WO2007095842A1

WO2007095842A1 - Use of ginkgolic acids in preparing biotic pesticide for killing snails and preventing schistomiasis

Info

Publication number: WO2007095842A1
Application number: PCT/CN2007/000461
Authority: WO
Inventors: Jiahu Pan; Zuohua Mao; Peizhong Yu; Delong Xie; Qi Gao; Xiaohai Pan; Kai Sun
Original assignee: Fudan University
Priority date: 2006-02-21
Filing date: 2007-02-15
Publication date: 2007-08-30

Abstract

The invention provides a new use of 6-alkylsalicylic acids known as ginkgolic acids in preparing biotic pesticide. Particularly, it is directed to the use of ginkgolic acids for killing snails and preventing schistomiasis. The invention makes the most of ginkgo leaves. The invention also relates to a killing-snail agent comprising ginkgolic acids, by which high activity and low toxicity can be achieved. The invention provides furthermore a pesticidal composition for killing snails, which comprises 20-50% ginkgolic acids, 0.1-10%niclosamide, 0.01-1%copper sulfate, and adjuvants. The composition can kill effectively Oncomelania snails as the only host of schistomiasis in china, thus controlling schistomiasis.

Description

Use of ginkgolic acid in preparing biopesticide for killing snails and preventing schistosomiasis

The invention belongs to the field of biological pesticides, and relates to a new use of 6-alkylsalicyl ic acids (6-ASAs), commonly known as ginkgolic acids (GA) in the preparation of biological pesticides, and specifically relates to ginkgolic acid. Use for killing snails, preventing schistosomiasis and as a new biopesticide

The invention also relates to a highly effective and low toxicity acaricidal preparation containing ginkgolic acid. Background technique

The genus Corynebacterium is the only intermediate host of schistosomiasis.

After the snails of the schistosomiasis invade the snail, they can develop into the sputum sputum, which can proliferate thousands of times in the snail, and continuously produce the tail scorpion in the water or moist soil surface, and even infect humans and other animals, so that the schistosomiasis The spread and spread of the disease. Elimination of snails can cut off the transmission of schistosomiasis. Therefore, killing snails is an internationally recognized important measure to prevent schistosomiasis.

Schistosomiasis is prevalent in temperate, subtropical, and tropical regions. In 2001, it is estimated that the global population of schistosomiasis is about 200 million people, 600 million people are threatened, and 76 countries are affected, the vast majority of which are developing countries. The disease has seriously harmed the people of these countries. Health and economic development. There are 12 provinces and 418 counties in China with schistosomiasis epidemics, and about 1 million schistosomiasis patients. The total population of the endemic area is about 100 million people, with a snail area of 3787 km ² . Although China's large-scale control of schistosomiasis in the 1950s and 1960s has effectively controlled the epidemic, in recent years, schistosomiasis has begun to rebound due to factors such as floods, reservoirs, dam construction, and population movements. For example, there are almost all nails in the Dongting Lake area of Hunan Province, and swimming has been banned. The Yueyang, Changde and Yiyang areas along the lake are the hardest hit areas. Therefore, the prevention and control of schistosomiasis has become one of the strategic goals of national sustainable development.

The use of molluscicide is one of the quickest and most effective ways to eliminate or reduce the infection and spread of schistosomiasis.

To date, the use of chemically synthesized acaricides has been the primary means of controlling snails. Sodium pentachlorophenolate has been used in many countries and has a good snail-killing effect, but it has broad-spectrum toxicity to non-target organisms, especially fish and shrimp cultures, and has caused serious environmental pollution. It has long been listed as a banned product by the international community. Niclosamidum has a good anti-snail effect and is recognized as a snail-killing agent in the world. However, it is expensive and has limited use in large areas in developing countries. It can also cause damage to aquatic organisms such as fish when used at high concentrations. It can also lead to increased resistance of target organisms.

In view of the drawbacks of chemical synthesis of snail-killing agents, the international community has given great efforts to develop natural and plant-derived acaricides. Concern. Therefore, it is necessary and urgent to research and develop a new type of molluscicide with good effect, low cost and biodegradability.

Ginkgo biloba acid is another active ingredient other than flavonoids and lactones in ginkgo, which is found in ginkgo leaves and outer seed coats. Ginkgoic acid (6-hydrocarbylsalicylate) is a class of alkyl or alkenylphenolic compounds.

Ginkgo biloba is an important component of ginkgo leaves and their fruits and outer seeds. Ginkgo is one of the oldest species on earth, and it is the "living fossil" left over from the Jurassic. Studies on the existing ginkgo population in China show that its longevity is 2 to 3 thousand years, which is a rare "longevity family" in nature. One of the important reasons for the longevity of ginkgo is that it has significant resistance to common pests and diseases of plants. Ginkgo acid is an important member of its own defense system. Ginkgo biloba is a deciduous tree species. Every year, a large number of ginkgo leaves and outer seed coats degrade and circulate in nature, without damaging the ecological environment. Thus, ginkgolic acid is environmentally compatible and biodegradable.

Ginkgo biloba accounts for 1 to 2% of the dry weight of Ginkgo biloba leaves, and accounts for 3 to 5% of Ginkgo biloba exocarp (dry). In China, the amount of ginkgo leaf extract is 200 tons, and the amount of ginkgo acid in the ginkgo leaf extract is about 2. 2%, that is, the total amount of ginkgo acid removed from the ginkgo leaf extract is about 4. 4 tons. The ginkgo fruit explants (dry) discarded each year amounted to 12,000 tons, including about 420 tons of ginkgolic acid. It can be seen that a considerable amount (more than 400 tons) of ginkgolic acid is discarded every year. The research and development of ginkgolic acid can comprehensively utilize ginkgo leaves and outer seed coats, turning waste into treasure, and generating enormous social and economic benefits.

Now use "6-alkylsalicylic acids" or "ginkgolic acids" and "molluscicide" as keywords, and search for major international patent databases such as US patents, European patents, and WIPO patents, and no related patents have been found.

