EP1940232A1

EP1940232A1 - Method and a composition based on quinoa saponins (chenopodium quinoa) intended for the control of aquatic fresh water snails

Info

Publication number: EP1940232A1
Application number: EP06750486A
Authority: EP
Inventors: Ricardo San Martin Gamboa
Original assignee: Dictuc SA; Hiley Paul
Current assignee: Dictuc SA; Hiley Paul
Priority date: 2005-07-13
Filing date: 2006-04-18
Publication date: 2008-07-09
Also published as: WO2007008268A1

Abstract

Extract from quino husk comprising partially hydrolyzed saponins and use thereof for the control of fresh water snails. The extraxt is obtained by alkaline or enzymatic hydrolysis of an aqueous quinoa husk extract.

Description

METHOD AND A COMPOSITION BASED ON QUINOA SAPONINS (CHENOPODIUM QUINOA) INTENDED FOR THE CONTROL OF AQUATIC FRESH WATER SNAILS

DESCRIPTION

The present invention relates to a method and a composition based on quinoa saponins {Chenopodium quinoa) intended for the control of aquatic fresh water snails such as Pomacea spp., Isidorella spp., Biomphalaria spp, Lymnaea spp., etc.. The method of the invention considers that the saponins are obtained from quinoa husk and are partially hydrolyzed in basic medium or with enzymes, increasing significantly their molluscicidal activity. The invention also considers a method for the control of fresh water snails, which comprises the application of a composition based on quinoa saponins partially hydrolyzed, in which the dose results in the death of the snails but not in the death non-target organism, such as fish, frogs, etc., allowing the safe application of the method on flooded plantations such as rice fields, as well as in rivers and lakes. The composition comprises liquid and powders which does not attack beneficial forms of aquatic life like fishes Prior Art

At a global level, fresh water snails cause great losses in agriculture and health human problems. Since its introduction in Asia in 1985, for instance, the golden apple snail (Pomacea canaliculata) has caused losses beyond US $ 1,000 million in rice crops. Likewise, there are several aquatic snails that transmit schistosomiasis (e.g. Biomphalaria spp.), which is, after malaria, the second important tropical disease for humans, affecting more than 200 million people. For the control of these snails, synthetic chemical molluscicides are mainly used, but are very expensive and extremely toxic to other living form and to the environment. For example, niclosamide, a widely recommended compound for the control of the golden apple snail in rice fields (Pomacea spp), is effective at doses from 0.5 - 1.0 mg/L (Bayer, 2005). However, the product kills 50% of tropical fish (LC₅o) as carps with only 0.14 mg/L (World Health Organization, WHO 2002. WHO Specifications and Evaluations for Public Health pesticides, Niclosamide). This means that no fish must be in the rice fields while the product is applied nor must the water be discharged into the rivers or lakes nearby. Furthermore, the cost of niclosamide is US $ 80-100/hectare, which is unaffordable for many farmers.

Based on this, there is the need to develop new products, preferably natural products, with low cost and minimum environmental impact. In the present invention the use of saponins from the Andean quinoa cereal (Chenopodium quinoa) is intended for the control of fresh water snails. Saponins are natural tensoactives present in many plants. One of its main properties is that they foam abundantly in aqueous solutions. They are also capable of permeating plant walls and microorganisms due to their association with sterols that are present in the cell membranes. Their chemical structure consists in a hydrophobic nucleus (sapogenin), to which sugar chains of hydrophilic nature are bound. There are two main types of saponins depending on the chemical structure of sapogenin: triterpenic saponins (where the sapogenin is a triterpene) and steroidal saponins (where the sapogenin is a steroid). The major industrial sources of steroidal and triterpenic saponins are Yucca shidigera and Quillaja saponaria Molina extracts, respectively. The biological and chemical activities of the saponins are directly related to the number of sugar chains bound to the sapogenin. Saponins with sapogenins that have two sugar chains are called bi-desmosidic saponins; those that have one sugar chain attached to the sapogenin are called mono-desmosidic saponins. Generally mono-desmosidic saponins show molluscicidal and fungal activity, while bi-desmosidic saponins have much lower activities. However, bi-desmosidic saponins have good tensoactive properties and produce abundant foam.

