US 200701965.17A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/01965.17 A1 San Martin (43) Pub. Date: Aug. 23, 2007

(54) MODIFIED SAPONIN MOLLUSCICIDE Related U.S. Application Data (63) Continuation-in-part of application No. PCT/US06/ (75) Inventor: Ricardo Manuel San Martin, Santiago 14458, filed on Apr. 18, 2006. (CL)CL (30) Foreign Application Priority Data Correspondence Address: Jul. 13, 2005 (CL)...... 1745-2005 MYERS WOLIN, LLC Publication Classification 100 HEADQUARTERS PLAZA North Tower, 6th Floor (51) Int. Cl. MORRISTOWN, NJ 07960-6834 (US) AOIN 45/00 (2006.01) AOIN 65/00 (2006.01) AOIN 43/04 (2006.01) (73) Assignee: DICTUC S.A., Santiago (CL) (52) U.S. Cl...... 424/725; 514/33; 514/26 (57) ABSTRACT (21) Appl. No.: 11/740,275 Saponins from plant materials are alkaline modified and (22) Filed: Apr. 25, 2007 used as molluscicides. Patent Application Publication Aug. 23, 2007 Sheet 1 of 5 US 2007/01965.17 A1

Ratio husk-water from 1:3-5 Temperature 20-35° C from 0.5 to 1 hr

Alkaline treatment NaOH 0.4-0.6N Temperature 90-100° C from 1 to 2 hr pH from 12 to 13

Neutralization pH from 6.5 to 7.5

Hot Air Dry 5-10% w/w moisture content

Figure 1: Major processing steps - Powder product Patent Application Publication Aug. 23, 2007 Sheet 2 of 5 US 2007/01965.17 A1

Quinoa husk Extraction Ratio husk-water from 1:8-10 Water Temperature 20-30°C from 0.5-1 h

Removal of proteins Bentonite and/or Bentonite 1-5 g/L and/or Acid Acid pH 3.5-4.0

Washing of solids Water Ratio husk-water from 1:5-8 20-30°C from 0.5-1 hr Liquid

Solid residue Concentration 200 to 350 g solids/L

Alkaline treatment NaOH 0.4-0.6N Temperature 90-100°C from 1.5-2.5 h pH from 12 to 13

Neutralization pH from 6.5 to 7.5

Packaging

Figure 2 : Major processing steps - Liquid product Patent Application Publication Aug. 23, 2007 Sheet 3 of 5 US 2007/01965.17 A1

1 kgQuinoa husk Extraction Ratio husk-water 1:3 Temperature 20 °C for 0.5 hr

80 g NaOH + Alkaline treatment 2 L Water Ratio husk-water 1:5 NaOH 0.4-N Temperature 95 °C for 2hr

Neutralization Temperature 20 °C pH 7

Air Dry 16 hr at 70 °C

Figure 3: Example Processing steps - Powder product Patent Application Publication Aug. 23, 2007 Sheet 4 of 5 US 2007/01965.17 A1

Figure 4b

O. F. m t; E. i. -- 3:as - www.xxxx-xx-x--

Figure 4 : RP-HPLC chromatogram of duinoa saponins. Absorbance at 210 nm vs. injection time (min) . Figure 4a: Crude acqueous eXtra Ct, Figure 4b : a CueOuS eXtra Cl purified with ultrafiltration membranes. Peaks A, B and C correspond to main Culin Oa Sap OnlinS. Patent Application Publication Aug. 23, 2007 Sheet 5 of 5 US 2007/01965.17 A1

- is E.. Y - 8. - : st

- c n o r, r secrys-----e.is - , ...&rs.s- & k ‘y...sier,t

Figure 5: RP-HPLC chromatograms of quinoa husks treated with NaOH. Absorbance at 210 nm vs. injection time (min). US 2007/01965.17 A1 Aug. 23, 2007