Using the words "ginkgo biloba", "snail snail" and "6-hydrocarbyl salicylate" as keywords, a Chinese patent CN1792173A was retrieved from the Chinese patent database, "-plant nail snail and its preparation" Method and method of use ", 'application date, · February 30, 2005, public date: June 28, 2006), but the patent application is related to the fat-soluble extract of Ginkgo biloba exocarp (respectively oil Ether, ethyl acetate, ethanol extraction), only two words marked "containing ginkgolic acid", but there is no evidence in the specification and examples to show the correlation between ginkgolic acid and snail activity, nor any Chemical composition identification and content analysis of this extract. The fat-soluble crude extract may contain dozens of fat-soluble components. Therefore, this patent does not have the necessary technical features to support ginkgolic acid as its snail-killing active ingredient.

Extraction and identification of ginkgolic acids, see the following paper:

1. van Beek TA, ffintermans MS. Preparation isolation and dual column high-performance liquid chromatography of ginkgolic acids from Ginkgo biloba. J Chromatogr A. 2001 ; 930: 109~117 2, van Beek, TA. Chemical analysis of Ginkgo biloba leaves and extracts. J Chromatogr A. 2002; 967 (1): 21-55

3, Fuzzati, N, Pace, R, Villa, F. A simple HPLC-UV method for the assay of ginkgolic acids in Ginkgo biloba extracts. Fitoterapia 2003; 74 (3): 247-56

4, Yang Liu-qing, Wu Xiang-yang, Chen Jun. Determination of ginkgolic acids by high performance liquid chromatography. Yaoxue Xuebao 2002; 37 (7): 555-558

In summary, 6-hydrocarbyl salicylic acid is a naturally occurring class of compounds in plants (represented by ginkgo), and there is no report on the activity of killing snails in its biological activity. Summary of the invention

The object of the present invention is to provide a new use of ginkgolic acid in the preparation of biological pesticides, in particular to the use of ginkgolic acid in the preparation of biological pesticides for killing harmful mollusks, in particular to the preparation of ginkgolic acid for killing snails and preventing schistosomiasis. Use in pesticides.

It is still another object of the present invention to provide a composite molluscing agent containing ginkgolic acid.

The invention has been confirmed by experiments that ginkgolic acid can effectively kill the only intermediate host of the widely-populated schistosomiasis, snail and ginkgolic acid, which can be used for the prevention and treatment of schistosomiasis.

The invention opens up a new application of ginkgolic acid to kill snails and prevent schistosomiasis, and not only can change waste into treasure, but also comprehensively utilize ginkgo leaves, and further prepare novel biological molluscicide to prevent schistosomiasis.

The ginkgolic acid (6-hydrocarbyl salicylic acid compound) according to the present invention is an alkyl group or an alkenyl phenolic acid compound, and has a structure of the formula (I), wherein R is a lipid chain and is a main activity for killing snails. Ingredients, mainly in the leaves and outer seed coats of deciduous tree species, Ginkgo biloba, can enter the natural world to degrade and circulate without causing environmental pollution. The structure of ginkgolic acid is different from known chemical synthetic snail-killing agents or plant-derived snail-killing structures (such as semi-indole, triterpenoids, isoflavones, etc.), which are simple in structure, easy to synthesize or structurally modified. The number of carbon (C) atoms on the R group may be 8 to 20 (C8 to 20); the number of double bonds on the R group may be 0 to 4, such as: C13: 0, C13: 1, etc.; The carbon (C) atom on the group may be substituted by other atoms, such as 0, S, N or a halogen element.

The ginkgolic acid is chemically separated from the ginkgo leaf and the ginkgo biloba and its crude extract or pure Obtained in the extract, or recovered from the waste remaining in the extraction process of Ginkgo biloba extract, comprehensively utilized, or obtained from extracts of other parts of the plant containing the compound; or obtained by chemical synthesis , synthesized by salicylic acid as raw material; or further analyzed, identified and controlled by high performance liquid chromatography (HPLC) and other related analytical techniques.

The drug comprising the compound of the present invention may be a crude extract or a pure extract derived from the leaves, outer seed coats and fruits thereof of ginkgo, or may be derived from extracts of various parts containing the compounds of other plants.

The medicament comprising the compound of the present invention can be used alone as a snail-killing agent, or can be used in combination with other snail-killing agents to enhance the snail-killing effect or reduce its toxic side effects and reduce the cost.

The pharmaceutical composition comprising the compound of the present invention may be in a solid form such as a powder, a granule, a liposome or the like. The solid carrier can be a non-toxic, biodegradable, hydrophilic or hydrophobic material. The pharmaceutical composition comprising the compound of the present invention may be in a liquid form such as a solution, an emulsion, a suspension or the like. The liquid carrier can be water or a hydrophilic or hydrophobic organic solvent and a mixture thereof in various proportions in water.

Pharmaceutical compositions comprising a compound of the invention can be sprayed onto water or soil containing snails. It can be sprayed once every 3 to 4 days to enhance the killing effect on the snail.

The invention provides a high-efficiency and low-toxic composite snail-killing agent containing ginkgolic acid, which is mainly composed of ginkgolic acid, and is composed of niclosamide, copper ions and auxiliary materials in the following weight percentage, ginkgolic acid: 20~50%, Niclosamide: 0.1 to 10%, copper sulfate: 0.01 to 1%, and the remainder is an auxiliary material.

The copper ion is a snail-killing synergist which is produced by reacting copper sulfate with sodium hydroxide.

The ginkgolic acid-containing composite snail-killing agent of the present invention can be prepared into a microemulsion by a suitable preparation method of three active ingredients. The microemulsion is relatively stable and easy to produce, store, transport and use.

The preparation containing the formulation of the preparation of the present invention may be in the form of a concentrated liquid preparation such as a microemulsion, an emulsion or the like, or a concentrated solid preparation such as a powder, a granule, a liposome or the like.

The ginkgolic acid-containing composite molluscicide of the present invention is prepared by the following method:

The ginkgolic acid and niclosamide are first dissolved in the co-solvent-ethanol according to the following weight percentage, and the non-ionic emulsifier-Tween 80 is added; and the configured 1 equivalent of sodium hydroxide is added to the quantitative copper sulfate. Prepared into a copper hydroxide colloid; then mixed by a high-speed emulsifier and added with water to prepare a microemulsion; or

The quantitative niclosamide wettable powder is directly dissolved in water, and the ginkgolic acid is dissolved in ethanol and added to Tween 80, and then the two are mixed with the copper hydroxide colloid by a high-speed emulsifier, and water is added to prepare a microemulsion. .

