CN103788074B - The Synthesis and applications of radix aconiti agrestis crude extract and effective constituent - Google Patents

Abstract

The present invention relates to a kind of radix aconiti agrestis crude extract and be therefrom separated the application of compound neoechinulin A in Antiphytoviral obtained, radix aconiti agrestis crude extract of the present invention is for raw material with dry radix aconiti agrestis (Aconitum carmichaeli Debx.), be the alcohol immersion of 70 ~ 95% with concentration after pulverizing and sieving, soak solution obtains ethanol extract through concentrating under reduced pressure, add a small amount of water-dispersion, use sherwood oil successively, ethyl acetate and n-butanol extraction, remaining water layer, the corresponding medicinal extract that the various extraction liquid concentrating under reduced pressure of gained obtain, compound neoechinulin A follows the tracks of to be separated from radix aconiti agrestis ethyl acetate layer by activity to obtain, radix aconiti agrestis crude extract and compound neoechinulin A are to Cucumber Mosaic Virus, tobacco mosaic virus disease, the plant viruses such as southern rice black-streaked dwarf virus disease and oryza virus 3 have good preventive and therapeutic effect.

Description

Preparation and Application of Aconitum Aconitum Crude Extract and Active Components

technical field

The invention relates to the application of a crude extract of Aconitum radix and the compound neoechinulin A isolated therefrom in anti-plant virus, belonging to the field of botanical pesticides in plant protection.

Background technique

In recent years, the hazards of plant virus diseases have become increasingly serious, causing huge economic losses to the agricultural production belt. According to reports, only cucumber mosaic virus, tobacco mosaic virus, rice stripe leaf blight virus and southern rice black streak Tobacco mosaic virus diseases such as dwarf virus disease have caused losses of more than US$ 100 million. At present, there is no very effective method for the prevention and treatment of viral diseases, and the existing commercial medicines for the prevention and treatment of crop viral diseases are few in variety, and the control effect is not ideal enough. Studies have found that the chemical components contained in many natural plants have the characteristics of high efficiency and low toxicity. Finding lead compounds with higher activity from plants has become one of the important ways to develop plant virus inhibitors.

Aconitum aconitum is the dry root tuber of the Ranunculaceae plant Aconitum, aliases: five poisonous roots, heartbroken grass, duck head, medicinal Artemisia annua, chicken head grass, and hundred step grass. Aconitum kusnezoffii has many plant bases, including Aconitum kusnezoffii Reichb, Aconitum Carmichaeli Debx., Aconitum Chinense Paxt, Aconitum Vilmorinianum Kowar , and Aconitum ( Aconitum Delavyl Franch) etc. are mainly wild varieties. There are about 350 species of Aconitum, mainly distributed in Asia, Europe and North America. There are about 167 species in my country, most of which are distributed in the alpine areas of northern Yunnan, western Sichuan, and eastern Tibet. According to literature reports, the main chemical components of Aconitum Aconitum are alkaloids, flavonoids, sterols, and volatile oils, among which diterpene alkaloids are the majority.

In 2013, Huang Liejun et al reported the anti-tobacco mosaic virus (TMV) activity of 13 diterpene alkaloids isolated from Aconitum recemulosum Franch, at a concentration of 30 μg /mL , the anti-TMV activities of compounds racemulosine A, sachaconitine, circinasine A, talatisamine, 8-decetylyunaconitine and 14-acetylyunaconitine were better than the positive control drug Ningnanmycin, and the anti-TMV activity of compounds racemulosine A, sachaconitine, circinasine A and 14-acetylyunaconitine The dyeing activity is particularly remarkable, and the inhibition rate is above 60%. Compounds racemulosine A and talatisamine have significantly better inhibitory activity on TMV proliferation than positive control drugs. (Huang Liejun, Ge Yonghui, Liu Kaixing, Zhang Jianxin, Hao Xiaojiang, Chemical constituents of Aconitum and its activity against tobacco mosaic virus disease, Pesticides, 2013, 52(4), 295-297.) This shows that Aconitum plants are resistant to plant Viruses have a lot of research and development space.

Contents of the invention

The object of the present invention is to provide the preparation and application of the crude extract of Aconitum radix and the compound neoechinulin A. The inventors of the present invention have found that the crude extract of Aconitum aconitum has a good inhibitory effect on plant virus diseases such as cucumber mosaic virus disease, rice stripe leaf blight virus and southern rice black-streaked dwarf virus disease. The crude extract of Aconitum radix was separated, and the compound neoechinulin A, which has a good inhibitory effect on plant virus diseases, was obtained from the ethyl acetate layer.

