CN104738102B - Chinese gooseberry root bark extract is used for the application for preparing anti-plant virus agent - Google Patents

Abstract

It is used for the application for preparing anti-plant virus agent the invention discloses Chinese gooseberry root bark extract, take Chinese gooseberry root skin, crushed after drying, through certain process, it is prepared into the antiviral soluble liquid of Kiwi berry root skin, aqua and microemulsion, available for preventing and treating tobacco mosaic virus disease (Tobacco mosaic virus in tobacco, capsicum, tomato, cucurbita pepo, potatoes and other crops, TMV), Cucumber Mosaic Virus (Cucumber mosaic virus,) and potato virus disease (Potato Virus X, PVX CMV；Potato Virus Y, PVY) etc. various plants virosis；Not only antiviral effect is good for the anti-plant virus agent of the present invention, and to environment, people, animal, pest natural enemy and other beneficial organisms safety.Anti-plant virus agent preparation technology is simple, and cost is cheap, suitable popularization and application.

Description

Application of root bark extract of Actinidia sinensis in the preparation of anti-plant virus agent

technical field

The invention belongs to the field of plants and applications thereof, in particular to the application of the root bark extract of Actinidia sinensis for resisting plant viruses.

Background technique

Plant virus is an intracellular parasitic pathogen composed of nucleic acid and protein with infectious activity. According to the eighth report of the International Committee on Taxonomy of Viruses (ICTV), there are 1122 plant virus species found in the world. Plant diseases caused by plant viruses are known as "plant cancers". The annual loss caused by plant viruses in the world is about 40 billion U.S. dollars, and the damage caused by tobacco mosaic virus (TMV) alone exceeds 100 million U.S. dollars.

Due to the strict parasitic nature of plant viruses, they parasitize in plant cells or even the nucleus to perform nucleic acid (RNA or DNA) replication and protein shell synthesis to form new virus particles. This determines that the prevention and treatment of plant virus diseases is difficult, and general medicines have no effect. At the beginning of the 20th century, Allard reported that there were TMV inhibitory substances in pokeweed juice, and then the focus of preventing and controlling plant viruses gradually shifted to the direction of natural plant extracts. Especially since the 1990s, people have successively found many inhibitory substances against plant viruses from natural products, which provides a basis for the development of efficient, specific and safe natural inhibitors of plant viruses.

Actinidia chinensis Planch. is a plant of the genus Actinidia in the family Actinidia. Its root is also known as vine pear root. It is a unique species in China and is mainly distributed in Shaanxi, Jiangxi, Hunan, Hubei, Fujian, Zhejiang, Henan, Anhui and other provinces in China. The whole plant of Chinese kiwifruit can be used medicinally. Traditional Chinese medicine believes that its roots and root bark are cold in nature and bitter in taste. Dysentery, filariasis, gastric cancer, breast cancer and other diseases. Modern pharmacological studies have found that Chinese kiwifruit root has the functions of scavenging active oxygen free radicals, anti-virus, protecting liver, inhibiting tumor cell growth, and improving immunity.

The reported chemical components of Actinidia sinensis are mainly flavonoids, triterpenes, steroids, anthraquinones, phenolic acids, sugars and other types of compounds, among which triterpenoids are the main components. The pharmacological activities of the discovered active compounds are mainly manifested in medical activities, such as anti-tumor, enhancing the immune function of the body, protecting the liver, anti-oxidation, scavenging free radicals, and anti-rotavirus. So far, there is no report on the effect of Actinidia sinensis root bark extract and its metabolites on agricultural production diseases, insects and weeds.

Contents of the invention

The object of the present invention is to use the root bark extract of Actinidia chinensis Planch. as a raw material to develop a plant-derived anti-plant virus agent with high anti-plant virus effect, no pollution, and low price.

In order to achieve the above object, the technical scheme that the present invention takes is:

Application of root bark extract of Actinidia sinensis for preparation of anti-plant virus agent.

Specifically, the root bark extract of Actinidia sinensis is a substance obtained after alcohol extraction of the crushed root bark of Actinidia sinensis.

More specifically, the particle size of the crushed root bark of Actinidia sinensis is 1-5mm, the solvent used for alcohol extraction is ethanol, and the alcohol extraction temperature is 50-70°C.

Also, the solvent used for alcohol extraction is ethanol with a mass fraction of 95%, and the alcohol extraction time is 18-20 hours.