Ginkgo biloba 20~50%,

Niclosamide: 0.1 to 10%, Copper sulphate ·· o.oi~i%,

Sodium hydroxide: 0. 002~0. 2%,

Tween 80: 1~5%,

Water ·· 10~40%

The other is ethanol.

The above ginkgolic acid is extracted from the outer coat of Ginkgo biloba, and includes the following five 6-hydrocarbyl salicylic acid monomer compounds: R = C ₁₃ H ₂₇ (GA-C _{13 : o} ), R = C ₁₆ H ₃₁ (GA) - C _{15 :} .), R = C ₁₅ H ₂₉ (GA- C _{15 : 1} ), R = C ₁₇ H ₃₃ (GA-C _{17 : 1} ), R = C ₁₇ H ₃₁ (GA-C _{n : 2} ). Its mother nucleus is a salicylic acid structure, and the benzene ring has a side chain of 13, 15 or 17 decyl or alkenyl groups.

According to the invention, the snail killing test is carried out according to the immersion method in the WHO "Snail Preparation Laboratory Final Screening Method". The results showed that the effect of ginkgolic acid on killing snails has the following characteristics compared with niclosamide: (1) Ginkgoic acid can effectively inhibit the snail climbing and prevent it from escaping and killing; (2) Ginkgo biloba kills snails The effect is faster, and the death of the snail mainly occurs within 24 hours.

The results of LC50 determination of ginkgolidine killing snails showed that the order of killing snails was: GA-C _{13 : o} > GA-C _{15 : I} > GA- C _{15 :} . 〉 GA-C _{n : i} > GA- C _{17 : 2} . The snail-killing activity was mainly in the former three. The 24-hour LC50 was 13.17 mg/L, 18.57 mg/L, and 26.33 mg/L, respectively. The content of these three in ginkgoic acid was about 70%. The synergistic effect of the body makes the ginkgolic acid achieve a better snail killing effect.

The above research results show that

Among them, the main monomeric compound of ginkgolic acid and the LC50 of guaric acid to kill snails are within the range of the snail-killing agent required by WHO (<100 mg/L). The snail-killing dose of the present invention is from l to 100 mg/L. The dose used will not cause harm to humans and freshwater fish;

Among them, the compatibility of the snail-clopidogrel wettable powder with ginkgolic acid can produce a synergistic effect, greatly reducing the dosage, and the dosage of niclosamide contained is 10~1000 ug/L. The dose used reduces its toxicity to freshwater fish, meets environmental requirements and reduces costs.

Among them, the snail-killing agent-copper ion, by compatibility with ginkgolic acid and niclosamide, can enhance the inhibition of the snail snail climb rate and increase its killing effect, and the dosage is 1~ ppm. The dosage used meets economic and environmental requirements.

The snail-killing agent of the present invention is subjected to a fish toxicity test according to the "Temporary Regulations for Fish Toxicity Test" formulated by the Yangtze River Fisheries Research Institute, and the results show that there is no obvious toxic effect on freshwater fish.

The preparation comprising the snail-killing agent of the present invention may be diluted with water to a suitable concentration at the time of use, and sprayed on the water or soil containing the snail. It can be sprayed once every 3 to 4 days to enhance the killing effect on the snail. DRAWINGS

Figure 1. HPLC chromatogram showing the composition and ratio of each monomer in the total extract of 6-hydrocarbyl salicylic acid:

C13 : 0 = GA-C _13: o (17. 6%) , C15 : l = GA-C _15:1 (52. 3%), C17 : 2 = GA-C _17:2 (3. 6%) , C15: 0 =

GA-C _16: o (3.2%), C17: l - GA- C _17:1 (23. 3%).

Figure 2. Mass spectrometric identification of each monomeric compound in the total extract of 6-light-based salicylic acid.

Fig.3,6-Effects of total hydrocarbyl salicylic acid extracts on the climb rate of snails at different times

Figure 4. Effect of niclosamide on snail climbing rate at different times

Example 1 The effect of ginkgolic acid (C13: 0) component on killing snails

1. Extraction of ginkgolic acid:

100 g of dried and pulverized Ginkgo biloba exocarp was added to petroleum ether (boiling range 60~90 °C) for 1 h in a ratio of 1:5, filtered, and the filter residue was repeatedly extracted twice, and the mixture was extracted and evaporated. A crude extract of the outer seed coat is obtained.

The extract was dissolved in a small amount of petroleum ether and applied to a silica gel column (120 g silica gel, silica gel column was Φ 32 X 400 mm glass column, wet packed column), with petroleum ether: diethyl ether: formic acid = 89: 11 : 1 (V/ V/ V) elution, the fluorescence is detected by thin layer chromatography at 254 nm, the ginkgolic acid component is collected, and concentrated to dryness; the column is repeated once, and the obtained ginkgolic acid component is washed to the middle. The mixture was concentrated to dryness or dried over anhydrous Na ₂ S04 to give a mixture.

2. Purification and analysis of ginkgolic acid (reverse HPLC method):

15cm C18 RP-HPLC column was connected with a 20cm silver-loaded cation HPLC exchange column, using methanol: water = 93: 7 (acidified with 0.1% formic acid) as mobile phase, UV detection wavelength was 311 nm, ginkgolic acid can be used The fractions were isolated and purified and compared to standards for quantitative determination. The purity of C13:0 prepared by this method can reach over 95%.

3. Use the ginkgolic acid (C13: 0) component to soak the snails:

The ginkgolic acid (C13: 0) component was purified by the above-mentioned white crystals with a purity of 95%, dissolved in a small amount of absolute ethanol, and diluted with tap water cooled by boiling to different concentrations: 50 mg/L, 25 mg/L, 12.5 mg. /L, 6.25 mg / L 3.13 mg / L (the final concentration of ethanol is <0.5%). The niclosamide (produced by Huainan Pharmaceutical Factory) was used as a positive control drug, and the niclosamide ethanolamine wettable powder was diluted with boiling water cooled by boiling to the desired concentration. At the same time, tap water cooled after boiling was used as a control with 0.5% ethanol solution.