Quinoa saponins are mainly bi-desmosidic, with sugar chains attached at C-3 and C-28. They concentrate in the outer husk of the grain, which is removed prior to consumption, and is a sub- product without commercial value. The quinoa husk has the following advantages: 1) low cost (it is a by product); 2) abundant (it is found in Bolivia and Peru, and more recently in Chile); 3) high saponin content (25-30%); 4) Quinoa saponins have recently been approved by the US Environmental Protection Agency for its use against pathogenic fungi, bacteria, and viral plant diseases (Saponins of Chenopodium quinoa. Biopesticides Registration Action Document PC Code 097094. U.S. Environmental Protection Agency. Office of Pesticide Programs. Biopesticides and Pollution Prevention Division, 2005). This facilitates registration of the product in other countries.

In the present invention, the method considers that the quinoa soponins are partially hydrolyzed in basic medium or with enzymes. This eliminates sugar in C-28, turning the saponins into mono- desmosidic, increasing thus their molluscicidal activity in a significant way. A major advantage of the present invention is that the mono-desmosidic quinoa saponins have a moderate toxicity to non-target organisms such as fish. This means that the lethal concentrations for snails are not lethal for fish. Thus the product is safe for its use in fresh water systems that have fish and other forms of beneficial aquatic life.

According to the literature, quinoa has at least 16 types of triterpenic saponins, within which the bidesmosidic saponins prevail (Woldemichael, G. and Wink, M. 2001. Identification and biological activities of triterpenoid saponins from Chenopodium quinoa. J. Agric. Food Chem. 49: 2327-2332.), that are mainly concentrated in the husk, with a high content of the molluscicidal triterpenes oleanolic acid and hederagenin.

The production of extracts rich in saponins from quinoa husk has been studied with various solvents. The USP patent 6,355,249 reports saponin extraction yields of 20-25% w/w using ethanol/water mixtures. Furthermore, the patent describes the production of quinoa sapogenins by acid hydrolysis of the saponins. Studies carried out at Catholic University of Chile (Department of Chemical and Bioprocess Engineering) show that the aqueous extraction of the quinoa husk (variety Real, Bolivia) yields up to 55% of solids that can be extracted with a saponin content of 50-60% w/w. This represents 25-30% of saponins in the husk (Campos, C. 2003. Extracciόn de Saponinas de Ia Cascara de Quinoa. B.S.c. Thesis, Faculty of Engineering, Catholic University of Chile).

The use of saponins for the control of aquatic snails is well known, especially at a laboratory level. The pioneer work of Hostettmann et al. (Hostettmann, K., Kizu, H. and Tomimori, T., 1982. Molluscicidal properties various saponins. Planta Medica, 44, 34-35), determined that the molluscicidal activity was mainly associated with mono-desmosidic saponins. This action is possibly related to saponin bonds to gill membranes, which causes an increase in the permeability and thus an important loss of physiological electrolytes (Hostettmann and Marston, 1995. Saponins. Cambridge University Press). This molluscicidal activity has encouraged since 1980's various studies for the characterization of existing saponins in vegetable species such as Phytolacca, Swartzia among others (Borel C, Hostettmann, K., 1987. Molluscicidal Saponins from Swartzia madagascariensis DESVAUX. Helvetica Chimica Acta 70, 570-576. Dorzas A.C., Hostettmann, K., 1986. Further Saponins from Phytolacca dodecandra L'Herit. Helvetica Chimica Acta VoI 69 2038-2047. Dorzas A.C., Hostettmann, M., Hostettmann K., 1988. Molluscicidal Saponins from Sesbania sesban. Planta Medica, 225-227. Treyvaud V, Marston A, Wahyo D, Hostettmann K., 2000. Molluscicidal saponins from Phytolacca icosandra. Phytochemistry 55, 603-609).

