obtained in the step b with distilled water, degreasing by adding petroleum ether with the volume of 2 times, repeating the operation for 3 times, and collecting a degreased suspension;

d. c, extracting the degreased suspension in the step c by using chloroform with the volume of 2 times, repeating the operation for 3 times, combining the extracting solutions, and concentrating by using a rotary evaporator to obtain an extract;

e. c, performing chromatography on the extract obtained in the step d by using a 100-200 mesh silica gel column, eluting by using a chloroform-methanol mixed solution with the volume ratio of 98:2, collecting the eluent, concentrating and drying to obtain yellow oily matter;

f. purifying the yellow oily matter obtained in the step e by a Sephadex LH-20 column, eluting by using chloroform-methanol solution with the volume ratio of 1:1, collecting eluent, concentrating and drying to obtain the compound methoxyl shou chrysanthemum with the purity of 95%.

The application of the methoxy cinerarin obtained by the method in preparing herbicide.

The invention relates to a method for extracting a compound methoxy cinerarin from Italian xanthium and application thereof, wherein the extracted compound methoxy cinerarin has a structural formula as follows:

the methoxy cineraria extract obtained by the method is prepared into different concentrations, and is used singly or mixed with other pesticides before or during seedling stage, thus having potential commercial application value. Dissolving with common 95% ethanol or acetone, diluting 10000-20000 times before use, and spraying at a concentration of 1000-2000 μg/ml before or after emergence of weeds.

The method for extracting the compound methoxy cineraria from the Italian xanthium and the application thereof have the beneficial effects that:

1. the methoxy cineraria extract from Italian xanthium provided by the invention has obvious inhibition on the growth of 3 tested plants, namely monocotyledonous plants such as bluegrass and pennisetum alopecuroides, and the seedling of dicotyledonous plants such as amaranthus retroflexus, and the root growth inhibition rate of the methoxy cineraria reaches 97% when the concentration is 1000 mug/ml; the root length inhibition rate of the pennisetum is 52 percent, and the bluegrass is 43 percent. In addition, the roots of the tested plants were distorted and brown in color, suggesting that they had been severely poisoned. High inhibition rate to seedlings: the amaranth on the back branch reaches 93 percent, the pennisetum 40 percent and the bluegrass 15 percent. In addition, the seedlings also undergo a distortion phenomenon.

2. The methoxy cineraria extract from Italian xanthium as herbicide provided by the invention is suitable for preventing and killing farmland weeds. Because the inhibition effect of the tetrandra branch amaranth on the tetrandra branch amaranth is far superior to the effect of other common weeds, the methoxy cineraria is more suitable for preventing and removing the tetrarhizoma branch amaranth. The methoxy cineraria extract from Italian xanthium is a natural component of plant source, can be rapidly degraded in nature, does not pollute the environment, is easy to apply in large quantities, and has important potential application value for sustainable development of agriculture. In addition, the compound can be extracted from the external invasive harmful plants Italian xanthium and Liujia, and the two plants grow flourishingly in the field. The height of Italian xanthium plants can reach more than two meters, the biomass is large, and the extraction of natural herbicide from the Italian xanthium plants is beneficial to environmental protection, and the comprehensive utilization of harmful plants can be realized, so that the Italian xanthium plants can be obtained by two purposes.

Drawings

FIG. 1 is a graph showing the growth regulating effect of methoxy cineraria on the growth of two monocotyledonous plants Poa pratensis and pennisetum alopecuroides and two dicotyledonous plants Amaranthus retroflexus, with the effect concentrations being 10, 50, 200, 1000. Mu.g/ml, respectively.

FIG. 2 is a graph showing the high growth regulating effect of methoxy cineraria on two monocotyledonous plants, poa pratensis and pennisetum alopecuroides and two dicotyledonous plants, amaranthus retroflexus and alfalfa, at concentrations of 10, 50, 200, 1000. Mu.g/ml, respectively.

FIG. 3 is a graph of methoxypsoralen inhibition of growth of two monocotyledonous plants Poa pratensis and pennisetum alopecuroides and two dicotyledonous plants Amaranthus retroflexus and alfalfa root length and seedling height.