Snail: taken from the epidemic area of schistosomiasis in Yueyang, Hunan. Male snails were removed by group eschar and cultured in the laboratory for 72 hours. The snails with a screwing age of 8 to 9 are selected for use.

Add 30 ral of the different liquids prepared above in the 25 ral beaker (ie, fill up to the cup). Place 10 snails in each beaker. Cover the nylon mesh with the cup to prevent the snail from climbing out. For the snails immersed in the corresponding liquid and placed in the beaker on the horizontal tabletop, observe the death of the snail at room temperature (22 ° 1 ° 0, respectively at 24 h and 48 h. Observe by acupuncture combined with tapping method) Its activity, determine whether it is dead.

The experimental results are shown in Table 1. In the water and ethanol control group, the activity of the snail was not significantly weakened, and the snail activity of the ginkgolic acid (C13: 0) component decreased, and there was basically no climbing. Table 1: Effect of ginkgolic acid (C13: 0) component on snail snail (48h)

The above experiments were repeated and the results were basically the same. Ginkgo acid (C13: 0) is calculated to kill snails

LD50 = 14.51 mg/L (R = 0.9549) Example 2 The effect of ginkgolic acid (C15: 1) on killing snails

1. Extraction of ginkgolic acid (C15: 1):

The above crude extract of the outer seed coat is dissolved in a small amount of petroleum ether and applied to a silica gel column (120 g of silica gel, a silica gel column is a glass column of Φ32 X 400 awake, and the column is wet-packed), and petroleum ether: diethyl ether: formic acid is used. =89: 11: 1 (V/V/V) elution, the fluorescence was detected by thin layer chromatography at 254 nm, and the fraction with the largest spot was collected and concentrated to dryness to obtain ginkgolic acid (C15: 1). Component.

2. Analysis and purity determination (reverse HPLC method):

A 15 cm C18 RP-HPLC column was coupled to a 20 cm silver-loaded cation HPLC column using methanol:water = 93:7 (acidified with 0.1% formic acid) as the mobile phase. The UV detection wavelength was 311 nra, for the above ginkgolic acid ( C15: 1) The component is subjected to identification and is compared with the standard for purity determination. The purity of the component of the ginkgolic acid (C15:1) was 60.63%.

3. Use the ginkgolic acid (C15: 1) component to soak the snails: '

The above obtained ginkgolic acid (C15: l) component was dissolved in a small amount of absolute ethanol, and diluted with tap water cooled by boiling to different concentrations: 50 mg / L, 25 mg / L, 12.5 mg / L, 6.25 mg / L 3.13mg/L (the final concentration of ethanol is <0.5%) o The niclosamide (produced by Huainan Pharmaceutical Factory) is the positive control drug, and the niclosamide ethanolamine wettable powder is diluted with the tap water cooled by boiling. To the desired concentration. At the same time, tap water cooled after boiling was used as a control with 0.5% ethanol solution.

Snail: taken from the epidemic area of schistosomiasis in Yueyang, Hunan. The male snails were removed by the group sputum method and cultured in the laboratory for 72 hours. The snails with a screwing age of 8 to 9 are selected for use.

Add 30 ml of the different preparations prepared above (ie top up to the cup) in a 25 ml beaker and place 10 snails in each beaker. Cover the nylon mesh with the cup to prevent the snail from climbing out.

For the snails immersed in the corresponding liquid and placed in the beaker on the horizontal tabletop, observe the death of the snail at room temperature (22 ° 1 ° 0, 24 h and 48 h respectively). Observe by acupuncture combined with tapping Its activity, determine whether it is dead.

The experimental results are shown in Table 2. The activity of snails in the water and ethanol control group was not significantly reduced. The snail activity of the ginkgolic acid (C15: 1) component decreased, and there was basically no climbing.

Table 2: Effect of ginkgolic acid (C15: 1) component on snail snail (48h)

The above experiments were repeated and the results were basically the same. The LD50 of the ginkgolidine (C15: 1) (60.63%) component was determined to kill snails: 20.46 mg/L (R = 0.9568). Example 3 The killing effect of the crude extract of Ginkgo biloba exocarp on the snails

1. Preparation of crude extract of Ginkgo biloba exocarp:

100 g of dried and pulverized Ginkgo biloba exocarp was added to petroleum ether (boiling range 60-90 °C) for 1 h in a ratio of 1:5, filtered, and the filter residue was extracted twice, combined and extracted, and petroleum ether was distilled off. A crude extract of the outer seed coat is obtained.

2. Analysis of the content of ginkgolic acid in the crude extract of Ginkgo biloba exocarp (reverse HPLC method):

The total ginkgolic acid content of the crude extract of Ginkgo biloba exocarp was determined by the above RP-HPLC method to be 5.46%.

3. The effect of the extract of Ginkgo biloba exocarp extract on the snails - The crude extract of Ginkgo biloba exocarp is dissolved in a small amount of absolute ethanol and diluted with tap water cooled by boiling until the crude extract content is 62.5 mg. /L (final concentration of ethanol <0.5%). Taking niclosamide (produced by Huainan Pharmaceutical Factory) as a positive control drug, the niclosamide ethanolamine wettable powder is diluted with boiled tap water to the desired concentration. At the same time, tap water cooled after boiling was used as a control with 0.5% ethanol solution.

Add 30 ral of the different preparations prepared above in a 25 ml beaker (ie top up to the cup). Place 10 snails in each beaker and cover the nylon mesh to prevent the snails from climbing out. For the snails immersed in the corresponding liquid and placed in the beaker on the horizontal tabletop, observe the death of the snail at room temperature (22 ° 1 ° 0, 24 h and 48 h respectively). Observe by acupuncture combined with tapping Its activity, determine whether it is dead.