MODIFIED SAPONN MOLLUSCCDE industry and with the intent of providing a valuable protein source for farmers, who primarily live on a rice diet. It is 0001. This disclosure is a continuation in part of pending now over 20 years since Golden Apple Snails (Pomacea PCT/US2006/014458 filed Apr. 18, 2006 and claims priority canaliculata) were introduced in Asian countries from its from Chilean application No. 1745-2005 filed Jul. 13, 2005, native habitat in Northern Argentina and Southern Brazil the entire disclosures of which are incorporated by reference with the intention of using them for human consumption. herein. Unfortunately, the imported snails are able to transfer Angiostrongylus cantonensis (rat lungworm) like the native BACKGROUND OF THE INVENTION apple Snail population (Pila). This parasite spends a part of 0002) 1. Field of the Invention its life cycle in apple Snails and can infect humans when the Snails arent cooked thoroughly. 0003. This disclosure relates to the recovery of saponins from plant material. In particular, the invention relates to the 0010 Instead of becoming a food source the snails recovery of saponins from Chenopodium quinoa (Ouinoa) escaped and became a serious threat to rice production and (Chenopodiaceae) husk in commercially useful forms. It the environment. During the 1980s the introduced snails also relates to a method and a composition based on quinoa rapidly spread to Indonesia, Thailand, Cambodia, Hong saponins. The invention also relates to a method for the Kong, Southern China, Japan, the Philippines and Australia. control of fresh water mollusks, in particular, Snails, which In 1989 Pomacea Canaliculata was introduced in Hawaii to comprises the application of a composition based on alkaline serve as a food source and aquarium pet. Some Snails treated quinoa Saponins, in which the dose results in the escaped to the wild and turned into a serious pest in the taro death of the mollusk but not in the death of non-target and rice fields. organisms, such as fish, frogs, etc., thus allowing the safe 0011. As a result of the failure this program, the snails application of the method and product on flooded plantations were released to rivers and rice fields, where they prolifer Such as rice fields, as well as in rivers, ponds, lakes and other ated to Such an extent that they are now considered one of naturally occurring or artificial bodies of water. the 100 worst invasive alien species in the world (ISSG, 0004 The composition comprises liquid and powders 2006). which do not attack beneficial forms of aquatic life when 0012. The Golden Apple Snail, popularly known as used in appropriate amounts. This disclosure also relates to 'golden kuholPomacea canaliculata Lamarck, is one of methods of producing the disclosed compositions. the major pest problems in rice production. In 1989, the 0005 2. Description of Related Art Food and Agriculture Organization of the United Nations estimated that yield losses owing to this pest ranged from I. Snails 1% to 40% of the planted area in the Philippines, resulting 0006 Snails are members of the molluscan class Gas in a huge production loss (The Philippine Rice Research tropoda that have coiled shells. Snails are found in fresh Institute). water, saltwater, and terrestrial environments. Most are 0013 Fresh water snails cause great losses in agriculture herbivorous, though a few land species and many marine and health human problems. Since its introduction in Asia in species may be omnivores or carnivores. The majority of 1985, the golden apple Snail (Pomacea canaliculata) has Snails are not terrestrial. Snails with lungs belong to the caused losses in excess of US $1,000 million in rice crops. group Pulmonata, while those with gills form a paraphyletic Likewise, there are several aquatic snails that transmit group. Schistosomiasis (e.g. Biomphalaria spp.), which is, after 0007. Apple snails are tropical and sub-tropical freshwa malaria, the second most important tropical disease for ter snails of the family Ampullariidae (sometimes referred to humans, affecting more than 200 million people. as Pilidae). They are commonly divided into several genera. 0014. In the water of the rice fields the snails reproduce The genera Asolene, Felipponea, Marisa, and Pomacea are extremely quickly and eat young rice seedlings Voraciously, found in South America, Central America, the West Indies causing significant economical damage to farmers. In addi and the Southern U.S.A., while the genera Afropomus, tion to countries Such as Taiwan, Thailand, Malaysia, Indo Lanistes and Saulea are found in Africa. The genus Pila is nesia and The Philippines which are suffering from exten native in both Africa and Asia. sive damage due to this pest, other countries, such as 0008. Apple snails are the largest living freshwater snails. Australia, consider the pest as a serious potential menace to The Giant Apple Snail, Pomacea maculata (Family Amp its rice agriculture (Plant Health Australia, 2005, pp. 12-13). ullariidae), is the largest freshwater Snail known, with a 0015 The invasion of Pomacea in new habitats has been diameter of up to 15 cm and a mass of over 600 g. Apple shown to drastically alter the state and function of invaded Snails are exceptionally well adapted to tropical regions natural wetlands. When plants are consumed, nutrients in the characterized by periods of drought alternating with periods system are shunted to phytoplankton instead of the plants of high rainfall. One of the more typical adaptations of apple which creates dense algal blooms. Snails is the bronchial respiration system comparable with 0016) Pomacea and Marisa species have been introduced the gills of a fish located at the right side of the snail body in Africa and Asia to control Snails such as Planorbidae, which enables the snails to breathe under water as well as a Bulinus sp. and Biomphalaria sp. which serve as interme lung at the left side of the body to respirate air. diate hosts for trematoda parasites which can cause Swim 0009 Pomacea canaliculata is a common apple snail. In mers itch and schistosomiasis, a disease that affects over 200 the 1980s, snails of the genus Pomacea (Pomacea canali million people in tropical regions. Despite the fact the culata) were introduced in Taiwan to start an escargot tremadote parasites do not complete their life cycle in apple US 2007/01965.17 A1 Aug. 23, 2007