The crude extract of Aconiti officinale according to the present invention includes the total extract of Aconitum aconitum, the petroleum ether layer extract, the ethyl acetate layer extract, the n-butanol layer extract and the aqueous layer extract of Aconitum aconitum, It is characterized in that dry Aconitum carmichaeli Debx. is used as raw material, crushed and sieved, soaked in ethanol with a concentration of 70-95%, the soaking solution is concentrated under reduced pressure, and the ethanol extract obtained after recovering ethanol is called Aconitum carmichaeli Debx. Aconitum aconitum extract, then add a small amount of water to disperse the ethanol extract, extract with petroleum ether, ethyl acetate and n-butanol in sequence, and leave the water layer, and the obtained various extracts are concentrated under reduced pressure to obtain corresponding extracts, which are respectively called Aconitum aconitum. Petroleum ether layer extract, Aconiti officinale ethyl acetate layer extract, Aconitum officinale n-butanol layer extract, Aconitum officinale water layer extract, plus Aconitum officinale total extract, collectively referred to as Aconitum officinale crude extract. Take the extract from the ethyl acetate layer and dissolve it in ethyl acetate, mix the sample with 100-200 mesh silica gel, pack it into a column with 200-300 mesh silica gel, separate it by medium-pressure column chromatography, and use petroleum ether:ethyl acetate=1:0~0 :1 (V:V) eluent gradient elution, TLC spot plate tracking, collect the eluent with the same polarity, and get 6 components from Fr 2-1 to Fr 2-6. Among them, Fr 2-4 is mixed with 100~200 mesh silica gel, packed into a column with 200~300 mesh silica gel, separated by normal pressure column chromatography, washed with dichloromethane:methanol=1:0~1:1 (V:V) The dehydration gradient elution gave compound neoechinulin A, the structural formula is as follows:

.

The application of the crude extract and active ingredients of the present invention lies in the medicine or medicament for preventing and treating crop diseases.

When the crude extract and compound neoechinulin A of the present invention are used as medicine, they can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 1%-99% of the crude extract of Aconiti Radix or the compound neoechinulin A of the present invention, and the rest are pharmaceutically acceptable, non-toxic and inert carriers and/or excipients for humans and animals. The pharmaceutical carrier or excipient is one or more solid, semi-solid and liquid diluents, fillers and pharmaceutical preparation adjuvants.

In the context of the present invention, it can be a pharmaceutical composition characterized by comprising an effective amount of the crude extract of Aconitum radix or the compound neoechinulin A, and its purpose is to prevent and treat plant virus diseases in agriculture. The pharmaceutical composition contains as an active ingredient at least the crude extract of Aconitum radix or the compound neoechinulin A itself or its mixture with one or more pharmaceutically acceptable inert and non-toxic excipients or carriers.

The invention provides the application of the crude extract of Aconitum radix and the compound neoechinulin A in preventing and treating crop diseases.

The crops include vegetables and cereal crops.

The crop diseases controlled by the crude extract of Aconitum aconitum include cucumber mosaic virus disease, corn stripe leaf blight virus, potato virus X, potato virus Y, rice dwarf virus disease and southern rice black-streaked dwarf virus disease.

The crop diseases controlled by the compound neoechinulin A are cucumber mosaic virus, corn stripe virus, potato virus X, potato virus Y, rice dwarf virus and southern rice black-streaked dwarf virus.

Detailed ways

In order to better understand the essence of the invention, the technical content of the invention is described in detail below with examples, but the content of the present invention is not limited thereto.

Embodiment one

Preparation of crude Aconitum carmichaeli Debx.: crush 26 kg of dried Aconitum carmichaeli Debx., pass through a 40-mesh sieve, add 5 times the amount of 95% ethanol for cold soaking three times, each time for 3 days, and the soaking solution is passed through a rotary evaporator Concentrate under reduced pressure and recover ethanol to obtain 2.0 kg of ethanol extract; the ethanol extract is called the total extract of Aconitum aconitum, and the ethanol extract is dispersed with a small amount of water. Extract with ethyl acetate to obtain the ethyl acetate layer extract of Aconiti officinale, finally extract with n-butanol to obtain the n-butanol layer extract, and finally leave the water layer, and concentrate the extracts of each layer under reduced pressure to obtain the extract. All extracts including ethanol extracts are collectively referred to as Aconitum aconitum crude extract, and 26 kg of dried Aconitum aconitum is extracted by this method to obtain about 250 g of petroleum ether layer extract, which is called Aconitum aconitum petroleum ether layer extract. Get about 150 g of ethyl acetate layer extract, called Aconitum ethyl acetate layer extract. Get about 600 g of n-butanol layer extract, called Aconitum n-butanol layer extract. Obtain about 800 g of aqueous layer extract, called Aconitum aconitum aqueous layer extract.