The results were as follows: crude extract of Ginkgo biloba exocarp 62.5mg/L 24h snail mortality was 60%, and 48h mortality was 100%. In the water and ethanol control group, the activity of the snail was not significantly weakened, and the activity of the snail treated by the extract decreased, and there was basically no climbing. Example 4 Synergistic killing effect of ginkgolic acid (C15: 1) component and niclosamide on snail

1. Preparation of ginkgolic acid (C15: 1 ) component and niclosamide mixture

The purity of the ginkgolic acid (C15:1) component was determined to be 60.63% by HPLC. The ginkgolic acid (C15: l) component is dissolved in a small amount of absolute ethanol, diluted with tap water cooled by boiling, mixed with niclosamide ethanolamine powder which has been prepared by boiling water cooled, and then boiled. The post-cooled tap water is diluted to a final concentration of 10 mg/L and 0.5 mg/L, respectively, of the ginkgolic acid (C15:1) component and the niclosamide ethanolamine wettable powder.

2. The effect of ginkgolic acid (C15: 1 ) component and niclosamide mixture on snails

The niclosamide (produced by Huainan Pharmaceutical Factory) was used as a positive control drug, and the niclosamide ethanolamine wettable powder was diluted to the desired concentration with tap water cooled by boiling. At the same time, tap water cooled after boiling was used as a control with 0.5% ethanol solution.

Snail: taken from the epidemic area of schistosomiasis in Yueyang, Hunan. The male snails were removed by the group sputum method, and the snails were selected to be snails in the laboratory for 72 hours.

Add 30 ml of the different preparations prepared above in a 25 ml beaker (ie top up to the cup). Place 10 snails in each beaker and cover the nylon mesh to prevent the snail from climbing out.

For the snails immersed in the corresponding liquid and placed in the beaker on the horizontal tabletop, observe the death of the snail at room temperature (22±ΓΟ, at 24h and 48h respectively. Observe the activity by acupuncture combined with tapping method In the case, determine whether it is dead.

The results showed that in the mixture of ginkgolic acid (C15: 1) and niclosamide, the mortality rate of snails was 80% and the mortality rate was 100%. In the water and ethanol control group, the motility of the snails was not significantly reduced, and the snails treated by the extracts decreased, and there was basically no climbing. Example 5 Decoupling effect of ginkgolic acid on oxidative phosphorylation of snail mitochondria

1. Preparation of ginkgolic acid (C15: 1) components The purity of the ginkgolic acid (C15: 1 ) component was determined to be 60.63% by HPLC. The ginkgolic acid (C15: 1 ) component was dissolved in a small amount of absolute ethanol, and diluted with tap water cooled by boiling, and used.

2. Preparation of snail mitochondria and determination of oxidative phosphorylation

A group of energetic adult snails were taken and the male snails were removed by group sputum method and cultured in the laboratory for 72 hours. Select a screw with a screwing age of 8 to 9 to be used as a screw. The shell was broken by a broken snail method, and the head and foot of the snail were taken, and the mitochondria were prepared by centrifugation according to the literature method. The mitochondrial respiratory control rate (RCR) was determined by oxygen electrode method, and the change of phosphorus and the activity of ATPase were measured by the malachite green method to study the cleavage of mitochondrial oxidative phosphorylation by ginkgolic acid (C15: 1 ) component. Joint action.

The results showed that the ginkgolic acid (C15: 1) component dose-dependently increased the content of inorganic phosphorus and ATPase in the dose range (10 mg/L, 20 mg/L, 40 mg/L). , reducing its RCR, indicating that the ginkgolic acid (C15: 1) component has a good decoupling effect on oxidative phosphorylation of the snail mitochondria.

Example 6 Extraction, Isolation, Purification and Identification of Ginkgoic Acid (6-Hydroxy Salicylic Acid)

1. Extraction of 6-hydrocarbyl salicylic acid compounds

100 g of Ginkgo biloba exocarp was dried at 50 ° C and then mechanically pulverized, and petroleum ether (boiling range 60 to 90 ° C) was added in a ratio of 1:5 for ultrasonic extraction for 1 h, filtered, and the residue was repeatedly extracted twice, and the extract was combined. Recover petroleum ether to obtain crude extract of external seed coat' extract.

2. Separation and purification of 6-hydrocarbyl salicylic acid compounds

Dissolve the extract in small portions with petroleum ether and apply to the silica gel column (silica column is Φ 32 X 400 mm glass column, silica gel 60, 48~75 um, wet packed column), with petroleum ether: ether : Formic acid = 89: 11 : 1 (V / V) Elution, collect the fractions of the eluate, and use thin layer chromatography to detect the single fluorescent spots appearing at 254 nm, and with the standard (GA-C _{13 :} o) Control, collect the components of the spot that are exactly the same as the standard control, concentrate to dryness; repeat the column once to obtain the total extract component of 6-hydrocarbyl salicylic acid, concentrate to dryness, or dry with anhydrous N3⁄4S04 , the total extract of 6-hydrocarbyl salicylic acid has a purity of more than 90%.

Purification of each 6-hydrocarbyl salicylic acid monomer was carried out on a Water HPLC preparative column chromatography system. The total extract of 6-hydrocarbyl salicylic acid was dissolved in petroleum ether and applied to an Elit Kromasil C18 column (4.6 x 300 mm, 5 um). The mobile phase was methanol: water: trifluoroacetic acid = 85: 15: 0. 01 (V/V), UV detection wavelength is 310 nm, flow rate is 2.0 ml/min, column temperature is 45 ^a C. The obtained eluate containing each component was concentrated and dried. The purity of each 6-hydrocarbyl salicylic acid monomer prepared in this way can be more than 95%. Figure 1 is a HPLC chart showing the composition and ratio of each monomer in the total extract of 6-hydrocarbyl salicylic acid: C13 : 0 = GA-C _13:0 (17. 6%) , C15 : l = GA- C _{15 :1} (52.3%), C17 :2 = GA_C _17:2 (3. 6%), C15 : 0 = GA-C _15:0 (3.2%), C17 : l = GA-C _{17: 1} (23.3%). 3. Identification of 6-hydrocarbyl salicylic acid compounds

Identification of each 6-hydrocarbyl salicylic acid monomer was carried out on an Agilent 1100 liquid-mass line. Detected with the G1946D MS detector and diode array detector. HPLC column is Agilent Zorbax SB-C18 reverse phase column (150 x 2. 1 awake, 5 um), mobile phase is methanol: water (containing 0.1% trifluoroacetic acid) = 87: 13 (V/V), UV The detection wavelength was 310 nm, the flow rate was 0.3 ral/min, and the column temperature was 35 °C.