Snails, apple Snails themselves can carry these parasites and turned to commercial molluscicides such as niclosamide and nematodes of the genus Angiostrongylus. Angiostrongylus metaldehyde which are effective against golden apple Snails cantonensis can afflict humans and cause eosinophilic men that are directly hit. Their efficacy lasts 2-3 days. The ingoenchephalitis. synthetic chemical molluscicides typically used to control 0017. There are many species of apple snails, among these Snails are very expensive and extremely toxic to other which are Pomacea (Pomacea canaliculata, Lamarck, living organisms and to the environment. 1819) Bright Orange Pomacea (Pomacea insularum, 0025 Niclosamide is a widely recommended compound D'Orbigny, 1839) Pink Pomacea (Pomacea lineata, Spix, for the control of the golden apple snail in rice fields 1827) Pink Pomacea (Pomacea doliodes, Reeve, 1856) Pink (Pomacea spp). Niclosamide 250EC at half the label rec Pomacea (Pomacea haustrum, Reeve, 1856)and Green ommendation (0.5 l/ha) killed about 80% of the golden Pomacea (Pomacea gigas/maculata, Perry, 1810) apple Snails that were sprayed. Niclosamide kills more 0018 Newly-transplanted rice seedlings are vulnerable to native snails than metaldehyde formulations. It is effective at golden apple Snail damage for up to 15 days after trans doses from 0.5-1.0 mg/L (Bayer, 2005). However, the prod planting or from 4 days to 30 days after sowing for direct uct kills 50% of tropical fish (LCs) as carp when applied at seeded rice. Golden apple Snails devour the base of young a level of as low as 0.14 mg/L (World Health Organization, seedlings. They may even consume the young plants in a WHO 2002. WHO Specifications and Evaluations for Public whole paddy overnight. Health pesticides, Niclosamide). This means that no fish must be in the rice fields while the product is applied nor 0019. The apple snail lays its eggs on any vegetation, must the water be discharged into the rivers or lakes nearby. leaves, and objects (e.g. twigs, stakes, Stones, etc.) above the Furthermore, the cost of niclosamide is USS 40-80/hectare, water Surface. The eggs hatch in 7-14 days. The hatchlings which is unaffordable for many farmers. are voracious feeders growing and maturing quickly. Golden apple snails reproduce rapidly, laying up to 1000-1200 eggs III. Zebra Mussel and Other Mollusks in a month. 0026 Saponins are effective in the control of golden 0020. They live in ponds, swamps, irrigated fields, canals apple snails members of the family Ampullariidae or Pilidae, and water-togged areas. Golden apple snails feed on a wide part of Prosobranchia subclass. range of plants such as algae, azola, duck weed, water 0027 Saponins also effectively control snails in the Plan hyacinth, rice seedlings, and other Succulent leafy plants. orbidae (Biomphalaria ssp and Isidorella ssp.) and Lym The Snails feed by Scraping plant Surface with its rough nacidae (Lynnaea ssp.) families, both part of Pulmonata tongue so they prefer young Soft plant parts. Subclass. 0021 Multiple attempts have been made to eradicate of 0028 Control effects in the Bivalvia class (bivalves and invasive Snails, in particular apple Snails. Many farmers clams) have also been reported, with respect to Blue mussel have resorted to the massive use of synthetic molluscicides or Mytilus edilus, part of the Mytilidae family, subclass that are expensive and broad spectrum, affecting non-target Pteriomorphia. organisms including human beings. 0029 Saponins also control Zebra mussel or Dreissena II. Controls polymorpha, Dreissenoidea family, Heterodonta Subclass. 0022. There are few naturally occurring biological con Since the arrival of the Zebra mussel, a number of chemicals trol agents. Redants feed on the eggs; ducks eat the flesh and with previously known or newly discovered molluscicidal young Snails. Techniques for managing apple Snails include properties have been proposed for deployment against this land preparation by handpicking golden apple snails from highly invasive organism but none have been able to satisfy rice paddies, introducing plants that contain toxic Substances the requirement of an effective, selective, low cost material. against golden apple Snails. Examples are gugo (bark) 0030) Zebra mussels are well adapted to water tempera Entada phaseikaudes K Meer, tubangkamisa (leaves), tures (12°C. to 32° C. (55°F. to 90°0 F)), pH range (6.5 to Sambong (leaves) Blumea balsamifera), tuba-tuba (leaves), >8), and turbidity levels that can be found in the Great Lakes gabihan (leaves) Monochoria vaginalis), tobacco (leaves) and many U.S. riverine environments. Females release up to Nicotiana tabacum L. calamansi (leaves) Citrus micro 30,000 planktonic (free-swimming) larvae, called velligers, carpa Bunge), tubli (roots), makabuhay (leaves) Tinospora which move with water currents and grow up to 1.3 cm (0.5 rumphii Boerl), and red pepper (fruit). in.) in the first half year. These settle in colonies and attach 0023 Recommendations have included placing a wire or to firm surfaces by means of secreted strands called byssal woven bamboo screen on the main irrigation water inlet and threads. Densities can reach 500,000 per square meter outlet to prevent the entry of hatchlings and adults and (46,500 or more per square foot), and individual life spans facilitate collection of trapped golden apple Snails; utilizing are 3 to 5 years. Zebra mussels are filter feeders, opening bamboo stakes on water-logged areas in the paddies or near their shells to allow ingestion of particulates. When their canals to attract adults for egg laying to facilitate hand sensitive chemoreceptors alert them to certain toxins in the collection and crushing of the egg masses easy; draining the environment, they have the ability to maintain shell closure rice field occasionally to limit snail mobility and feeding for up to 2 weeks and thereby remain immune to certain activity and herding ducks in rice paddies immediately after biocide contact. Not all molluscicides evoke this response, harvest to eat the snails however. 0024. These techniques are highly labor intensive and are 0031 Chemicals identified for Zebra mussel control have unsuited to many areas. In addition they are not highly been derived mainly from water treatment compounds and effective in eradicating the Snails. Thus, many farmers have antifouling biocides and biodispersants. Chlorine has been US 2007/01965.17 A1 Aug. 23, 2007 used for nearly a hundred years in drinking water disinfec 0037 toxins have been tested for use in controlling zebra tion, where its properties and behavior in effluent are well mussels (Taylor and Zheng 1995, 1997). In addition, anti known, and it has been the primary chemical for Zebra biotic materials excreted by other aquatic organisms to keep mussel control in Europe. In contrast, molluscicidal prop them free of biofouling are relatively common, and these are erties have been associated only recently with endothall, a being investigated for their ability to prevent settling when compound used for several decades as an aquatic herbicide. applied as extracts or as a component in coatings. However, Investigation of toxicity to both the target and nontarget none of these has become commercially available. Com organisms in the aquatic environment is the first step in the pounds that are toxic to mussels are also potentially toxic to ongoing effort to identify more compounds that will be other life forms, and they must be tested and handled as effective against Zebra mussel. While oxidizers, and particu carefully as other molluscicides. larly the various forms of chlorine, continue to be the most 0038. One such natural compound, Endod, is a plant commonly used of the chemical controls, additional com toxin product that includes chemicals called Lemmatoxins pounds have been registered; and more continue to be tested derived from the fruit of the African soap berry tree Phyto in the search for environmentally sound and effective treat lacca dodecandra. Two U.S. patents for its use as mollus ment of this pest. cicides have been awarded. Lemmatoxins have been shown 0032. The major types of oxidants frequently used for to be lethal to Zebra mussels at concentrations higher than 15 chemical control of biofouling and available as generic mg L', while lower concentrations inhibited attachment chemicals for molluscicide use are chlorine, chlorine diox and aggregation of adult mussels (Lemma et al. 1991, Lee, ide, chloramines, such as monochloramine, oZone, hydrogen Lemma, and Bennett 1993). Toxicological studies have been peroxide, bromine and permanganates such as potassium done on nontarget mammals (Hietanen 1997). permanganate. In general they have similar modes of action based on the oxidation of organic matter, which leads to 0.039 However, there still exists a need for an effective, non-selective toxic and lethal effects. Oxidizers also present selective molluscicide effective against Zebra mussels. problems because of their corrosive effects on metals. IV. Saponins 0033 While chlorine dominates all chemical use for 0040 Saponins are glycosides of steroids, steroid alka Zebra mussels, there has been concern that there will be loids or triterpenes found in plants, especially in the plant additional restrictions on its discharge in the future due to its skins where they form a waxy protective coating. There are nonselectivity and its formation of undesirable by-products two main types of saponins depending on the chemical such as trihalomethanes (THMs) and chloramines upon structure of Sapogenin: triterpenic saponins (where the Sapo coming into contact with organic compounds in open water. genin is a triterpene) and steroidal saponins (where the 0034) Most nonoxidizing molluscicides were originally Sapogenin is a steroid). A third type of Saponin, the alkaloid developed for bacterial disinfection and algae control in saponins is sometimes identified as a separate class. The water treatment systems and include organic film-forming saponins dissolve in water to form a stable Soapy froth antifouling compounds, gill membrane toxins, and nonor thought to be due to their amphiphilic nature. ganics. The proprietary formulations have a higher per 0041. The triterpenoid saponins have an oleanane struc Volume cost than oxidizing chemicals but remain cost ture and one or more glycosides, the glycosides being bound effective due to lower use rates and rapid toxicity. They often to the triterpenoid at the 3 position and/or at the 28 position. can provide better control of adult mussels due to the Saponins are composed of a ring structure (the aglycone) to inability of mussels to detect them; because shells remain which is attached one or more Sugar chains. The glycoside open, shorter exposures are required. Most are easy to apply Sugars found naturally in quinoa include arabinose, glucose, and do not present corrosion problems for metal compo galactose, Xylose and glucuronic acid. A sapogenin is the nentS. triterpenoid alone without glycosides attached at either the 3 0035 Numerous pesticide compounds have been sug or the 28 position. gested or investigated for Zebra mussel control and are 0042. The aglycone can be asteroid triterpenoid or a discussed in a variety of research and product information steroidalalkaloid and the number of Sugars attached to the literature. However, most are currently not in common use, glycosidic bonds vary greatly. either because they are less effective on Zebra mussels, harmful to native bivalves as well as Zebra mussel and 0043 Saponins are natural tensoactives present in many therefore limited to use in contained systems or have not yet plants. One of their main properties is that they foam been registered for Zebra mussel control. abundantly in aqueous solutions. They are also capable of permeating plant walls and microorganisms due to their 0.036 Some of the proposed compounds are Clonitralid association with Sterols that are present in the cell mem (5-chloro-n-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide branes. Their chemical structure consists in a hydrophobic compound with 2-aminoethanol (1:1) (9ci)); Niclosamide nucleus (Sapogenin) to which Sugar chains of hydrophilic (Bayluscide 70) (2-aminoethanol salt of 2,5-dichloro-4, nitro-salicylanilide); Bayer 73 (Bayluscide) (2,5-dichloro nature are bound. 4'.nitro-salicylanilide), Zinc oxide: Rotenone (1.2.12.12a 0044 Saponins have been identified in Aloe, amaranth, tetrahydro-2-iso-propenyl 8,9-dimethoxy-1bensopyrano asparagus (as protodioscin), chickweed, Bacopa monnieri, 3.4 furo 2.3-b1benzo pyran-6 (6aH) one): Salicylanilide Chlorophytum species, Chlorogalum species, soap plants, I (Sal I) (2,5-dichloro-3-tert-butyl-6-methyl-4-nitrosalicy Conkers/horse chestnuts, tuberous cucurbit species, digitalis lanilide); and TFM (Lamprecid) (3-trifluoromethyl-4-nitro (as digitonin), echinodermata, fenugreek, grape skin1. phenol). A number of compounds derived from natural gotu kola, Gypsophila (Baby's Breath), jiaogulan, liquorice, Sources Such as plant mullein, olives, panax (as ginsenoside), Ouillaia Saponaria US 2007/01965.17 A1 Aug. 23, 2007

