AU2015394961A1 - Use of plant crude extracts of Pueraria peduncularis for preparation of molluscicides - Google Patents

Abstract

Provided is a use of plant crude extracts from Pueraria peduncularis for preparation of pesticides against pomacea canaliculata and oncomelania snails, the method of extracting crude extracts from Pueraria peduncularis comprising the steps of: (1) drying the roots of Pueraria peduncularis at 50°C and grinding the dried roots of Pueraria peduncularis into crude pieces of a size ranging from 1 mm-5 mm; (2) using ether petroleum and chloroform as extraction solvents to extract the crude pieces obtained in step (1) in a sequential order, the weight ratio of the crude pieces to the extraction solvents being 1:3, and subsequently using methanol, 1-butanol, ethanol or isopropyl alcohol, or a combination thereof, as an extraction solvent to carry out the extraction at 40-70°C for 12-24 hours three times; and concentrating and drying the extracted liquid to obtain a concentrate, namely crude extracts of Pueraria peduncularis.

Description

THE APPLICATION OF THE CRUDE EXTRACT FROM PUERARIA PED UNCULARISIS (GRAH. EX BENTH.) BENTH. FOR PREPARING MOLLUSCICIDES I FIELD OF THE INVENTION]

The present invention belongs to the field of pesticide. More specifically, the present invention relates to the application of the crude extract from Pueraria peduncularisis (Grah. ex Benth.) Benth., for preparing molluscicides against Pomacea canaliculata and Oncomelania hupensis Gredler. iBACKGROUND OF THE INVENTION] P. canaliculata and O. hupensis are the main harmful snails in China. P. canaliculata has been an invasive species in China since the 1980s and, due to its rapid reproduction rate, quickly spread to most of the southern provinces of China. It can cause great harm to rice production by feeding on rice plants, and more seriously can transmit the parasitic nematode Angiostrongylus cantonensis, which is the most common cause of eosinophilic meningitis in humans. O. hupensis, the intermediate host of Schistosoma japonicum, is widely distributed in most provinces of China. Schistosomiasis continues to be a significant parasitic disease and is severely affecting human health. Accordingly, control of these two snails is very important and carries significant implications for agriculture production and epidemic prevention.

At present, chemical control is one of the most important methods to regulate harmful snails mainly through the application of a few molluscicides, such as niclosamide and metaldehyde. Among them, niclosamide is the only recommended molluscicide by WHO since 1972, which has a good killing effect of adult snails, young snails and the eggs, and has a long effect. It has low toxicity to human and animal while has strong toxicity to fish. Niclosamide at the effective concentration for killing snails can cause massive death of aquatic organisms and upward climbing phenomenon of P. canaliculata , which seriously influences its molluscicidal effect. 1 he extract trom the iruit ot Balanites aegyptiace ot Balanjtaceae plant was tirst confirmed to have noticeably toxic effects on snails by Archibald R.G in 1933. Since then, an increasing number of plants have gradually been found to have abundant active substances for snails’ control, and most of the active molluscicidal compounds can markedly inhibit the upward climbing of the snails and are relatively safe for aquatic organisms. These findings point out a new direction for the development of molluscicide.

Pueraria peduncularis Grah., which belongs to the Pueraria family within the Leguminosae, is the peculiar Chinese medicinal herb in China and distributed widely in Tibet, Yunnan, Sichuan, Guangxi and other places. Research on Pueraria peduncularisis (Grah. ex Benth.) Benth generally focuses on its medical activity, such as heat-clearing, eruption-promoting, thirst-quenching and insecticidal activity and so on. The extracts of this species are reported to have some degree of bioactivity against Rhizoctonia solani, Venturia nashicola, and Fusarium semitectum. It has also been shown that P. peduncularis extracts have a toxic effect on the com borer, cabbage caterpillar, and on aphids. But few studies have evaluated its potential as a control agent of snails, and the molluscicidal mechanism and the molluscicidal component are still unknown. I SUMMARY OF III I INVENTION]

One object of the present invention is to provide a botanical molluscicide, which uses the crude extract from roots of Pueraria peduncularisis (Grah. ex Benth.) Benth. produced by a specific extraction method as the material, for controlling P. canaliculata and O. hupensis.