Meanwhile, the plant virus includes tobacco mosaic virus, cucumber mosaic virus and potato virus.

An anti-plant virus agent, the dosage form of the anti-plant virus agent is a microemulsion, and the microemulsion is prepared by the following raw materials by weight percentage:

Chinese kiwifruit root bark extract: 15%-30%, organic solvent: 0-55%, surfactant: 5%-20%, antifreeze agent: 5%-10%, the balance is water, the weight percentage of raw materials and for 100%.

Or, the dosage form of the anti-plant virus agent is a soluble liquid, and the soluble liquid is prepared by weight percentage from the following raw materials:

15%-30% of kiwifruit root bark extract, 0%-55% organic solvent, 5%-30% surface active agent, ethanol as the balance, and the sum of the weight percentages of the raw materials is 100%.

Or, the dosage form of this anti-plant virus agent is water preparation, and described water preparation is prepared by weight percentage by following raw material:

Chinese kiwifruit root bark extract: 10%-20%, organic solvent: 0%-40%, surfactant: 5%-30%, antifreeze agent: 5%-10%, the balance is water, the weight percentage of raw materials The sum is 100%.

The kiwifruit root bark anti-plant virus agent made by the present invention has the following advantages:

(1) The present invention finds that the root bark extract of Actinidia sinensis has significant preventive effect on plant viruses by studying the extracts of kiwifruit of different varieties, which is obviously better than the preventive effect of other varieties of kiwifruit extracts on plant viruses;

(2) In addition, the inventor also carried out anti-plant virus activity research on the extracts of different parts of the Actinidia chinensis plant, and found that only the root bark part of the Actinidia chinensis extract has a significant preventive effect on plant viruses, and other parts even have no effect on plant viruses. Has anti-plant virus activity;

(3) The anti-plant virus activity of the kiwi fruit extract of the present invention has a broad spectrum, and can be used to prevent and treat tobacco mosaic virus (Tobacco mosaic virus, TMV) and cucumber mosaic virus on crops such as tobacco, capsicum, tomato, zucchini, and potato. Various plant virus diseases such as Cucumber mosaic virus (CMV) and potato virus disease (Potato Virus X, PVX; Potato Virus Y, PVY).

detailed description

The plant-derived anti-plant virus agent of the present invention uses the perennial Actinidia sinensis root bark extract as an active substance, adds a certain proportion of auxiliary agents for formulation processing, and can obtain an environmentally friendly preparation microemulsion, water formulation or soluble liquid formulation. The root bark extract of Actinidia sinensis is the substance after alcohol extraction, and the optional alcohol extraction solvent is ethanol, which has no adverse effect on the antiviral effect of the plant extract.

Plant virus of the present invention comprises: tobacco mosaic virus (Tobacco mosaic virus, TMV), cucumber mosaic virus (Cucumber mosaic virus, CMV) and potato virus disease (Potato Virus X, PVX; Potato Virus Y, PVY ).

The extraction method of Chinese kiwifruit root bark extract of the present invention comprises:

A. Grinding the root bark of Actinidia chinensis to a coarse pulverization of 1-5mm with a plant tissue pulverizer;

B. The coarse pulverized matter obtained in step A is extracted with 95% ethanol, the extraction temperature is 50-70° C., and the extraction time is 18-20 hours, and the extract is vacuum-concentrated to be equivalent to 1 kg dry powder/kg concentrate.

The organic solvent of the present invention is one or a mixture of two or more of ethyl acetate, cyclohexanone, methyl oleate, ethanol, methanol, acetone and dimethylformamide;

The emulsifiers are alkylbenzene sulfonates, alkylphenol polyoxyethylene ethers, polystyrene phenol polyoxyethylene ethers, styroylphenol polyoxyethylene ethers, styrylphenol polyoxyethylene ethers One or more mixtures of polyoxypropylene ethers and their similar substances, Span series, Tween and agricultural anion-phosphates;

The surfactants are alkylbenzene sulfonates, alkylphenol polyoxyethylene ethers, styrylphenol polyoxyethylene ethers, styrylphenol polyoxyethylene ethers polyoxypropylene ethers and the like One or a mixture of two or more substances, adducts of castor oil and ethylene oxide, and aralkylphenol polyoxyethylene ether formaldehyde condensation products;

Described antifreezing agent is ethylene glycol or glycerol.