The most important works relates to the use of Phytolacca dodecandra extracts in Africa (particularly Ethiopia), for the control of snails of Biomphalaria, Bulinus and Oncomelania genus, which transmit the schistosomiasis disease. This is the second most important disease in tropical countries, which affects over 200 million people. The P. dodecandra plant contains fruits (berry type) with 25% of saponins. The molluscicidal saponins are oleanolic acid glycosides and are active from 1.5 to 3 ppm. These saponins have been used for the control of snails that transmit schistosomiasis, but only in rural areas and in a craft way. Despite that the control of aquatic fresh water snails with saponins is well known, until now there are no economical formulations that are attractive. This is probably due to the fact that saponins with high molluscicidal activity come from plants that are not commercially cultivated or have low saponin content, which raises the price of the product.

Another problem is that the molluscicidal saponins also are very toxic to non-target organisms such as fish. For instance, currently for the golden apple snail control in Asia, tea seed cake (a by product of tea seed oil production in China), that contains around 7% w/w of saponins is used. This product is very economical (US $ 0.35/kg, US $ 16-27/hectare), but like the synthetic chemical products, its use is restricted because it is extremely toxic for fish and the environment at low doses (1-2 mg saponin/L).

The USP Patent 6,649,182 describes a method to protect plantules of rice against golden apple snails. To this purpose the roots of the plantules are impregnated with chemical molluscicides, as well as saponins extracted from the tea seed cake. The patent do no relates to the direct control of snails by the addition of saponins to the rice field water nor to the use of partially hydrolyzed saponins as it is in the invention.

Regarding the state-of-the-art, the present invention allows for the production of highly molluscicidal saponins from an abundant and low cost row material. Furthermore, the product is safe for human beings and the environment. Detailed Description of the Invention

The method of the invention considers the production of a composition based on partially hydrolyzed quinoa saponins which does not attack beneficial forms of aquatic life like fishes. The preparation of a liquid formulation considers a first phase of extraction with water. For this, the quinoa husk is contacted with water by stirring at a temperature from 20 to 90° C for 0.1 to 3 h. Once the initial extraction is finished, the husk is separated from the liquid extract by different alternate forms such as decantation, filtration with the help of diatomaceous earth or centrifugation. The husk already extracted is contacted with water again in similar conditions as in the initial extraction in order to maximize the recovery of the saponins. Once the second extraction is finished, the husk extract is separated in the same way as in the initial extraction. If desired, more than two extractions can be performed to maximize saponin recovery. The extracts are combined and treated with base at pH from 8 to 12 and heat (50-100° C) for 1-3 h, to give partially hydrolyzed saponins, which have mono-desmosidic features. Once the partial hydrolysis is finished, the extract is cooled at room temperature and concentrated acid is added to bring the extract to pH 3-7.5. The extract is then concentrated by evaporation at atmospheric pressure or vacuum until the desired concentration of solids is reached in the final product, which can be from 5 to 50% of solids. The final extract can be filtrated with the help of diatomeaceous earth or by centrifugation or decantation to eliminate impurities before packing. In this phase, excipients or preservatives can also be added to improve its stability.

As an alternate method to obtain partially hydrolyzed saponins as liquid formulation, hydrolytic enzymes that allow the conversion of bi-desmosidic saponins into mono-desmosidic saponins can be used. For instance, beta-glucosidase enzymes can be used, which hydrolyze the glucose group present in C-28. For this purpose the husk is extracted with water as described above, and the liquid extract is recovered, to which enzymes in optimal conditions of temperature, time and pH are added. The degree of hydrolysis can be determined by saponins analysis by reverse phase HPLC or another analytical method. Once the hydrolysis is finished, the extract is subjected to the concentration, filtration and addition of excipients and preservatives phases already described.