Embodiment two

The separation of compound neoechinulin A: take the ethyl acetate layer extract and dissolve it with ethyl acetate, mix the sample with 100~200 mesh silica gel, pack the column with 200~300 mesh silica gel, separate by medium pressure column chromatography, and use petroleum ether: ethyl acetate = 1:0~0:1 (V:V) eluent gradient elution, TLC spot plate tracking, collect the eluent with the same polarity, and get 6 components from Fr 2-1 to Fr 2-6. Wherein Fr 2-4 is mixed with 100~200 mesh silica gel sample, 200~300 mesh silica gel column is packed, normal pressure column chromatography separates, with dichloromethane:methanol=1:0~1:1 (V:V) washing The compound neoechinulin A (20 mg) was obtained by gradient elution.

Neoechinulin A, yellow amorphous powder, C ₁₉ H ₂₁ O ₂ N ₃ ; ¹ H NMR (CD ₃ OD, 500 MHz, ppm) δ : 1.53(3H, d, J =6.9 Hz, H-15), 1.55(6H , s, H-19, H-20), 4.23(1H, q, J =20.7 Hz, H-12), 5.10(2H, dd, J =17.8, 10.9 Hz, H-18), 6.11(1H, dd, J =16.9, 9.8 Hz, H-17), 7.07(1H, t, J =14.9 Hz, H-5), 7.13(1H, t, J =14.9 Hz, H-6), 7.21(1H, s, H-8), 7.25(1H, d, J =8.0 Hz, H-7), 7.42(1H, d, J =8.0 Hz, H-4); ¹³ C NMR (CD ₃ OD, 125 MHz, ppm) δ : 20.8(C-15), 28.3(C-19), 28.3(C-20), 40.6(C-16), 52.7(C-12), 104.4(C-9), 112.7(C- 18), 112.8(C-4), 114.5(C-8), 120.0(C-7), 121.3(C-5), 122.7(C-6), 124.8(C-2), 127.5(C-7a ), 137.0(C-3), 146.1(C-1), 146.4(C-17), 162.4(C-10), 168.8(C-13). (1. Yagi R, Doi M. Isolation of an Antioxidative Substance Produced by Aspergillus repens[J]. Bioscience, Biotechnology, and Biochemistry. 1999, 63(5): 932-933. Neoechinulin A [J]. Natural Product Research and Development. 2007(19): 48-50.)

Embodiment Three

Anti-cucumber mosaic virus activity test of the crude extract of Aconitum aconitum and its compound neoechinulin A

1 Living body therapeutic effect

Select amaranthus quinoa with consistent growth, dilute the crude extract of cucumber mosaic virus (CMV) to an appropriate concentration with phosphate buffer, and inoculate it on the leaves of the right age sprinkled with emery (whole leaf inoculation of virus) with a brush. Rinse with clean water afterwards. After the leaves were dry (after 1 h), the drug solution was applied to the right half leaf, and the corresponding dose of solvent was applied to the left half leaf as a control. Afterwards, they were cultured in a light incubator with moisture at a temperature of 25±1°C, observed and recorded the number of dead spots after 5-7 days.

2 Survey and statistics of experimental results

After culturing for 5-7 days, the half leaves of the blank control showed obvious dead spots. Record the number of dead spots on the left and right half leaves of each leaf, and calculate the inhibition rate of plant viruses according to the formula, that is, the relative effect.

Where: Y is the inhibition rate of crude extract or compound to plant virus

C is the number of dead spots in the control group (left half leaf), unit: piece

A is the number of dead spots in the sample treatment group (right half leaf), unit: piece.

Table 1 In vivo therapeutic effect of Aconitum Aconitum crude extract and compound neoechinulin A on CMV infection

The test results showed that the crude extract of Aconiti officinalis and the compound neoechinulin A also had a certain inhibitory effect on cucumber mosaic virus, and the therapeutic activity of neoechinulin A on CMV was equivalent to that of the control drug Ningnanmycin.

Embodiment Four

Anti-tobacco mosaic virus activity test of the crude extract of Aconitum aconitum and its compound neoechinulin A

1 Living body therapeutic effect

Select tobacco with consistent growth, dilute the TMV crude extract to an appropriate concentration with phosphate buffer, and inoculate it on the leaves of the right age sprinkled with carborundum by manual friction with a brush (the whole leaf is inoculated with the virus), and rinse with water after inoculation. After the leaves were dry (after 1 h), the drug solution was applied to the right half leaf, and the corresponding dose of solvent was applied to the left half leaf as a control. Then, they were cultured in a light incubator with moisture at a temperature of 25±1°C. After 3-4 days, the number of dead spots was observed and recorded.