In order to clarify the particularity of the antiviral activity of the root bark of Actinidia sinensis and to further explore the effective parts of the activity, the inventors have done several works in three aspects in advance.

1. Differences in the antiviral activity of other plants in the same genus of Actinidia sinensis root bark:

The inventors compared the antiviral activity of the root bark extracts of Actinidia chinensis and other plants of the genus Actinidia that are closely related to it, and the results showed that the antiviral activity of the root bark extracts of Actinidia chinensis was significantly better than other varieties. The results are shown in Table 1.

Table 1 In vitro inactivation effect of several kiwifruit root bark extracts on TMV

Note: 1. The test agents are all 20% root bark extract microemulsion;

2. The data in the table is the average value of 3 repetitions; in the column of "control effect", different letters after the same number mean that there is a significant difference at the level of α=0.05.

2. Determination of antiviral active parts of Actinidia sinensis:

The inventor measured the in vitro passivation activity of extracts from different parts of Actinidia sinensis to TMV. The results showed that the antiviral activity of the extracts from different parts was very different. At the same concentration, the root bark extract had significant activity, and the roots, stems , leaves do not show activity, and the whole plant shows a certain activity, which may also be due to the effect of antiviral activity in the root bark (Table 2). Accordingly, the inventor speculates that the antiviral activity in Actinidia sinensis is concentrated in the root bark. Therefore, the present invention determines that the root bark extract of Actinidia sinensis is the main raw material for antiviral activity.

Table 2 In vitro inactivation effect of extracts from different parts of Actinidia sinensis on TMV

Note: 1. "-" indicates no passivation activity.

2. The data in the table is the average value of 3 repetitions; in the column of "control effect", different letters after the same number mean that there is a significant difference at the level of α=0.05.

3. Broad-spectrum antiviral activity of kiwifruit root bark extract:

The inventor has measured the in vitro inactivation activity of the root bark extract of Actinidia sinensis to several common plant virus diseases, and the results show that it has certain activity to TMV, CMV, PVX and PVY, which shows that the root bark of Actinidia sinensis Contains broad-spectrum anti-plant virus active ingredients.

Table 3 In vitro inactivation effect of Actinidia sinensis root bark extract on several plant virus diseases

Note: The data in the table are the average value of 3 repetitions; in the column of "control effect", different letters after the same number mean that there is a significant difference at the level of α=0.05, the same below.

On the basis of the above experiments, in order to further develop the antiviral active preparation of the root bark extract of Actinidia sinensis, it was made into microemulsion, soluble liquid and water respectively. The inventor's test proves that 20% kiwifruit root bark microemulsion, 20% kiwifruit root bark soluble liquid and 10% kiwifruit root bark water have good control effects on tobacco mosaic virus (TMV), wherein 20% kiwifruit root bark Root bark microemulsion had the best control effect. In order to better understand the essence of the invention, the following examples are used to describe the technical content of the invention in detail, but the invention is not limited to these examples.

Four, specific preparation preparation examples:

Embodiment 1: the preparation of 20% kiwifruit root bark microemulsion

Weigh 20kg of kiwifruit root bark concentrate, dissolve it in 30kg of ethyl acetate, add 15kg of calcium dodecylbenzenesulfonate, 5kg of styrene acid polyoxyethylene ether, and 5kg of ethylene glycol, mix under high-speed stirring, and heat to about 40°C; add 25kg of deionized water dropwise under high-speed stirring, and control the dropping speed of the water to keep the temperature at about 40°C; Emulsion 100kg. The stability and appearance of the preparation meet the requirements of commercial preparations.

Embodiment 2: the preparation of 20% kiwi fruit root bark soluble liquid

Weigh 20kg of kiwifruit root bark concentrate, dissolve it in 30kg of ethyl acetate, add 5kg of alkylphenol polyoxyethylene ether, 1kg of alkyl polyglycoside A, and make up to 100kg with ethanol, mix well, and stir at a speed of 800～ Stir at 1000 rpm for 10 to 30 minutes to prepare 100 kg of kiwifruit root bark soluble liquid. The stability and appearance of the preparation meet the requirements of commercial preparations.

Embodiment 3: the preparation of 10% kiwifruit root bark water

Weigh 10kg of kiwifruit root bark concentrate, dissolve it in 30kg of ethyl acetate, then add 5kg of alkylphenol polyoxyethylene ether, 1kg of alkyl polyglycoside A, 5kg of ethylene glycol, make up to 100kg with water, mix well, Stirring at a stirring speed of 800-1000 rpm for 10-30 minutes can prepare 100 kg of kiwifruit root bark soluble liquid. The stability and appearance of the preparation meet the requirements of commercial preparations.