[Technical Plan]

The present invention is achieved by the following technical plan.

The present invention relates to a botanical molluscicide, the said botanical molluscicide contains the crude extract from roots of Pueraria peduncularisis (Grah. ex Benth.) Benth., solvents and surfactants. The content of the crude extract from roots of P. peduncularis , solvent and surfactants is 1U-jU/o (w/wj, Zv-jO /o (w/w) and j-όν/ο (w/w) respectively.

The said crude extract from roots of P. peduncularis can be obtained by the method as follows:

Step 1: The root samples of P. peduncularis are dried using a drying oven at 50 °C and ground to 1-5 mm using a powder grinder, to obtain coarse powder of P. peduncularis;

Step 2: The coarse powder obtained in Step 1 is successively extracted with petroleum ether and chloroform (the said coarse powder and the extraction solvent by weight ratio of 1:3), and then the residue is extracted three times with one or more solvent(s) selected from methanol, ethanol, n-butanol or iso-propanol (the said coarse powder and the extraction solvent by weight ratio of 1:2) at the temperature of 40-70°C for 12-24h. Finally, the combined extracts are concentrated and dried, thus obtaining the crude extract of P. peduncularis root.

Preferably, the dosage of the crude extract of P. peduncularis root is 10-30% by weight, and more preferably 15%.

The said solvent of the molluscicide can be one or more solvent(s) selected from cyclohexanone, ethyl acetate, methanol, n-butanol, ethanol, iso-propanol and water, and n-butanol is preferable.

The said surfactant of the molluscicide can be one or more surfactant(s) selected from styrylphenol phenol formaldehyde resin polyoxethylene ether, styrylphenol phenol formaldehyde resin polyoxyethylene polyoxypropylene ether, lauryl alcohol polyoxyethylene ether, phenylethyl polyoxyethylene ether, alkylphenol polyoxyethylene ether and calcium dodecyl benzene sulfonate, and the mixture of alkylphenol polyoxyethylene ether and calcium dodecyl benzene sulfonate in a 1:3 radio (w/w) as an emulsifier is preferable.

Preferably, the above-mentioned molluscicide is made into a microemulsion by the following steps: mix the above-mentioned extract from roots of P. peduncularis, the solvent and the surfactant according to the formulation in the modulation reactor at the temperature of 40-60°C for 1 h , stir uniformly, then a microemulsion of moitusciciae containing r. peduncularis is obtained.

Usage: the microemulsion of molluscicide containing P. peduncularis extract can be used for the spray only after dilution.

[Beneficial Effects]

The molluscicide of the present invention has good killing effect of snails, significant inhibition to the upward climbing of snails, safety to non-target organisms and environment as a plant original bio-molluscicide, and low drug resistance due to the diversified active substances. Additionally, P. peduncularis, which grows fast and has abundant resource, is favorable for popularization and application in snails control.

[embodiments!

Example 1: Preparation of 15% of P. peduncularis extract ME (microemulsion)

The root samples of P peduncularis are dried using a drying oven at 50°C and ground to 1-5 mm using a powder grinder. The obtained coarse powder is successively extracted with petroleum ether and chloroform (the weight ratio of coarse powder to extraction solvent being 1:3, w/w), and then the residue is extracted three times with one or more solvent(s) selected from methanol, ethanol, n-butanol and iso-propanol (the weight ratio of coarse powder to extraction solvent being 1:2, w/w) at 40-70 °C for 12-24 h. Finally, the combined extracts are concentrated and dried, thereby obtaining the crude extract of P. peduncularis root. 15 kg of crude extract of P. peduncularis root is suspended in 20 kg of n-butanol and the suspension is mixed with 5kg of alkylphenol polyoxyethylene ether and 15kg of calcium dodecyl benzene sulfonate. Then dropwise add 45 kg deionized water into the mixture while stirring at 45 °C , and keep stirring for 1 h after adding the deionized water. As a result, 100 kg of 15% P. peduncularis extract ME is obtained and its key performance indicators such as stability and appearance come up to national standards.