From both preferred modalities described for the liquid extract it is possible to obtain a liquid composition that comprises such aqueous quinoa extract with a concentration from 1 to 40% partially hydrolyzed quinoa saponins and an excipient. This composition is applied in effective amounts in order to control fresh water snails. The effective amount should range from 4 to 200/kg/hectare.

The preparation of a solid formulation comprises contacting the quinoa husk with water in a ratio of 1 part by weight of husk with 1-10 parts by weight of water, at a temperature from 20 to 90° C and 0.1-3 h, with stirring. The mixture is then treated with base at pH from 8 to 12 with the addition of NaOH or another basic agent and heat from 50 to 100° C for 0.1-3 h, to obtain partially hydrolyzed saponins with mono-desmosidic features. Once the partial hydrolysis is finished, the mixture is cooled at room temperature, and concentrated acid is added to bring it to pH 3-7.5. The mixture is then dried at a temperature that may range from 20 to 80° C.

As an alternate method to obtain partially hydrolyzed saponins as solid formulation, hydrolytic enzymes that allow the conversion of bi-desmosidic saponins into mono-desmosidic saponins can be used. For instance, beta-glucosidase enzymes can be used, which hydrolyzes the glucose group present in C-28. For this purpose the husk is extracted with water as it has been described, and the enzymes in optimal conditions of temperature, time and pH are added. The degree of hydrolysis can be determined by saponins analysis by reverse phase HPLC or another analytical method. Once the hydrolysis is finished, the mixture is dried at a temperature that may range from 20 to 80° C.

From both preferred modalities described for the solid formulation it is possible to obtain a composition that comprises a solid quinoa formulation with a concentration from 1 to 40% of partially hydrolyzed quinoa saponins and an excipient. This composition is applied in effective amounts in order to control fresh water snails. The effective amount should range from 4 to 200/kg/hectare.

For the control of aquatic fresh water snails, any of the two formulations liquid or dry under any of their preferred modalities of extraction should be added at saponin concentration from 5 to 20 ppm to the water to be treated.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius and, all parts and percentages are by weight, unless otherwise indicated. Example

The example of the invention consisted in the application of different compositions based on quinoa saponins for the control of golden apple snail (Pomacea canaliculata), to measure then their effect on both snails and fish.

The objective of the example was to determine the product concentrations that kill 100% of snails in 24 h: LCi₀₀ (24 h), and 50% of fish in 24, 48, and 96 h: LC₅₀(24 h), LC₅₀(48 h), and LC₅₀(96

h).

For both fish and snails the following products obtained from Bolivian quinoa Real husk were assessed: 1) powdered quinoa husk (30% of saponins); 2) partially hydrolyzed powdered of quinoa husk (27% of saponins); 3) quinoa liquid extract , 3.4% of saponins); 4) liquid extract of partially hydrolyzed saponins ( 3.0% of saponins). Furthermore, for comparison tea seed cake (7% w/w saponins) obtained from Zhejiang Orient Tea Development (Hangzhou, China) was assessed.

The saponin toxicology in snails was assessed with golden apple snails (Pomacea canaliculata). For this purpose, 20 to 35 mm adult snails, collected in a nursery located in IsIa de Maipo (Chile) were used. The snails were maintained in 25 x 20 x 15 cm. glass aquaria with lids, 7,5 liters of capacity each, and water was treated with a chlorine neutralizing product, 2 drops every liter of water. This product is harmless for fish, snails and plants. Each aquarium was filled with 2.5 L of tap water. The snails were kept at 20-22°C using electrical heaters (3 Watt resistance), while checking the water temperature every 12h with a mercury thermometer for aquaria. The water pH was checked every 24 h with a pH-meter (Orion, model 420A) keeping the parameter considered appropriate for the snails (pH 7-8).