The mass spectrum was obtained by negative ion mode and recorded in the Λ?/250~450 interval. The capillary column temperature was 300 ^e C and the gas flow rate was 10 L/min. The injection voltage in the negative ESI mode is 3500 V. See Figure 6-6 for the mass spectrometric identification of each monomeric compound in the total hydrocarbyl salicylate extract.

Example 7 Extraction by other routes 6-hydrocarbyl salicylic acid total extract

In addition to the above-mentioned total extraction of 6-hydrocarbyl salicylic acid by direct extraction from an organic solvent, other routes can be used to extract and prepare a total extract of 6-hydrocarbyl salicylic acid. For example, the acidity of 6-hydrocarbylsalicylic acid is extracted by the following alkali extraction method.

1 kg of Ginkgo biloba exocarp was dried at 50 ° C and then mechanically pulverized. Add NaOH solution (1 ～ 5%) in a ratio of 1:3, ultrasonically extract for 1 h, filter, filter residue was extracted twice, and the extract was combined. The precipitate was precipitated by a hydrochloric acid solution of _P H = 1 to 4, and extracted with chloroform to recover chloroform to obtain a crude extract of the outer seed coat. Then, separation was carried out by the above column chromatography to obtain a total extract of 6-hydrocarbyl salicylic acid. HPLC analysis showed that the content and proportion of each monomer component in the total extract of 6-hydrocarbyl salicylic acid obtained by this route were similar to those of the total extract of 6-hydrocarbyl salicylate obtained by the above method.

Example 8 Determination of the activity of each monomeric compound of 6-hydrocarbyl salicylic acid to kill snails

Each of the 6-hydrocarbyl salicylic acid monomer compounds is isolated and purified by the above method, all of which are white crystals with a purity of >95%, dissolved in a small amount of absolute ethanol (final concentration <0.5%), and added with a small amount of emulsifier or cosolvent. (Tween-80, final ethanol concentration <1%), and then diluted to different concentrations with tap water cooled after boiling: 50mg / L, 25mg / L, 12.5mg / L, 6.25mg / L, 3.13mg / L. The niclosamide ethanolamine wettable powder (manufactured by Huainan Pharmaceutical Factory) was used as a positive control drug, and it was diluted with boiling water cooled by boiling to the desired concentration. At the same time, tap water cooled after boiling was used as a control with 0.5% ethanol solution.

Snail: collected from the epidemic area of schistosomiasis in Yueyang County, Hunan Province. The male snails were removed by the group sputum method, and the snails with the snail age of 8 to 9 were selected in the laboratory for 72 ho.

For the snails immersed in the corresponding liquid and placed in the beaker on the horizontal tabletop, observe the snail climb rate at room temperature (22 ± ,, 5, 10, 15, 30, 60, 120 minutes, respectively, and record Snails at 12 h, 24 h and 48 h The death situation. The activity was observed by acupuncture combined with tapping to determine whether it died.

The experimental results were analyzed and the LD50 and LD90 of each 6-hydrocarbyl salicylate monomer were calculated at 12h, 24h and 48h. The activity of the snails was compared by the LD50 value of each 6-hydrocarbyl salicylic acid monomer at 24 h. (See Table 1) LD _5Q and LD ₉₀ values for killing snail activity of each 6-hydrocarbyl salicylic acid monomer

(mg-L ¹ ) GA-C13.0 GA-Ci5; i GA-C-i5: o GA-Ci7; i GA-C ₁₇ : ₂

LC ₅₀ (12 h) 37.76 58.32 62.72 - 62.47

64.92 88.51 52.17 - 93.01

LC ₅₀ (24 h) 20.79 27.28 33.76 51.89 59.10

LC _go (24 h) 38.95 59.95 52.17 61.47 83.46

LC ₅₀ (48 h) 5.06 3.27 15.40 13.69 17.53

LC ₉₀ (48 h) 9.50 4.49 30.12 30.09 26.08

The order of strength of 6-hydrocarbyl salicylic acid monomer compounds to kill snails is:

GA C ₁₃ 0 > GA-C ₁₅ > GA-C ₁₅ > GA-C ₁₇ , > GA- C _l7 : ₂ . Take GA-C ₁₃ :. It is the most active compound and can be used as a quality control indicator for this type of molluscicide. Example 9 Determination of the activity of 6-hydrocarbyl salicylic acid total extracts for killing snails

The total extract of 6-hydrocarbyl salicylic acid can be obtained by extraction and separation by the above method, and its purity is >90%. Dissolve with a small amount of absolute ethanol (final concentration <0.5%), add a small amount of emulsifier or co-solvent (Tween-80, final ethanol concentration <1%), and dilute to different concentrations with tap water cooled by boiling: 50mg/ L 25mg/L 12. 5mg/L 6. 25mg/L 3. 13mg/L with niclosamide ethanolamine wettable powder (produced by Huainan Pharmaceutical Factory) as a positive control drug, which is cooled after boiling: tap water Dilute to the desired concentration. At the same time, a tap water cooled after boiling was used as a control with a 0.5% ethanol solution.

Snail: collected from the epidemic area of schistosomiasis in Yueyang County, Hunan Province. The male snails were removed by the group sputum method, and the snails were selected to be snails in the laboratory for 72 ho.

For the snails immersed in the corresponding liquid and placed in the beaker on the horizontal tabletop, observe the death of the snail at room temperature (22 ΓΟ, 12 h, 24 h and 48 h respectively). Observe the activity by acupuncture combined with tapping In the case, determine whether it is dead.

The experimental results were analyzed and the LD50 and LD90 of the total extracts of 6-mercaptosalicylic acid were calculated for 12h, 24h and 48h. (See Table 2)

Example 10 Effect of total extract of 6-hydrocarbyl salicylic acid on the climb rate of snail

The total extract of 6-hydrocarbyl salicylic acid prepared above is dissolved in a small amount of absolute ethanol (final concentration < 0.5%), and a small amount of emulsifier or cosolvent (Tween-80, final concentration of ethanol <1%) is added. Dilute with tap water cooled after boiling to different concentrations: 50 mg / L, 25rag / L, 12. 5 mg / L, 6. 25 mg / L, 3. 13 mg / L. The niclosamide ethanolamine wettable powder (manufactured by Huainan Pharmaceutical Factory) was used as a positive control drug, and it was diluted with boiled and cooled tap water to the desired concentration. At the same time, tap water cooled after boiling was used as a control with 0.5% ethanol solution.