Example 2: Molluscicidal activity of the crude extract from P. peduncularis root against P. canaliculata and O. hupensis 1. Animal material

Pomacea canaliculata: adult snails in two size classes (15 mm ± 2 mm and 25 mm ± 2 mm in shell diameter), artificially raised by Biorational Pesticide Laboratory of Sichuan Agricultural University, are selected

Oncomelania hupensis: adult snails with 7-8 spirals, artificially raised by Biorational Pesticide Laboratory of Sichuan Agricultural University, are selected 2. Tested molluscicide

The crude extract from P. peduncularis root prepared according to the above mentioned method is produced and provided by Biorational Pesticide Laboratory of Sichuan Agricultural University.

The water extract from P. peduncularis: the root samples of P. peduncularis are ground using a powder grinder, and the obtained root powder is extracted three times using deionized water (the weight ratio of the powder to water being 1:3, w/w) at 40-70 °C for 12-24 h. Then the combined extracts are concentrated and dried, thus obtaining the water extract from P. peduncularis. It was made and provided by Biorational Pesticide Laboratory of Sichuan Agricultural University. 50% niclosamide WP is commercially available in the market. 3. Assay methods

The molluscicidal activity of the crude extract from P. peduncularis root against P. canaliculata and O. hupensis are examined by immersion method.

The crude extracts from P. peduncularis root with five different concentrations are poured into the 500 mL glass beaker separately, with every glass beaker being added 300 mL of distilled water , and the water extract from P. peduncularis and niclosamide are used as positive controls, distilled water as a negative control. Twenty snails (P. canalicidata with shell diameter 15 ± 2 mm and 25± 2 mm; O. hupensis with 7 spirals ).) are placed in each glass beaker of 500 mL, covered with a clean nylon net and incubated at 25°C in thermostat-controlled water baths. The assay has three replicates per treatment. The mortality percentages are recorded after 24, 48 and 72 h. A regression equation is devised using the ratio of extract concentration and snail corrected mortality percentages to calculate the lethal concentration. 4. Test results

The toxicity of the crude extract from P. peduncularis against P. canaliculata and O. hupensis are shown in Table 1 and Table 2, respectively.

The crude extract from P. peduncularis has high molluscicidal activity against P. canaliculata (Table 1). For the smaller snails (15 ± 2 mm shell diameter), the LC50 values are 58.0835 mg-L'1, 9.8505 mg-L'1 and 5.5112 mg-L'1 after 24 h, 48 h and 72 h treatment, respectively. The LC50 values of the water extract from P. peduncularis are 588.5323 mg-L'1, 249.3690 mg-L'1 and 227.2630 mg-L'1, and the niclosamide are 0.7251 mg-L'1, 0.5145 mg-L'1 and 0.4198 mg-L'1 for the same treatment times.

For the larger snails (25 ± 2 mm shell diameter), the LC50 values after 24 h, 48 h and 72 h are 85.5309 mg-L'1, 14.3924 mg-L'1 and 12.3832 mg-L'1, respectively. The LC5o values of the water extract from P. peduncularis are 723.0034 mg-L'1, 280.2784 mg-L'1 and 267.8989 mg-L'1, and the niclosamide are 0.9185 mg-L'1, 0.5686 mg-L'1 and 0.4847 mg-L'1 for the same treatment times.

From Table 2, the results show that the crude extract from P. peduncularis is highly toxic against O. hupensis and the LC50 values are 12.4862mg-L'1, 1.0444 mg-L'1 and 0.9268 mg-L'1 after 24 h, 48 h and 72 h treatment, respectively. The LC50 values of the water extract from P. peduncularis are 588.1544 mg-L'1, 115.2999 mg-L'1 and 101.6794 mg-L'1, and the niclosamide are 0.1671 mg-L'1, 0.1360 mg-L'1 and 0.0969 mg-L'1 for the same treatment times.

Accordingly, the molluscicidal activities of the crude extract from P. peduncularis against P. canaliculata and O. hupensis after 48 h and 72 h treatment are significantly higher than that of 24 h treatment, which indicates that the crude extract effectively controls the snails after 48 h treatment. Moreover, the crude extract from P. peduncularis has much higher LC50 values than the water extract, and this demonstrates that the solubility of molluscicidal active compounds in water are very low.