Before starting the tests, snails were acclimatized to lab conditions for a week, receiving food every 12 h, with Nutrafin Max complete food in flakes. They were exposed to a 6 h cycle con artificial light (25 W) every 24 h. Four snails were placed in each aquarium and each experiment was carried out three times. In order to determine LCio_O(24h), the snails were observed at 24 h, recording the eventual clinical signs y mortalities, assessed by stimuli with a stainless steal needle and detection or lack of movement. After 24 h, snails classified as dead were placed in a separate container with fresh water and checked for recovery. Only those snails that did not recover within additional 24 h in fresh water were reported dead.

The saponin toxicology in fish was assessed in 2-3 g goldfish (Carassius auratus auratus) using the products and the aquarium system previously described. This time 5 liters of water per aquarium were used, temperature 16-18 ⁰C. Four fish were used in each aquarium and each experiment was carried out three times. In some experiments 7-8 g tilapias were used, kept al 21- 22⁰C. Every 24 h during 96 h, the number of dead fish per aquarium was recorded.

Results

Table 1 shows the results of the experiments for the different product concentrations. Table 2 summarizes lethal concentrations for snails and fish. Tea seed cake, in 24 h and at very low doses of saponins (1.5 ppm of saponins) kills 100% of fish, and only 66% of snails. This makes it impossible to use this product in rice fields, rivers and lakes nearby, because the doses and periods to kill snails is more lethal for fish. This is similar to niclosamide behavior, which kills fish at lower concentrations than snails.

In the case of quinoa husk, the concentration of 40 ppm of saponin (the biggest essayed) does not cause the death neither in snails nor in fish. Partially hydrolyzed quinoa husk, however, as stated by the method and composition of this invention, has a LC_1Oo= 7-10 ppm of saponins for snails. At this concentration, goldfish and tilapia do not die in 96h, which makes it safe to use it.

Liquid composition without partial hydrolysis of the saponins does not kill neither snails nor fish at 40 ppm of saponins in 24 h, but it does in 48 h. The partially hydrolyzed saponins in liquid composition has a LC_JOO ⁼ 7-10 ppm of saponins for snails, and this concentration it does not kill fish in 96h. Still, at a dosage of 15 ppm of saponins, it is not lethal for goldfish or tilapia in 96 h.

These results confirm the importance of the present invention: The composition based on partially hydrolyzed quinoa saponins is lethal for snails at lower dosages than for fish. This makes it possible for use it safely in flooded rice fields, and it is also safe for rivers and lakes. The composition and method to treat fresh water snails have the advantage of being of natural origins and economical, because it is an industrial by product derivative. Table 1: Quinoa saponin toxicology in snails and fish.

Table 2: Saponin concentration (in ppm) of lethal quinoa for snails. LCioo (24 h), and for fish: LC₅o(48h).

The entire disclosures of all applications, patents and publications, cited herein and of corresponding Chilean application No. 1745-2005, filed July 13, 2005 are incorporated by reference herein.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims

WHAT IS CLAIMED IS:

1. Partially hydrolyzed saponins obtained from quinoa husk.

2. Partially hydrolyzed saponins according to claim 1 , which are predominantly mono-desmosidic.

3. Partially hydrolyzed saponins according to claim 1 , obtained by a process comprising contacting quinoa husk with water at elevated temperature, removing quinoa husk, adding a basic agent to effect partial hydrolyzation, neutralizing and optionally concentrating to produce a solid.

4. Partially hydrolyzed saponins according to claim 1 , obtained by a process comprising: a. contacting quinoa husk with water in a ratio of 1 part by weight by 1 -10 parts by weight of water at a temperature from 20° to 90°C, from 0.1 to 3h, b. removing the quinoa husk from liquid extract, c. subjecting the amount of quinoa husk subjected to the extraction phase of phase a), to a second phase of extraction to subsequently

( subject it to the same husk removal phase described in phase b), d. subjecting such extract to a partially saponin extraction phase by adding NaOH or another basic agent in order to bring such extract to pH 8-12, e. subjecting such extract simultaneously to such partial hydrolysis phase, to a heating phase at a temperature from 50 to 100°C, f. subjecting the hydrolyzed extract to a neutralization phase with acid, to bring the extract to pH 3-7,5, g. concentrating the hydrolyzed extract until the desired solid concentration is reached in the final product.