(bois de Panama, a member of the Rosaceae family), countries, which affects over 200 million people. The P. quinoa, rambutan, Soapberry and many other members of the dodecandra plant contains fruits (berry type) with 25% of family Sapindaceae, including buckeyes, Saponaria (Soap saponins. The molluscicidal saponins are oleanolic acid wort, Bouncing Betty), soybeans, Tribulus terrestris (as glycosides and are active from 1.5 to 3 ppm. These saponins protodioscin), wild yam, yucca, and many other plants used have been used for the control of snails that transmit in medicine or as food items. Schistosomiasis, but only in rural areas and in a craft way. 0045. The major industrial sources of steroidal and trit 0.052 U.S. Pat. No. 6,649,182 describes a method of erpenic saponins are Yucca Shidigera and Ouillaia protecting plantules of rice against golden apple snails. For saponaria Molina extracts. this purpose the roots of the plantules are impregnated with 0046) The saponins from Quillaja Saponaria are used in chemical molluscicides, as well as Saponins extracted from veterinary vaccines as adjuvant (e.g., foot-and-mouth dis the tea seed cake. The patent does not disclose the control of ease vaccines). Saponins are also mild detergents and are snails by the addition of saponins to the rice field water nor used commercially as well as for research. the use of partially hydrolyzed saponins. 0047 The biological and chemical activities of the 0053 Despite the fact that the control of aquatic fresh saponins are directly related to the number of Sugar chains water Snails with Saponins is well known, until now there are bound to the Sapogenin. Saponins with Sapogenins that have no economical formulations that are effective and selective. two Sugar chains are called bi-desmosidic saponins; those This is probably due to the fact that saponins with high that have one Sugar chain attached to the Sapogenin are molluscicidal activity come from plants that are not com called mono-desmosidic saponins. Generally mono-desmo mercially cultivated or have low Saponin content, which sidic saponins show molluscicidal and fungal activity, while increases the price of the product. bi-desmosidic saponins have much lower activities. How 0054 Another problem is that the molluscicidal saponins ever, bi-desmosidic saponins have good tensoactive proper also are very toxic to non-target organisms such as fish. For ties and produce abundant foam. instance, currently for the golden apple Snail control in Asia, 0.048. The high levels of saponins found in certain plants tea seed cake (a by product of tea seed oil production in have long been thought to be responsible for the medicinal China), that contains around 7% w/w of Saponins is used. effects of some of these plants (Waller, G. R. and K. This product is very economical (US $ 0.35/kg, US S16 Yamasaki, Saponins used in Traditional and Modern Medi 27/hectare) but like the chemical products its use is cine, Advances in Experimental Medicine and Biology, restricted because it is extremely toxic to fish and the Vol.404, 1996, New York: Plenum Press). The presence of environment at low does of 1-2 mg Saponin/L. high levels of Saponins in the seeds of plants such as Quinoa 0055 Traditionally, the saponin content in plant extracts (Chenopodium quinoa) has restricted the use of the human has been determined by bioassay or by GLC analysis of the consumption of this grain. Sapogenins derived by hydrolysis of the Saponins (Ridout et 0049. The use of saponins for the control of aquatic snails al., J. Sci. Food Agric. 54:165 (1991)). is well known, especially at a laboratory level. The pioneer V. Quinoa work of Hostettmann et al. (Hostettmann, K., Kizu, H. and Tomimori, T., 1982. Molluscicidal properties various 0056 Chenopodium quinoa (“Ouinoa) is a grain crop saponins. Planta Medica, 44, 34-35), determined that the which has been cultivated in South America for a number of molluscicidal activity was mainly associated with mono years. Many saponins have been characterized from Quinoa desmosidic saponins. This action is possibly related to which have a single Sugar at position 28, and between one saponin bonds to gill membranes, which causes an increase and three Sugars at position 3 of the aglycone. in the permeability and thus an important loss of physiologi cal electrolytes (Hostettmann and Marston, 1995. Saponins. 0057 Quinoa husk is the material obtained in a commer Cambridge University Press). cial mill used to de-husk quinoa for human consumption. 0058. It is known that quinoa husks contain at least 16 0050. This molluscicidal activity has encouraged various different triterpenic saponins (Woldemichael and Wink, studies since the 1980s for the characterization of existing 2001). The main saponins are bidesmosidic glycosides of saponins in vegetable species such as Phytolacca, Swartzia oleanolic acid, hederagenin and phytolaccagenic acid (Dini among others (Bore, C., Hostettmann, K., 1987. Mollusci et al. 2001*; Woldemichael and Wink, 2001; Zhu et al. cidal Saponins from Swartzia madagascariensis DES 2002). The type of Saponin present is important, since VAUX. Helvetica Chimica Acta 70, 570-576. Dorzas A. C., saponins derived from oleanolic acid and hederagenin Hostettmann, K., 1986. Further Saponins from Phytolacca exhibit high molluscicidal activity (Hostettmann and Mar dodecandra L'Herit. Helvetica Chimica Acta Vol 69 2038 ston, 1995, p 261). 2047. Dorzas A. C., Hostettmann, M., Hostettmann K., 1988. Molluscicidal Saponins from Sesbania Sesban. Planta 0059 Quinoa husks represent about 8-12% w/w of the Medica, 225-227. Trey vaud V. Marston A, Wahyo D, grain and are considered a by-product with no commercial Hostettmann K., 2000. Molluscicidal saponins from Phyto value. The Saponin content depends on the quinoa variety: lacca icosandra. Phytochemistry 55, 603-609). so called "Sweet quinoas contain lower amounts of 0051. The most important work relates to the use of saponins than “bitterquinoas. Bitter varieties show more Phytolacca dodecandra extracts in Africa (particularly resistance to pests and are more widely cultivated. Ethiopia), for the control of snails of Biomphalaria, Bulinus 0060. The saponins concentrate in the outer husk of the and Oncomelania genus, which transmit the Schistosomiasis grain, which is removed prior to consumption, and is a disease. This is the second most important disease in tropical by-product without commercial value. (Woldemichael, G. US 2007/01965.17 A1 Aug. 23, 2007