Table 1 Toxicity of the crude extract from P. peduncularis on P. canaliculata

Example 3: Mollusicicdal activity of 15% P. peduncularis extract ME against P. canaliculata in the field 1. Tested molluscicide 15% P. peduncularis extract ME: prepared as described in Example 1 by Biorational Pesticide Laboratory of Sichuan Agricultural University. 50% niclosamide WP: commercially available in the market. 30% P. peduncularis water extract EW (emulsion in water): the water extract from P. peduncularis is prepared as described in Example 2, and then, 30% of water extract from P. peduncularis, 5% of alkylphenol polyoxyethylene ether and 65% of distilled water (w/w) are mixed unifonnly to make 30% of P. peduncularis water extract EW. It is prepared and provided by Biorational Pesticide Laboratory of Sichuan Agricultural University. 2. Tested crop

Oryza sativa (the rice variety is Gangyou 606). 3. Control object

Pomacea canaliculata. 4. Assay methods

The main soil type of the experimental field is clay soil and it is in middling soil fertility. Area of the field is 5m2, and 100 clusters of rice seedlings are transplanted to each plot. Each plot is separated by dirt and plastic film and keeps the depth of water to 3-5cm. 7 days after transplanting, artificially stock 100 snails (P canaliculata: 25 ± 2 mm in shell diameter) into each plot. 15% of P. peduncularis extract ME with the dosage of 2 kg/hm2, 4 kg/hm2 and 6 kg/hm2 are sprayed into the water in the field, and λ 30% of P. peduncularis water extract EW with dosage of 10 kg/hm and 50% niclosamide WP with dosage of 0.5kg/hm2 are used as positive controls, distilled water as a negative control. For each assay, there are four replicates and a randomized block arrangement. The number of living snails is counted after 1 d, 3 d, 5 d and 7 d of treatment. Then, the control efficiency is calculated and the significant difference analysis is conducted. 5. Test results 15% of P. peduncularis extract ME with the dosage of 2 kg/hm2 has less toxic effect on P. canaliculata compared with the other two dosages (Table 3). While the dosage is 4 kg/hm or more, 15% P. peduncularis extract ME exhibits high toxicity against P. canaliculata, and there are no statistically significant correlations in molluscicidal activity between treatments and controls after 3 d, 5 d and 7 d of treatment except 1 d of treatment. Accordingly, the results show that 15% P. peduncularis extract ME with the dosage of 4 kg/hm" or above has highly toxic effect against P. canaliculata but the toxic effect develops relatively slow which is going to take a while to act. Moreover, the control efficiency of 15% of P. peduncularis extract ME against the snails are significantly higher than that of 30% of P. peduncularis water extract EW at any treatment times which indicates that using the water extract of P. peduncularis to control P canaliculata doesn’t seem to be working.

Table 3 Toxicity of 15% P. peduncularis extract ME on P. canaliculata in the field

Example 4: Mollusicicdal activity of 15% of P. peduncularis extract ME against 0. hupensis in the field 1. Tested molluscicide 15% P. peduncularis extract ME: prepared as described in Example 1 by Biorational Pesticide Laboratory of Sichuan Agricultural University. 50% niclosamide WP: commercially available in the market. 30% P. peduncularis water extract EW (emulsion in water): the water extract from P. peduncularis is prepared as described in Example 2, and then, 30% of water extract from P. peduncularis, 5% of alkylphenol polyoxyethylene ether and 65% of distilled water (w/w) are mixed uniformly to make 30% P. peduncularis water extract EW. It is prepared and provided by Biorational Pesticide Laboratory of Sichuan Agricultural University. 2. Control object