5. A composition comprising an aqueous quinoa extract with a concentration from 1 to 40% of partially hydrolyzed quinoa saponins and an excipient.

6. A composition according to claim 5, comprising saponins obtained from quinoa husk, in the form of a solid quinoa formulation with a concentration from 1 to 40% of partially hydrolyzed quinoa saponins and an excipient.

7. A method of controlling fresh water snails, comprising applying an effective amount of a composition containing partially hydrolyzed quinoa saponins to fresh water where such snails are found.

8. A method according to claim 6, comprising applying said composition to flooded plantations, rivers or lakes.

9. A method according to Claim 7, wherein the effective amount is 5 - 20 ppm of saponins in aqueous solution.

10. A method according to Claim 7, wherein the composition comprises an aqueous quinoa extract with a concentration from 1 to 40% of partially hydrolyzed quinoa saponins and an excipient.

11. A method according to Claim 7, comprising controlling Pomacea ssp, Isidorella ssp, Biomphalaria ssp, Lymnaea spp.

12. A method according to Claim 7, comprising controlling Pomacea canaliculata.

13. A method for producing a composition containing saponins extracted from quinoa husk, comprising: a. contacting quinoa husk with water in a ratio of 1 part by weight by 1- 10 parts by weight of water at a temperature from 20° to 90⁰C, from 0.1 to 3h, b. removing the quinoa husk from liquid extract, c. subjecting the quinoa husk subjected to the extraction phase of phase a), to a second phase of extraction to subsequently subject it to the same husk removal phase described in phase b), d. subjecting such extract to a partial saponin extraction phase by adding NaOH or another basic agent in order to bring such extract to pH 8-12, e. subjecting such extract simultaneously to such partial hydrolysis phase, to a heating phase at a temperature from 50 to 100°C, f. subjecting the hydrolyzed extract to a neutralization phase with acid, to bring the extract to pH 3-7,5, g. concentrating the hydrolyzed extract until the desired solid concentration is reached in the final product.

14. A method according to Claim 13, wherein phase b) of quinoa husk removal from the liquid extract is carried out by decantation, filtration or centrifugation.

15. A method according to Claim 13, wherein phase g) of extract concentration is carried out at atmospheric pressure or in vacuo.

16. A method according to Claim 13, comprising filtering, centrifuging or decanting a final product in order to eliminate impurities before packing.

17. A method according to Claim 16, wherein filtration is carried out with diatomeaceous earth.

18. A method according to Claim 13, wherein in phase e) a decanted and filtrated extract is partially hydrolyzed using hydrolytic enzymes.

19. A method according to Claim 18, wherein said enzymes hydrolyze the sugar group located at the saponin C-28 carbon.

20. A method for producing a powdered composition containing saponins extracted from quinoa husk, comprising: a. contacting quinoa husk with water in a ratio of 1 part by weight of husk with 1-10 parts by weight of water at a temperature from 20° to 90°C, from 0.1 to 3h, b. subjecting the husk mixture with water to a heating phase for 0.1 to 3 hours at a temperature from 50 to 100° C, and adjusting such mixture to pH 8 to 12 in order to extract partially hydrolyzed saponins, c. cooling said mixture at room temperature and adding concentrated acid to bring it to pH 3-7.5, d. drying such mixture at a temperature of 20 to 80° C.

21. A method according to Claim 20, wherein water adjustment in phase b) is carried out by the addition of NaOH or another basic agent.

22. A method according to Claim 20, wherein in phase c) concentrated acid is concentrated HCl.

23. A method according to Claim 20, wherein in phase b) partially hydrolyzed saponins are extracted using hydrolytic enzymes.

24. A method according to Claim 23, wherein said enzymes hydrolyze the sugar group located at the saponin C-28 carbon.