and Wink, M. 2001. Identification and biological activities F., et al. Chem. Pharm. Bull., 38:375 (1990)); however, the of triterpenoid saponins from Chenopodium quinoa. J. procedures used in these investigations for the recovery of Agric. Food Chem. 49: 2327-2332.). The husk contains a the Saponins are not practical and applicable for commercial high content of the molluscicidal triterpenes oleanolic acid scale for production. The studies of Mizui et al. (Mizui, F., and hederagenin. et al. Chem. Pharm. Bull., 38:375 (1990)) and others have 0061 Quinoa husk has the following advantages: 1) low shown that the Saponins found in quinoa are of the triterpene cost (it is a by-product); 2) abundant (it is found in Bolivia type. and Peru, and more recently in Chile); 3) high saponin 0067. The prior art for isolation of saponins from quinoa content (25-30%); and 4) Quinoa saponins have recently falls into two categories: a) an aqueous extraction route been approved by the US Environmental Protection Agency typically as described in Estrada et al., U.S. Pat. No. for its use against pathogenic fungi, bacteria, and viral plant 5,597,807 and U.S. Pat. No. 5,688,772; and b) a more diseases (Saponins of Chenopodium quinoa. Biopesticides traditional hot alcohol solvent (Mizui, F., et al. Chem. Registration Action Document PC Code 097094. U.S. Envi Pharm. Bull., 36:1415 (1988); Mizui, F., et al. Chem. ronmental Protection Agency. Office of Pesticide Programs. Pharm. Bull., 38:375 (1990). Estrada et al. U.S. Pat. No. Biopesticides and Pollution Prevention Division, 2005). 5,688,772 teaches that water extracts of quinoa (10 g of hulls This facilitates registration of the product in other countries. extracted by 2x100 ml of water) contain all or most of the 0062) The production of extracts rich in saponins from saponins present in quinoa. These extracts, used for their quinoa husk utilizing various solvents has been studied. U.S. molluscicidal activity, are not selective in control and kill Pat. No. 6,355.249 reports saponin extraction yields of many desirable aquatic species at the levels required to kill 20-25% w/w using ethanol/water mixtures. Furthermore, the the intended mollusks. patent describes the production of quinoa Sapogenins by 0068 Therefore, there exists the need to develop a prod acid hydrolysis of the Saponins. Studies carried out at uct which is effective for its intended use in controlling Catholic University of Chile (Department of Chemical and mollusks but is selective in its effect on non-target species. Bioprocess Engineering) show that the aqueous extraction of the quinoa husk (variety Real, Bolivia) yields up to 55% of 0069. In addition, there exists the need to develop new Solids that can be extracted with a saponin content of products, preferably natural products, with low cost and 50-60% w/w. This represents 25-30% of saponins in the minimum environmental impact. husk (Campos, C. 2003. Extraccion de Saponinas de la 0070. It is an object of this invention to provide a low cost Cascara de Quinoa. B.S.c. Thesis, Faculty of Engineering, product that has molluscicidal properties. Catholic University of Chile). 0071. It is another object of this invention to provide a 0063 Quinoa originates from the Andes region of South molluscicide that is selective in effect and does not harm America where it was a staple grain in pre-Spanish Conquest aquatic species other than mollusks. times. Traditional use declined after the Spanish Conquest (Galwey, N. W., et al., Food Sci. Nutr; 42F:245, 1990) and 0072. It is yet another object of this invention to provide cultivation and use of the grain was not widespread until a a molluscicidal product whose use does not harm human recent revival due to western interest in this crop as a high beings or the environment. lysine, high protein grain for human consumption (De Bruin, A. J. Food Sci., 26:872, 1964). The principal obstacle to BRIEF SUMMARY OF THE INVENTION wider human consumption of this grain is the bitter taste of the saponin content of the grain. In traditional use, the 0073. This need for a selective molluscicide has been met saponin content of the grain was reduced to acceptable by the present discovery of a method of modifying Saponins. levels by washing the grain in running water. These saponins The present specification discloses techniques for the pro have been shown to be anti-nutritive in animal studies (Gee, duction of highly molluscicidal saponins from abundant and J. M., et al., J. Sci. Food Agric., 63:201, 1993). low cost raw materials. 0064. Since the revival of interest in Quinoa, a number of 0074. In the present invention the use of saponins from attempts have been made to devise practical methods to the Andean quinea cereal (Chenopodium quinoa) is pre reduce the Saponin content (Amaya-Farfan, J., et al., ferred and is used to control populations of fresh water Removal of Saponins from quinoa (Chenopodium quinoa Snails, mussels and other mollusks. Willd) grain by milling, 5th Int. Congr. Food Sci. Technol. 0075 Modified saponins were tested against GAS and Kyoto, Japan (1978); Gee, J. M., et al., J. Sci. Food Agric. non-target organisms, such as fish, at a laboratory scale. The 63:201,1993; Reichert, R. D., et al., Cereal Chem. 63:471, tests were performed with saponins in their native state and 1986; Galwey, N.W., et al., Food Sci. Nutr. 42F:245, 1990; also after treatment under alkaline conditions. Tests against Rios, M. L. T., et al., Arch Latinoamer Nutr. 28:253, 1978; GAS were also carried out in rice fields in Northern Argen Ridout, C. L., et al., J. Sci. Food Agric. 54:165, 1991), tina and The Philippines during 2005 and 2006. including combinations of milling and washing. In all cases the saponin rich fraction was considered to be a waste 0076 A major advantage of the present invention is that product and was discarded. the Saponins modified according to the instant process have only a moderate toxicity to non-target organisms such as 0065. Many interesting physiological and pharmacologi fish. This means that product concentrations that are lethal cal effects have been attributed to saponins and/or the for the target species, such as Snails, mussels or other corresponding Sapogenins. mollusks are not lethal for fish. Thus the product is safe for 0.066 The chemical nature of the saponins found in its use in fresh water systems where fish or other forms of quinoa has been the Subject of several investigations (Mizui, beneficial aquatic life are present. US 2007/01965.17 A1 Aug. 23, 2007