Oncomelania hupensis Gredler. 3. Assay methods

The small ditch with high density of O. hupensis is divided into equal sections •3 and the water volume of each section is approximately 3-5 m . 15% P. peduncularis extract ME with the dosage of 3 g/m3, 5 g/m3 and 7 g/m3 are sprayed into the water, •3 and 30% P. peduncularis water extract EW with dosage of 10 g/m and 50% niclosamide WP with dosage of 0.5 g/m3 are used as positive controls, distilled water as a negative control. For each assay, there are four replicates and a randomized block arrangement. The number of living snails is counted after 1 d, 3 d, 5 d and 7 d of treatment. Then, 100 snails of each section are chosen randomly to calculate the control efficiency. Dead snails are identified by the lack of any reaction to outside stimulation. 4. Test results 15% P. peduncularis extract ME with the dosage of 3 g/m3 has less toxic effect on O. hupensis compared with the other two dosages (Table 4). While the dosage is 5g/m or more, 15% P. peduncularis extract ME exhibits high toxicity against O. hupensis, and there are no statistically significant correlations in molluscicidal activity between treatments and controls after 3 d, 5 d and 7 d of treatment except 1 d of treatment. Accordingly, the results show that 15% P. peduncularis extract ME with the dosage of 5 g/mJ or above has highly toxic effect against O. hupensis but the toxic effect develops relatively slow which is going to take a while to act. Moreover, the control efficiency of 15% P. peduncularis extract ME against the snails are significantly higher than that of 30% P. peduncularis water extract EW at any treatment times which indicates that using the water extract of P. peduncularis to control O. hupensis doesn’t seem to be working.

Table 4 Toxicity of 15% P. peduncularis extract ME on O. hupensis in the field

In this invention, the application of the crude extract from P. peduncularisis as molluscicide has been specifically described through the Examples. Those skilled in this field can use for reference the invention to achieve other goals by changing the raw materials and technical conditions, and all the associated changes should be made without departing from the present invention. Nevertheless, all similar replacements and modifications, which are obvious to those skilled in this field, should be considered within the scope of this present invention.

Claims (8)

1. A botanical molluscicide, characterized in that the said botanical molluscicide contains the crude extract from roots of Pueraria peduncularisis (Grah. ex Benth.) Benth., solvents and surfactants; the content of the crude extract from roots of P. peduncularis, solvent and surfactants is 10-50% (w/w), 20-50% (w/w) and 5-30% (w/w) respectively; The said crude extract from roots of P peduncularis can be obtained by the method as follows: Step 1: The root samples of P. peduncularis are dried using a drying oven at 50°C and ground to 1-5 mm using a powder grinder, to obtain coarse powder of P. peduncularis', Step 2: The coarse powder obtained in Step 1 is successively extracted with petroleum ether and chloroform (the said coarse powder and the extraction solvent by weight ratio of 1:3), and then the residue is extracted three times with one or more solvent(s) selected from methanol, ethanol, n-butanol or iso-propanol (the said coarse powder and the extraction solvent by weight ratio of 1:2) at the temperature of 40-70°C for 12-24h, and then the combined extracts are concentrated and dried, thereby obtaining the crude extract of P peduncularis root.

2. The botanical molluscicide according to claim 1, characterized in that the dosage of the crude extract of P. peduncularis root is 10-30% by weight.

3. The botanical molluscicide according to claim 1, characterized in that the dosage of the crude extract of P. peduncularis root is 15% by weight.

4. The botanical molluscicide according to any of claim 1-3, characterized in that the said solvent of the molluscicide is one or more solvent(s) selected from cyclohexanone, ethyl acetate, methanol, n-butanol, ethanol, iso-propanol and water.

5. The botanical molluscicide according to claim 4, characterized in that the said solvent of the molluscicide is n-butanol.

6. The botanical molluscicide according to any of claim 1-3, characterized in that the said surfactant of the molluscicide is one or more surfactant(s) selected from styrylphenol phenol formaldehyde resin polyoxethylene ether, styrylphenol phenol formaldehyde resin polyoxyethylene polyoxypropylene ether, lauryl alcohol polyoxyethylene ether, phenylethyl polyoxyethylene ether, alkylphenol polyoxyethylene ether and calcium dodecyl benzene sulfonate.

7. The botanical molluscicide according to claim 4, characterized in that the said surfactant of the molluscicide is the mixture of alkylphenol polyoxyethylene ether and calcium dodecyl benzene sulfonate in a 1:3 radio (w/w).

8. The botanical molluscicide according to any of claim 1-3, characterized in that the said molluscicide is made into a microemulsion by the following steps: mix the above-mentioned extract from roots of P. peduncularis, the solvent and the surfactant according to the formulation in the modulation reactor at the temperature of 40-60°C for 1 h , stir uniformly, thereby obtaining the microemulsion of molluscicide containing P. peduncularis.