Detailed Description

The present invention is further illustrated by the following examples, but is not limited thereto.

All of the raw materials, reagents and equipment selected for use in the present invention are well known in the art, and some other reagents and equipment known in the art are suitable for implementation.

Example 1

Extraction of methoxy cineraria:

a. collecting Italian xanthium plants in the mature period, separating fruits from other parts, airing and crushing for later use;

b. soaking the crushed Italian xanthium fruits in the step a with ethanol with the volume of 5 times of that of 95% for 7 days at room temperature, filtering the soaking solution to remove residues, and steaming the filtrate with a rotary evaporator until no alcohol smell exists to obtain an extract;

c. stirring the extract obtained in the step b with distilled water, adding petroleum ether with the same volume for degreasing, repeating for 3 times, and collecting supernatant to obtain a degreased suspension;

d. c, extracting the degreased suspension in the step 3 times by using chloroform with the same volume, combining the extracting solutions, and concentrating by using a rotary evaporator to obtain an extract;

e. c, performing chromatography on the extract obtained in the step d by using a 100-200 mesh silica gel column, eluting by using a chloroform-methanol mixed solution with the volume ratio of 98:2 to obtain an eluent, and concentrating the eluent to obtain yellow oily matter;

f. and e, subjecting the yellow oily matter obtained in the step e to Sephadex LH-20 column chromatography, and eluting with chloroform-methanol in a volume ratio of 1:1 to obtain the compound methoxy cineraria with the purity reaching 95%.

Example 2

Use of methoxyl shou Jun in preparing herbicide:

preparing 1000 mug/ml of methoxy cineraria extract obtained in example 1 by using 0.5% acetone, diluting the methoxy cineraria extract to 10 mug/ml, 50 mug/ml or 200 mug/ml by using distilled water in sequence, sequentially adding 5ml of methoxy cineraria extract solution with the concentration of 10 mug/ml, 50 mug/ml, 200 mug/ml and 1000 mug/ml into culture dishes with the diameter of 9cm respectively, paving 1 layer of filter paper on each culture dish, adding 20 full alfalfa, pennisetum, bluegrass and amaranth seeds into each culture dish respectively, making 3 repeats of each culture dish, sealing the culture dishes by using a sealing film, placing the culture dishes in a culture box with the temperature of 25 ℃, illuminating for 16 hours and measuring the seedling height and root length of seedlings in each culture dish after culturing for 5 days by using distilled water containing 0.5% acetone as a control;

the data statistics method comprises the following steps: the high data of each plant root length and seedling is processed by software such as SPSS22.0, EXCEL2017 and the like, analysis of variance (ANOVA) is performed by single factor analysis, and the difference significance among different treatment groups is compared by an LSD method;

results: the measurement result of the activity of the methoxy cinerarin solution on the growth of 4 receptor plant seedlings of alfalfa, pennisetum alopecuroides, bluegrass and amaranth seeds shows that the methoxy cinerarin has an inhibition effect on the growth of the tested plant seedlings, the higher the concentration is, the stronger the inhibition effect is, under the concentration of 200 mug/ml, the growth of dicotyledonous plant amaranth and monocotyledonous plant bluegrass and pennisetum alopecuroides seedlings is influenced, and the influence degree is enhanced along with the increase of the concentration; it is well known in the art that these weeds alfalfa, bluegrass, pennisetum and amaranthus retroflexus are all weed targets well known in the art that are generally difficult to eradicate using common herbicides; when the concentration is increased to 1000 mug/ml, the seedling heights and root lengths of the bluegrass, the pennisetum and the amaranthus retroflexus are obviously different from those of a control, the root length inhibition rate is 43%, 52% and 97% of that of the control in sequence, the roots of the seedlings are obviously distorted and deformed and become black brown, and the seedlings are suggested to be severely poisoned; the effect of the methoxy cineraria on the seedling height of the tested plant is similar to the effect on root growth, but because the methoxy cineraria directly acts on the root of the seedling, the effect intensity on the seedling height is slightly weaker than the effect on the root growth, and the high inhibition rate on the bluegrass, the pennisetum and the amaranthus retroflexus are 15%, 40% and 93% of the control in sequence; see fig. 2 and 3; from the high inhibition rate of the methoxy cineraria solution to the root length and seedling height of 4 plants, the effect of the methoxy cineraria solution on the growth of 4 plant receptors is that amaranthus retroflexus > pennisetum > bluegrass > alfalfa; because the inhibition effect of the tetrandra branch amaranth on the tetrandra branch amaranth is far superior to the effect of other common weeds, the methoxy cineraria is more suitable for developing into the tetrarhizoma branch amaranth herbicide.