BRIEF DESCRIPTION OF THE DRAWINGS 0089 Saponins sourced from quinoa hulls or husks are the preferred Saponin Source in the present invention. 0.077 FIG. 1 is a flow chart of the major processing steps for producing a powder product. 0090 Natural saponins have no effect or are of varying effectiveness in their extracted state and suffer from the 0078 FIG. 2 is a flow chart of the major processing steps problems of non-selectivity in that they kill significant for producing a liquid product. numbers of non-target organisms. Thus, an ongoing problem 0079 FIG. 3 is a flow chart of the example processing has been to maintain or increase the effectiveness of the steps for producing a powder product. extracted Saponins while at the same time minimizing det rimental effects on the environment and non-target species. 0080 FIG. 4 shows RP-HPLC chromatogram of quinoa saponins where FIG. 4a shows a chromatogram of crude 0091. The naturally occurring saponins when processed aqueous extract, and according to the procedures disclosed herein overcame the existing problems with the use or the saponins of the prior 0081 FIG. 4b is a chromatogram of aqueous extract art. purified with ultrafiltration membranes. 0092. The modified saponin molluscicide of the present 0082 FIG. 5 shows RP-HPLC chromatograms of quinoa invention is useful to limit or eradicate populations of husks treated with NaOH. mollusks of various types. DETAILED DESCRIPTION OF THE 0093. In one embodiment, it is especially useful to con INVENTION trol or eradicate populations of apple Snails, especially golden apple Snails. 0083. The present disclosure describes a selective mol luscicide that is able to kill mollusks in an aqueous envi 0094. In another embodiment it is useful in the control or ronment while not harming non-mollusk species; methods eradication of Zebra mussels. of producing Such a selective molluscicide and methods of 0095 Apple snails are only a small part of the snail utilizing Such a molluscicide. species that inhabit bodies of water around the world. The 0084. The molluscicide of the present invention utilizes a method and product of the present invention may also be saponin containing plant material Such as quinoa husk used to control or eradicate populations of other Snails, fresh (Chenopodium quinoa) as a raw material. The quinoa raw water Snails and other mollusks. material is treated to render the saponins present in the raw 0096. The modified saponin containing molluscicides of material more effective in their molluscicidal properties the present invention are useful against organism of the while at the same time diminishing or removing any detri Class Gastropoda and Class Bivalvia. More particularly, the mental effects the product may have on non-mollusk species. modified Saponin containing molluscicides of the present This is accomplished by treating the husks with alkali to invention are effective against members of the following: increase the molluscicidal properties of the Saponins. 0097 Class Gastropoda Subclass Pulmonata 0085. The molluscicide of the present invention is uti lized by introduction to the aqueous environment of the 0.098 Family Acroloxidae, genera Acroluxus, Pseu target species. dancylastrum 0.086 The product useful in the present invention is a 0099 Family Lymnaeidae, subfamilies: Lancinea and composition containing as an active ingredient a modified Lymnaeinae, genera Fisherola, Lanx , Lynnea, Galba, saponin obtained by treatment of a natural source of Stagnicola, Radix, Lymnea, and other genera. saponins with an aqueous alkaline Solution under conditions 0.100 Family Physidae, genera Physa, Physella, that modify a substantial proportion of the naturally occur Aplexia, Stenophysa Family Planorbidae, subfamilies ring saponins to a form that is both more effective in Rhodacmeinae, Buliniae and Planorbinae, subfamily Suppressing or eradicating populations of target mollusk Buliniae three tribes: Bulinini genera: Bulinus, Indopl species and which is also selective in that it has a minimal anorbis, Laevapex, Gundlachia; Physastrini genera: effect on non-mollusk animals. Glyptophysa, Physastra, Ameriana, Barnupia, Ferre sia, Pettancyclus, Isidorella, Bayardella, Patelloplan 0087 Many natural sources of saponins have been iden Orbis, Oppletora, Ancylastrum, Miratesta; Campro tified, including in Aloe, amaranth, asparagus (as protodios ceratini genera: Helisoma, Planorbarius; Subfamily cin), chickweed, Bacopa monnieri, Chlorophytum species, Planorbinae of five tribes: Planorbulini genera Planor Chlorogalum species, Soap plants, Conkers/horse chestnuts, bula, Menetis, Promenetus, Planorbella; tuberous cucurbit species, digitalis (as digitonin), echino Biomphalariini genera Biomphalaria, Drepanotrema: dermata, fenugreek, grape skin1 gotu kola, Gypsophila Planorbini genera: Planorbis, Afrogyrus, Afrogyrorbis, (Baby's Breath), jiaogulan, liquorice, mullein, olives, panax Anisus, Bathy Omphalus, Gyraulus, Choanomphalus; (as ginsenoside), Ouillaia saponaria (bois de Panama, mem ber of the Rosaceae family), quinoa, rambutan, Soapberry Segmentini genera: Segmentina, Hippeutis, Lentorbis, and many other members of the family Sapindaceae, includ Polypylis, Helicorbis; Ancylini genus: Ancylus ing buckeyes, Saponaria (Soapwort, Bouncing Betty), Soy 0101 Class Gastropoda Subclass Prosobranchia; beans, Tribulus terrestris (as protodioscin), wild yam, yucca. 0102 Family Thiaridae genera Aylacostoma, Cubae 0088. The major industrial sources of steroidal and trit domus, Dorissa, Fijidoma, Hemisinus, Melanatria, erpenic saponins are Yucca Shidigera and Ouillaia Melanoides, Pseudopotamis, Sermyla, Sermylasma, saponaria Molina extracts. Thiara, Tvlomelania. US 2007/01965.17 A1 Aug. 23, 2007