2 In vivo protection

Select the heart leaf tobacco with the same growth potential, apply the compound solution to the right half leaf, and apply the corresponding dose of solvent to the left half leaf as a control. And keep it moist in the light incubator, control the temperature at 25±1℃, dilute the TMV virus crude extract to an appropriate concentration with phosphate buffer solution after 12 hours, use a brush to artificially rub and inoculate it on the leaves of the right age sprinkled with emery (all Leaves inoculated with virus), rinsed with water after inoculation. Afterwards, they were kept moist and cultivated in a light incubator, and the number of scabs was observed and recorded after 3-4 days.

3 In vivo passivation

Select tobacco with the same growth potential, dilute the TMV virus solution to 6×10 ^-3 mg/mL with phosphate buffer solution, mix the drug solution with an equal volume of virus solution to passivate for 30 min, and inoculate the seedlings sprinkled with artificial rubbing with a brush. For the right half of the age-appropriate heart-leaf tobacco with corundum, the corresponding dose of solvent and virus solution was mixed and inoculated on the left half of the age-appropriate heart-leaf tobacco sprinkled with emery, and rinsed with water after inoculation. Afterwards, they were cultured in a light incubator with moisture at a temperature of 25±1°C. After 3-4 days, the number of dead spots was observed and recorded.

The number of dead spots in the control group (left half leaf) and the number of dead spots in the sample treatment group (right half leaf) can refer to the average number of repeated groups or the total number of dead spots repeated in each group. Each treatment uses its own other half as a control, and a group of commercial drug Ningnanmycin is set as a positive control. The preparation of the drug solution was as follows: 2 mg of the sample was dissolved in 40 μL of DMSO, and then 1% Tween was added to 4 mL with a concentration of 500 μg /mL.

4 Investigation and statistics of experimental results

After culturing for 3-4 days, the half leaves of the blank control showed obvious dead spots. Record the number of dead spots on the left and right half leaves of each leaf, and calculate the inhibition rate of plant viruses according to the formula, that is, the relative effect.

Where: Y is the inhibition rate of crude extract or compound to plant virus

C is the number of dead spots in the control group (left half leaf), unit: piece

A is the number of dead spots in the sample treatment group (right half leaf), unit: piece.

Table 2 The in vivo therapeutic, protective and passivation effects of the crude extract of Aconitum Aconitum and the compound neoechinulin A on TMV infection

The test results show that the crude extract of Aconitum aconitum and the compound neoechinulin A have a good inhibitory effect on tobacco mosaic virus, and the inactivation activity of the n-butanol layer extract of Aconitum aconitum and neoechinulin A on TMV is equivalent to that of the control drug Ningnanmycin .

Embodiment five

Activity test of compound neoechinulin A against southern rice black-streaked dwarf virus

Test conditions

1.1 Test target

The white-backed planthopper [ Sogatella furcifera (Horvath)] is kept in the Key Laboratory of Green Pesticides and Agricultural Bioengineering of the Ministry of Education of Guizhou University all year round. Southern rice black streaked dwarf virus (SRBSDV) has been preserved on infected rice for a long time by the Key Laboratory of Green Pesticides and Agricultural Bioengineering of the Ministry of Education, Guizhou University.

1.2 Tested rice

A two-line hybrid late rice variety, Xinliangyou 98.

Test design

2.1 Preparation of test materials

Soak the rice seeds in clean water for 5 minutes, remove the grains and clean them, put them into a fresh-keeping box with a certain capacity, cover them with black cloth, and put them in an artificial intelligence box for germination for about 48 hours (temperature 27±1 ^o C, relative to Humidity 70%~80%), after the seeds germinate, plant them in glass test tubes with a diameter of 4 cm and a height of 15 cm (put an appropriate amount of soil in the glass test tube), and use them for experiments when the seedlings grow to the stage of 2 leaves and 1 heart.

2. 2. Pharmacy

2.2.1 Test drug

The test agent is the original drug of neoechinulin A, as above.

2.2.2 Control drug

The control drug was Ningnanmycin, as above.