5. Indoor bioassay of 3 kinds of kiwifruit root bark antiviral agents against TMV:

(1) In vitro passivation effect on TMV

The half-leaf dead spot method was used to test, and Nicotiana glutinosa at the 5-6 leaf stage, which grew vigorously and had the same growth vigor, was selected as the dead spot host. With the leaf veins as the boundary, the left half leaf was inoculated with an equal volume mixture of medicine and virus, and the right half was inoculated with an equal volume mixture of distilled water and virus as a control. The virus inoculation concentration was 10 μg/mL, and the passivation time was 5 minutes. Rinse the surface of the inoculated leaves with water. Each treatment was inoculated with 4 leaves, and the experiment was repeated 3 times. After 3 days, the number of dead spots was counted, and the inhibition rate was calculated.

Inhibition rate (%)=(control number of dead spots-treatment number of dead spots)/control number of dead spots×100

The measurement results are shown in Table 4.

Table 4 The in vitro inactivation effect of three kinds of kiwifruit root bark antiviral agents on TMV

(2) Inhibitory effect on initial TMV infection

The half-leaf dead spot method was used to select the healthy and healthy 5-6-leaf stage tobacco as the dead spot host, and the virus was inoculated 6h, 12h, and 24h after applying the liquid medicine. The blank control was treated with water, and each treatment was inoculated with 4 leaf, repeated 3 times, counted the number of dead spots after 3 days, and calculated the inhibition rate. The results are shown in Table 5.

Table 5 Inhibitory effects of three antiviral agents on kiwifruit root bark on initial TMV infection

(3) Inhibitory effect on TMV proliferation

Using the half-leaf dead spot method, select tobacco heart leaves with consistent growth and health at the 5-6 leaf stage as the dead spot host, and apply the liquid medicine 6h, 12h, and 24h after inoculating the virus. The blank control is treated with water, and each treatment is inoculated with 4 leaf, repeated 3 times, counted the number of dead spots after 3 days, and calculated the inhibition rate. The results are shown in Table 6.

Table 6 Inhibitory effects of three antiviral agents from kiwifruit root bark on TMV proliferation

From Tables 4, 5, and 6, it can be seen that the three kinds of kiwifruit root bark antiviral agents have good effects on TMV passivation, initial infection, and proliferation inhibition. Among them, the effect of 20% kiwifruit root bark microemulsion is better than the other two formulations. Significantly higher than the control drug morpholinoguanidine. Ethyl copper.

(4) Pot experiment on TMV

1) Protective effect on ordinary smoke

The common tobacco (Nicotiana tabacum) with consistent growth and robustness at the 5-6 leaf stage was selected for testing. The experiment consisted of two groups, the prevention group (inoculated with TMV after 3 times of application) and the control group (only inoculated without application of drug). Spray three kinds of kiwifruit root bark antiviral agents with 200 times liquid spray, and spray with 500 times liquid of morpholinoguanidine and ethyl copper as a control agent to treat ordinary cigarettes, and spray once every 5 days for a total of three times. TMV was inoculated 24 hours after the last application, and the inoculation concentration was 1:20 (W/V). 14d and 21d after inoculation, the incidence was checked, and the disease index and control effect were counted. For each treatment of 60 tobacco seedlings, the experiment was repeated 3 times. Calculate the effectiveness.

Morbidity rate=(number of diseased plants in each treatment/total number of plants in treatment)×100%

Disease index=∑(number of disease grades×number of diseased plants)/(highest disease grade×total number of plants in each treatment)×100

Disease index growth rate = (disease index after spraying - disease index before spraying) / disease index before spraying × 100%

Control effect = (control disease finger - treatment disease finger) / control disease finger × 100%

The results of the protective effect assay are shown in Table 7.