For the snails immersed in the corresponding liquid and placed in the beaker on the horizontal tabletop, the snail climbing rate was observed at room temperature (22 1 Ό) at 15 min, 30 min, l h and 2 h, respectively. It can be seen from Fig. 3 and Fig. 4 that the total extract of 6-hydrocarbyl salicylic acid and niclosamide have significant differences in the climbing rate of snails. The total extract of 6-hydrocarbyl salicylate 30 miniB showed a dose-dependent inhibition of snail climbing, showing significant inhibition at lh, while niclosamide showed a tendency to inhibit from 2 h. Therefore, the climb rate of the snail can be used to reflect the effect of the snail on the climb rate. Example 11 Preparation of 6-hydrocarbyl salicylic acid composite molluscicide

The composite snail-killing agent is mainly composed of the above-mentioned 6-hydrocarbyl salicylic acid total extract, and is scientifically compatible with niclosamide, copper ions and other auxiliary materials according to the following weight percentages to form a new composite snail-killing agent:

6 -hydrocarbyl salicylic acid: 10~60%, niclosamide: 0. 1~20%, copper ion: 0. 01~5%, the rest are auxiliary solvents such as cosolvents, emulsifiers and stabilizers. The 6-hydrocarbyl salicylic acid is a total extract of 6-hydrocarbyl salicylic acid having a relatively constant content ratio of each monomer compound; niclosamide is a wettable powder; copper ion is a snail-killing synergist, ie A copper hydroxide colloid produced by the reaction of copper sulfate with sodium hydroxide.

First, the total extract of 6-hydrocarbyl salicylic acid is dissolved in a small amount of absolute ethanol (final concentration <0.5%), and a small amount of emulsifier or cosolvent (Tween-80, final concentration <1%) is added, and chlorine is added. Nitramide, copper hydroxide colloid and stabilizer are then thoroughly mixed by an idler emulsifier or a homomixer, and water is added to prepare a 0/W type microemulsion or emulsion. This method produces a high concentration of stock solution for production, storage, transportation and use. When used, it can be diluted with water to the desired concentration.

Example 12 Synergistic snail killing effect of total extract of 6-hydrocarbyl salicylic acid and niclosamide

6-hydrocarbyl salicylic acid total extract is dissolved in a small amount of absolute ethanol, added with a suitable emulsifier, by high-speed emulsification Prepare the emulsion (the final concentration of ethanol is <0.1%). The niclosamide wettable powder is formulated into a stock solution using tap water cooled after boiling. According to the ratio of Table 3, two liquids were separated and prepared into test liquids containing different concentrations of 6-hydrocarbyl salicylic acid total extract and niclosamide. At the same time, the tap water cooled after boiling was used as a blank control.

The snail-killing test was carried out according to the soaking method in the WHO "Spermicide Laboratory Final Screening Method".

Snail: From the epidemic area of schistosomiasis in Yueyang, Hunan. The male snails were removed by the group sputum method, and the snails with a snail age of 8 to 9 were selected in the laboratory for 72 o.

For the snails immersed in the corresponding liquid and placed in the beaker on the horizontal tabletop, observe and record the upswing rate of the snail for 1 hour at room temperature (22 和 and observe and record the death of the snail at 24 h and 48 h) The situation was observed by the acupuncture method combined with the tapping method to determine whether it died. Table 3 shows the results of the synergistic effect of niclosamide and 6-hydrocarbyl salicylate.

Synergistic snail killing effect of 6-hydrocarbyl salicylic acid extract and niclosamide

Group 1 2 3 4 5 6 7 8 Niclosamide (ug L) 100 50 25 12.5 6.25 3.13 0 0

6-ASAs (mg/L) 0 1.25 2.5 5 10 20 40 0

1 h climb rate (%) 90 70 90 90 70 10 10 100

24 h mortality (%) 70 10 20 30 40 60 90 0

48 h Mortality (%) 90 10 40 100 100 100 100 0 For snails soaked in the corresponding liquid and placed in a beaker on a horizontal tabletop, observe at room temperature (22 ± ΓΟ and see from the above results, with The concentration of niclosamide increased and the concentration of 6-hydrocarbyl salicylate decreased, and the snail climb rate was not affected. However, as the concentration of niclosamide decreased, the concentration of 6-hydrocarbyl salicylate increased. The snail snail climb rate was significantly reduced; at the same time, 6-hydrocarbyl salicylic acid increased the mortality of snails in a dose-dependent manner. Example 13 Copper ion on 6-hydrocarbyl salicylic acid and niclosamide snail killing effect Synergistic effect

The total extract of the quantitative 6-based salicylic acid is dissolved in the co-solvent-ethanol, and the niclosamide and the emulsifier-Tween 80 (1/10 of the amount of ethanol) are added; the other 1 equivalent of sodium hydroxide is configured. Adding quantitative copper sulfate, preparing copper hydroxide colloid and diluting to different concentrations; then preparing the test solution according to the ratio of Table 4 (the concentrations shown in the table are the final concentration), and then mixing the two by a high-speed emulsifier And added water to prepare a microemulsion. At the same time, tap water cooled after boiling was used as a blank control.

The snail-killing test was carried out according to the immersion method in the WHO "Spiral Laboratory Final Screening Method". Snail: collected from the epidemic area of schistosomiasis in Yueyang, Hunan. The male snails were removed by the group sputum method, and 72 snails were selected in the laboratory culture for 8~9 rotation.

For the snails immersed in the corresponding liquid and placed in the beaker on the horizontal tabletop, at room temperature (22+1 Ό), observe and record the snail climb rate for 1 hour and observe and record the death of the snail for 12 hours. The activity was observed by the acupuncture method combined with the tapping method to determine whether it died. Table 4 shows the synergistic effect of copper ions on the killing of snails by 6-hydrocarbyl salicylic acid and niclosamide in the preparation of the present invention.