0.103 Family Viviparidae, subfamily Viviparinae gen Description of Processes and Usage Rates era Viviparus, Tulatona; Subfamily Campelominae or Lioplacinae genera Campeloma, Lioplax; Subfamily 0.117) The final product may be in dry or aqueous form. Bellamyinae genera Bellamyia, Cipangopaludina, Depending on the form of the final product desired, the Notopala. extraction parameters and additional stages differ. 0.104 Family Neritidae genera Clypeolum, Fluvion Powder Type Product erita, Neritilia, Neritina, Septaria, Theodoxus. 0118. In general, quinoa husks as acquired from the de-hulling procedure without any preliminary preparation 0105 Family Pilidae genera Afropomlus, Asolene, are used for saponin extraction with water. The ratio of husks Felipponea, Lanistes, Marisa, Pila, Pomacea, Pomella, to water (weight/weight) is on the range of from about 1:1 Saulea, Turbinicola to about 1:10, preferably in the range of from about 1:1 to about 1:7 most preferably in the range of from about 1:3 to 0106 Class Bivalvia Subclass Pteriomorphia about 1:5. 0107 Family Mytillidae 0119) The extraction time is a function of both the 0108 Class Bivalvia Subclass Heterodonta temperature and the ratio of water to husks. In general, the extraction time is in the range of from about 0.1 to about 3 0109) Family Dreissenoides hours, preferably in the range of from about 0.5 to about 3 hours and most preferably in the range of from about 0.5 to 0110 Most particularity, the modified saponins are effec about one hour. tive to control members of the subclass Prosobranchia Family Pilidae, genera Pila and Pomacea. 0.120. The temperature also affects the extraction time. In general the extraction may be carried out at ambient tem 0111. The preferred source of the saponin active compo perature but at a temperature in the range of from about 20 nent is quinoa husk (Chenopodium quinoa) but the saponins to about 90° C. has been found to be satisfactory with a from other natural sources may also be used. temperature in the range of from about 20 to about 50° C. 0112 Quinoa husks are obtained as a byproduct of edible being preferable and a temperature in the range of from quinoa production where the Saponin rich husk of the quinoa about 20 to about 35° C. being most preferable. seed is removed from the seed to provide an edible product. 0121 Where a powder type final product is desired, the The husks area byproduct of quinoa production and thus are flow sheet for the production process, including the most available at a low or even negative cost. preferred process parameters, is shown in FIG. 1. 0113. The extraction process of the invention is prefer 0.122 Extraction: where a powder type final product is ably carried out on a commercial variety or cultivar of quinoa using a dry (non-green) part, and most preferably a desired the extraction parameters are shown in Table 1. husk product obtained by dry de-hulling to remove seed coats from commercial quinoa grain. Quinoa husk is com TABLE 1. mercially available and inexpensive (virtually a waste prod Extraction parameters for powder type product uct) resulting from the treatment of quinoa seed to form consumable flour. Quinoa husk is rich in Saponins (the husk Most contains approximately 50 times more Saponin than could be Dry Product Suitable Preferred Preferred recovered by washing the whole grain or extracting ground Ratio Guinoa Water (wiw) 1:1-10 1:1-7 1:3-5 seed) and can be obtained by dry milling of high Saponin Temp ( C.) 2O-90 20-50 20-35 content quinoa. However, if desired, other (preferably not Time (hrs) O. 1-3 O.S. 3 OS-1 green) parts of the quinoa plant may be used as starting materials for the saponin extraction, e.g. whole seeds, ground seeds, seed coats or quinoa flour. 0123 Alkaline treatment: the whole solution from the extraction step is treated with a basic reagent to modify the 0114. The husks are stripped from the quinoa seed by pH of the solution. Any convenient base may be utilized: conventional means. The husks need not be pre-treated by sodium hydroxide is preferred. This basic reagent is added physical or chemical means before use in the several diluted in water so the ratio of husks to alkaline solution is embodiments of the present invention, although communi in the range of 1:2. This makes a final solid to liquid ratio of tation by mechanical means to further reduce the particle about 1:5. size of the husks is a convenient method of reducing the extraction time. If reduction in particle size is desired it may 0.124. The concentration of the basic agent in the reaction be accomplished by any conventional means. mixture may vary from about 0.1 N to about 1 N. In particular, if the end product is to be a powder type product, 0115 When ready for use, either as received or as further the concentration is preferably in the range of from 0.3 N to reduced in size, the husks are mixed with water in a vessel 0.8 N, most preferably in the range of from about 0.4 N to of convenient size and the mixture stirred to aid extraction about 0.6 N. of the saponins from the husks into the water. 0.125 The pH of the solution is raised to a pH in the range 0116. The extraction step is carried out at a specified ratio of from about 8 to about 13, preferably to a range of from of husk to water and for specified times and temperatures about 10 to about 13, most preferably in the range of from Sufficient to extract the Saponins from the husks. about 12 to about 13. US 2007/01965.17 A1 Aug. 23, 2007