Conclusion:

the methoxy cineraria extract obtained by the method of the invention has no inhibiting effect on dicotyledon alfalfa, and has inhibiting effect on the growth of other 3 monocotyledon pennisetum, bluegrass and amaranthus retroflexus seeds at the concentration of 200 mug/ml; when the concentration reaches 1000 mug/ml, the root length inhibition rate of the amaranthus retroflexus on dicotyledonous plants reaches 97%, the root length inhibition rate of the amaranthus retroflexus on monocotyledonous plants is 52% of pennisetum, and the root length inhibition rate of the amaranthus praecox is 43%; in addition, the roots of the tested plants were distorted and brown in color, suggesting that they had been severely poisoned; the inhibition rate for the seedlings is as follows: 93% of amaranth, 40% of pennisetum, and 15% of bluegrass; the seedlings also have a distortion phenomenon; overall, the inhibition effect of methoxy cineraria on the amaranthus retroflexus is more obvious.

The methoxy cineraria extract is used as herbicide in different concentration, and has excellent herbicidal effect on other weeds.

The invention relates to a method for extracting a compound methoxy cineraria from Italian xanthium and application thereof, wherein the extracted methoxy cineraria is suitable for preventing and removing weeds in agricultural fields; because the influence on the bluegrass and the like is far smaller than the effect on the amaranthus retroflexus, the methoxy cineraria is more suitable for preventing and killing the amaranthus retroflexus weeds in farmlands. The methoxy cineraria extract is extracted from Italian xanthium which invades harmful plants in the outside, the plants grow luxuriantly in the field, the height of the plants can reach more than two meters, the biomass is high, and the extraction of herbicide from the plants is not only beneficial to environmental protection, but also comprehensive utilization of the harmful plants is realized, and the prevention and control of the harmful plants are facilitated. Methoxy cineraria is a natural component of plant source, can be rapidly decomposed in nature, does not pollute the environment, and can be applied to weed control in organic agriculture. Is beneficial to developing sustainable agriculture and simultaneously utilizes waste of harmful plants.

Claims (1)

1. Use of methoxyl cineraria extract from Italian xanthium in the preparation of herbicide, said herb being amaranthus retroflexus; the preparation method of the compound methoxy cineraria comprises the following steps:

a. collecting Italian xanthium plants in the mature period, separating fruits from other parts, airing and crushing for later use;

b. soaking the crushed Italian xanthium fruits in the step a with 5 times of ethanol with the concentration of 95% for 7 days at room temperature, repeating the operation for 3 times, filtering the soaking solution to remove residues, collecting filtrate, concentrating and drying the filtrate by a rotary evaporator to obtain an extract;

c. stirring the extract obtained in the step b with distilled water, degreasing by adding petroleum ether with the volume of 2 times, repeating the operation for 3 times, and collecting a degreased suspension;

d. c, extracting the degreased suspension in the step c by using chloroform with the volume of 2 times, repeating the operation for 3 times, combining the extracting solutions, and concentrating by using a rotary evaporator to obtain an extract;

e. c, performing chromatography on the extract obtained in the step d by using a 100-200 mesh silica gel column, eluting by using a chloroform-methanol mixed solution with the volume ratio of 98:2, collecting the eluent, concentrating and drying to obtain yellow oily matter;

f. purifying the yellow oily matter obtained in the step e by a Sephadex LH-20 column, eluting by using chloroform-methanol solution with the volume ratio of 1:1, collecting eluent, concentrating and drying to obtain the compound methoxyl shou chrysanthemum with the purity of 95%.