Table 4 Synergistic effect of copper ions on the snail-killing effect of 6-hydrocarbyl salicylic acid and niclosamide

Group 1 2 3 4 5 6 7

6-ASAs (mg L) 2.5 2.5 2.5 2.5 2.5 0 0 Niclosamide (ug/L) 50 50 50 50 50 0 .0

Cu ²⁺ (ppm) 8 4 2 1 0 2 0

1 h climb rate (%) 0 0 0 30 10 20 100

12 h Mortality (%) 70 50 30 10 0 0 0 From the above results, it can be seen that the addition of copper ions can effectively inhibit the snail snail climb rate and increase the snail snail death rate within 12 hours, which plays a very good synergistic effect. EXAMPLES, Toxicity Tests of Total Hydrocarbon Salicylic Acid Extracts and the above Compound Ceromers to Freshwater Fish

4000 mL of microemulsion containing different concentrations of 6-hydrocarbyl salicylic acid extract (5 mg/L, 10 mg/L, 20 mg/L,) was prepared as described above. Take 50 mL from it for culture of snails. The rest is used to culture freshwater fish - red carp fry. The fish toxicity test was carried out according to the “Interim Regulations on Fish Toxicity Test” formulated by the Yangtze River Fisheries Research Institute.

A composite microemulsion 4000 mL containing 6-hydrocarbyl salicylic acid (5 mg/L), niclosamide (50 ug/L), and copper ions (6 ppm) was prepared as described above. Take 50 mL from it for culture of snails. The rest is used to culture freshwater fish - red carp fry. The fish toxicity test was carried out according to the “Temporary Regulations for Fish Toxicity Test” formulated by the Yangtze River Fisheries Research Institute.

Snail: From the epidemic area of schistosomiasis in Yueyang, Hunan. The male snails were removed by the group sputum method, and the snails with the snail age of 8 to 9 were selected in the laboratory culture. Add 30 ml of the above solution to the 25 ml beaker (ie, fill up to the cup), put 10 snails, and cover the nylon mesh to prevent the snail from crawling out. For the snails immersed in the corresponding liquid and placed in the beaker on the horizontal tabletop, observe and record the death of the snails at room temperature (22+ΓΟ). Observe the activity by acupuncture combined with tapping method. Determine if it is dead.

Take 10 red mullet (length 7~8 cm) into the above prepared solution, and keep it at regular temperature, water temperature 20 ^Q C, After 72 hours of observation, the death of red snapper was recorded.

The results showed that the snail mortality was 60% in the 5 mg/L total extract of the 6-hydrocarbyl salicylate total extract, and the snail mortality was 100% in the other dose groups and the composite snail group. All the red snapper cultured in each group survived after the 72-hour observation period, and none died. This indicates that the total extract of 6-hydrocarbyl salicylic acid and the above-mentioned composite mollusc have low toxicity to freshwater fish, and have high efficiency and low toxicity.

Claims

1. The use of ginkgolic acid in killing snails of biological pesticides.

The use according to claim 1, characterized in that said ginkgolic acid (6-hydrocarbyl salicylic acid compound) is an active ingredient in ginkgo biloba and ginkgo biloba exocarp having a structure of the formula (I) ,

right

Wherein R is a fatty chain having a carbon number of 8 to 20 on the R group; and the number of double bonds on the R group is 0 to 4, such as: C13: 0, C13: 1, C13: 2, C13: 3 Any one of C13:; the carbon atom on the R group may be substituted by other atoms 0, S, N or a halogen element.

3. Use according to claim 2, characterized in that the medicament comprising ginkgolic acid is used alone as a snail-killing agent or in combination with other snail-killing agents.

4. Use according to claim 2, characterized in that the pharmaceutical composition comprising ginkgolic acid is in solid form or in liquid form, including any of powders, granules, liposomes, solutions, emulsions or suspensions.

5. A composite molluscicide according to claim 2, characterized in that the ginkgoic acid is the main drug, and further comprises niclosamide, copper ions and an auxiliary material, which are composed of the following weight percentages: ginkgolic acid : 20~50%, niclosamide: 0.1~ should, copper sulfate: 0.01~: L%, the remainder is the auxiliary material.

The ginkgolic acid-containing composite molluscing agent according to claim 5, wherein the ginkgolic acid comprises the following five 6-hydrocarbyl salicylic acid monomer compounds:

(GA-C _{15 : 1} ), R=C ₁₇ H ₃₃ (GA-C _{17 : 1} ) , R=C ₁₇ H ₃₁ (GA-C _{I7 : 2} ) , the mother nucleus is salicylic acid structure, benzene ring There is a side chain of 13, 15 or 17 alkyl or alkenyl groups; the copper ions are produced by the reaction of copper sulfate with sodium hydroxide.

A ginkgolic acid-containing composite snail-killing agent according to claim 5 or 6, wherein said composite snail-killing agent is formulated into a concentrated liquid preparation comprising a microemulsion or an emulsion.

A method for preparing a ginkgolic acid-containing composite molluscing agent according to claim 5 or 6 or 7, which comprises the steps of:

According to the following weight percentage, firstly, the quantitative ginkgolic acid and niclosamide are dissolved in the co-solvent-ethanol, and the non-ionic emulsifier-Tween 80 is added; the prepared 1 equivalent of sodium hydroxide is added to the quantitative copper sulfate to prepare Copper hydroxide a body; then mixing the two by an idler emulsifier and adding water to prepare a microemulsion; or

The quantitative niclosamide wettable powder is directly dissolved in water, and the ginkgolic acid is dissolved in ethanol and added to Tween 80, and then the two are mixed with the copper hydroxide colloid by an idle speed emulsifier, and water is added to prepare the micro Emulsion,

Ginkgolic acid: 20~50%

Niclosamide: 0.1 to 10%

Copper sulfate: 0.01~1%

Sodium hydroxide: 0. 002~0. 2%

Tween 80: 1 to 5%

Water: 10~40%

The balance is ethanol.

A ginkgolic acid-containing composite snail-killing agent according to claim 5 or 6, which is characterized in that said composite snail-killing agent further comprises a concentrated solid preparation comprising a powder, granule or liposome.