Table 7 Determination of the protective effect of three kinds of kiwifruit root bark antiviral agents on common cigarettes

2) Therapeutic effect on common smoke

The common tobacco (Nicotiana tabacum) with consistent growth and robustness at the 5-6 leaf stage was selected for testing. The experiment consisted of two groups: a treatment group (administered 3 times after inoculation with TMV) and a control group (only inoculated but not administered). The virus source TMV was inoculated, the inoculation concentration was 1:20 (W/V), and the drug was applied every 5 days after inoculation, and the drug was applied three times in total. The medicaments were 200-fold liquid sprays of three kinds of kiwifruit root bark antiviral agents, and 500-fold liquid sprays of the control drug morpholinoguanidine.Ethyl copper. On the 14th day and 21st day after the last spraying, the incidence was checked, and the disease index and control effect were counted. For each treatment of 60 tobacco seedlings, the experiment was repeated 3 times. Calculate the effectiveness. The results of the therapeutic effect assay are shown in Table 8.

Table 8 Determination results of the therapeutic effects of three kinds of kiwifruit root bark antiviral agents on common cigarettes

Pot test result shows (table 7, table 8), and 3 kinds of kiwifruit root bark antiviral agents show good preventive and therapeutic effect to tobacco mosaic virus disease, and its protection and therapeutic effect are all better than contrast agent, and wherein, 20% The protective effect of kiwifruit root bark microemulsion is about 25% higher than that of the control agent.

6. Field plot efficacy test of 3 kinds of kiwifruit root bark antiviral agents against TMV:

The plot test is randomly arranged and repeated 3 times. The area of the plot depends on the actual situation, but the area cannot be less than 30 square meters. The selection of the test site requires uniform fertility, consistent crop planting and management levels, and relatively uniform disease occurrence and damage; Protective lines shall be set up around the test area. Use 100, 200, and 400 times of 3 kinds of kiwifruit root bark antiviral agents for constant spraying on the leaves, and use 20% morpholine guanidine. Ethyl copper wettable powder 500 times as the control agent for constant spraying on the leaves, and set clear water Control; all test agents must be diluted twice. The medicine was sprayed since the early stage of the disease, and then sprayed once every 7 days, a total of 3 times. Before the first spraying and after the last spraying, the incidence rate and statistical disease index were investigated for 10 days, and the control effect was calculated. The results are shown in Table 9.

Table 9 Field plot efficacy test of three kinds of kiwifruit root bark antiviral agents in controlling tobacco virus disease

It can be seen from the above table that the three kiwifruit root bark antiviral agents have good control effects on tobacco mosaic virus, which are significantly better than the drug control virus A when diluted 100, 200, and 400 times. The difference in control effect is significant, and 20 The average preventive effect of % kiwifruit root bark microemulsion is better than the average control effect of 20% kiwifruit root bark soluble liquid and 10% kiwifruit root bark water, it can be seen that the kiwifruit root bark extract is prepared into microemulsion, which is more beneficial to plant viruses prevention and treatment.

Claims (4)

1. Chinese gooseberry root bark extract is used for the application for preparing anti-plant virus agent；

Described Chinese gooseberry root bark extract is the material that will be obtained after the progress alcohol extracting of Chinese gooseberry root skin crushed material；

The particle diameter of described Chinese gooseberry root skin crushed material is 1~5mm, and the solvent that alcohol extracting uses is ethanol, and alcohol extracting temperature is 50~70 DEG C；The solvent that alcohol extracting uses is the ethanol of mass fraction 95%, and the alcohol extracting time is 18~20 hours；

Described plant virus includes tobacco mosaic virus (TMV), cucumber mosaic virus and Potyvirus.

A kind of 2. anti-plant virus agent, it is characterised in that the formulation of the anti-plant virus agent is microemulsion, described microemulsion by Following raw materials by weight is prepared：

Chinese gooseberry root bark extract：15%~30%, organic solvent：0~55%, surfactant：5%~20%, prevent Freeze agent：5%~10%, surplus is water, and the percentage by weight sum of raw material is 100%.

3. a kind of anti-plant virus agent, it is characterised in that the formulation of the anti-plant virus agent is soluble liquid, and described is solvable Property liquor is prepared by following raw materials by weight：

Chinese gooseberry root bark extract 15%~30%, organic solvent：0%~55%, surfactant：5%~30%, it is remaining Measure as ethanol, the percentage by weight sum of raw material is 100%.

4. a kind of anti-plant virus agent, it is characterised in that the formulation of the anti-plant virus agent is aqua, and described aqua is by following Raw materials by weight is prepared：

Chinese gooseberry root bark extract：10%~20%, organic solvent：0%~40%, surfactant：5%~30%, Antifreezing agent：5%~10%, surplus is water, and the percentage by weight sum of raw material is 100%.