2.3 Test procedure

2.3.1 Rearing of white-backed planthopper

Pour the adults of white-backed planthoppers into the turnover box, use the insect sucker to select male and female white-backed planthoppers of the same age, and put them into the cultivated rice seedlings. Put about 100 pairs of white-backed planthoppers into each box of rice seedlings Adults, and then put them into an incubator with a temperature of 27±1oC, a relative humidity of 70%~80%, and a relative light of 66% for mating and reproduction. After the worms hatch and grow to the second instar, they are replaced with new ones. Rearing on rice seedlings, wait for the insects to grow to 3 instars, and use them for indoor bioactivity assays

2.3.2 Preparation of liquid medicine

Take a certain amount of the test agent in the EPP tube, the quality of the monomer compound depends on the needs of the test; select the solvent according to the physical and chemical properties of the test compound, generally the preferred solvent is acetone in the bioassay test of anti-plant virus agents, Followed by DMF and DMSO, the amount of solvent should be taken from less to more, just enough to dissolve the drug, generally controlled at the μL level; the acetone solution (DMF solution or DMSO, etc.) solution containing the test compound is vortexed Oscillate on the instrument to promote its complete dissolution; convert according to the required concentration of the drug to obtain the required amount of PBS (containing 0.05% Tween-80), put it in a beaker, stir on a magnetic particle stirrer, and add appropriate heating , the temperature should not be too high, preferably controlled below 35°C; under the stirring state of PBS, use a micropipette to take the solution of the test compound, and add it dropwise to PBS to make the dispersion state reach the most ideal state.

2.3.3 Poison-infected rice

Inject 2-3 instar poisonous nymphs into test tubes or 100 mL measuring cylinders using an insect extractor. According to the poisoning rate of white-backed planthoppers, 4-5 insects per tube and seedling were inserted into white-backed planthoppers, and the poison was fed for 48 days. After h, the white-backed planthoppers were sucked out with a suction tube.

2.3.4 Chemical treatment

According to the purpose of the experiment, the rice seedlings can be treated with chemicals at different treatment times. Southern rice black-streaked dwarf virus is an insect-borne virus. For the evaluation of the protective activity of antiviral agents: the rice seedlings were treated with the drug before feeding the poisoning on the tested rice seedlings, and then at intervals (6 d, 3 d, 1 d or 12 h), the tested rice seedlings were fed poisoned; for Evaluation of the therapeutic activity of antiviral agents: After inoculating the rice seedlings with poison for 48 hours, the rice seedlings were treated with the agents at certain intervals (6 days, 3 days, 1 day or 12 hours). The method of application can be the seedling soaking method.

3 Determination of control effect

    The total protein of rice plants was extracted, and the total protein was quantified by BCA method. The SRBSDV antibody uses the self-made P10 polyclonal antibody, and the dilution ratio is 1:3000. Alkaline phosphatase-labeled goat anti-rabbit antibody was used as the secondary antibody, and the dilution ratio was recommended in the kit instructions. Quantitative detection of virus was carried out by ELISA method. Coat the extracted total protein on a 96-well ELISA plate, and incubate at 37°C for 1 hr; add the primary antibody, dilute the antibody with 2% BSA (PBST buffer), and incubate at 37°C for 2 hr; use horseradish peroxide as the secondary antibody Enzyme or alkaline phosphatase-labeled goat anti-rabbit/mouse secondary antibody, the working concentration is according to the instructions, 100 μL/well; incubate at 37°C for 1-2 hr; wash 3-5 times with PBST after each step above; Finally, after color development using TMB color development and other color methods, the microplate reader reads the absorption value (OD) at a wavelength of 450 nm, and calculates the virus inhibition rate through the susceptible control and healthy control.

4 results

The test results show that neoechinulin A, a monomeric compound of Aconitum aconitum, has a better protective effect on southern rice black-streaked dwarf virus, and its protective activity against TMV is better than that of the control drug Ningnanmycin, with a control effect of 61.2%, while the control Mycin control effect was 48.2%.

Claims (5)

1. the application of the compound neoechinulin A shown in the following structural formula in anti-plant virus, . 2. The application of the compound neoechinulin A according to claim 1 in anti-plant virus is characterized in that it is used in the medicine or medicament in the prevention and treatment of crop diseases. 3. The application of the compound neoechinulin A according to claim 1 in anti-plant virus, characterized in that when the compound neoechinulin A is used as a medicine, it can be used directly or in the form of a pharmaceutical composition. 4. The application of the compound neoechinulin A according to claim 2 in anti-plant virus, characterized in that the crops are tobacco, vegetables and cereal crops. 5. the application of compound neoechinulin A according to claim 2 in anti-plant virus, it is characterized in that described crop disease is tobacco mosaic virus disease, cucumber mosaic virus disease, corn stripe leaf blight virus, potato X virus , Potato Y virus, rice dwarf virus disease and southern rice black-streaked dwarf virus disease.