0126 The alkaline aqueous solution is heated attempera 0.135 Where a liquid type final product is desired the ture in the range of from about 50 to about 100° C. flow sheet for the production process, including the most preferably to a temperature in the range of from about 70 to preferred process parameters is shown in FIG. 2. about 100° C., most preferably to a temperature in the range 0.136 Extraction: where a liquid final product is desired of from about 90 to about 100° C. the extraction parameters are shown in Table 3. 0127 Heating of the basic aqueous solution generally continues for a period of from about 0.5 to about 3 hours, TABLE 3 preferably for a period of from about 0.5 to about 2 hours, most preferably for a period of from about 1-2 hours. This Extraction parameters for liquid type product alkaline treatment converts the native quinoa Saponins to Most more hydrophobic saponin derivatives. Liquid Product Suitable Preferred Preferred 0128. Where a powder type final product is desired the Ratio Guinoa Water (wiw) 1:1-15 1:5-10 1:8-10 alkaline treatment parameters are shown in Table 2. Temp ( C.) 20-100 20-SO 20-30 Time (hrs) O. 1-3 OS-3 OS-1 TABLE 2 Alkaline treatment parameters for powder type product 0.137 Protein removal: following extraction, the whole mixture is treated with concentrated acid to precipitate any Dry Most proteins present in the Solution. This is accomplished by Product Suitable Preferred Preferred lowering the pH to 3 to 5, most preferable to pH 3.5 to 4, Base used NaOH NaOH NaOH which corresponds to the isoelectric point of quinoa O. 1-1 N O3-08 N O4-06 N saponins. This produces the precipitation of an amorphous pH 8-13 10-13 12-13 Temp ( C.) SO-100 70-100 90-100 protein fraction. An alternative procedure considers adding Time (hrs) OS-3 OS-2 1-2 bentonite to the Solution, since this compound adsorbs proteins rendering them insoluble. It has also the incidental use of inducing more rapid clarification. Bentonite can be 0129 Neutralization: once the alkaline treatment is fin added at 1-10 g/L, most preferable at 1 to 5 g/L. ished, the extract is cooled to room temperature and con 0138 Removal of insoluble solids: following extraction centrated acid is added to bring the extract to a pH in the and protein precipitation, the husks and the precipitated range of from about 3 to about 7.5, preferably in the range protein fraction are separated from the liquid extract by any of from about 4 to about 7.5, most preferably to a pH in the conventional means, such as by decantation, filtration with range of from about 6.5 to about 7.5. or without the help of diatomaceous earth or other agent or 0130 Drying: the extract is then dried with hot air to a centrifugation. This step must be done carefully so as to also moisture content of 5-10% w/w, followed by packaging. remove the starch fraction present in the extract. These starch granules may produce gelatinization when the extract 0131 Mollusk Control: This composition is useful in the control of fresh water snails and other mollusks. For the is further heated in the process (e.g. concentration by heat). control of aquatic fresh water mollusks, the inventive prod 0.139 Washing of solids: the solid fraction remaining uct is added at product concentration from about 10 to about after Solids removal may be re-extracted in a second extrac 40 ppm, most preferably from about 20 to about 35 ppm in tion step to recover any residual Saponins present in the the volume of water to be treated. occluded solution. For this, the solids are contacted with Liquid Type Product water under similar conditions to the initial extraction. 0.132. In general, quinea husks as acquired from the 0140. The second extraction step is conducted under the de-hulling procedure without any preliminary preparation following conditions. are used for saponin extraction with water. The ratio of husks to water (weight/weight) is on the range of from about 1:1 TABLE 4 to about 1:15, preferably in the range of from about 1:5 to Re-extraction parameters for liquid type product about 1:10 most preferably in the range of from about 1:8 to about 1:10. Most Liquid Product Suitable Preferred Preferred 0133. The extraction time is a function of both the Ratio Guinoa Water (wiw) 1:1-15 1:5-10 1:5-8 temperature and the ratio of water to husks. In general, the Temp ( C.) 20-80 20-50 20-30 extraction time is in the range of from about 0.1 to about 3 Time (hrs) O. 1-3 OS-2 OS-1 hours, preferably in the range of from about 0.5 to about 3 hours and most preferably in the range of from about 0.5 to about one hour. 0.141. Once the second extraction is finished, the solids 0134) The temperature also affects the extraction time. In and extract are separated in the same manner as in the initial general the extraction may be carried out at ambient tem extraction. If desired, more than two extractions can be perature but at a temperature in the range of from about 20 performed to further maximize saponin recovery. The to about 100° C. has been found to be satisfactory with a parameters of Such further extraction steps are equivalent to temperature in the range of from about 20 to about 50° C. those of the second extraction step. being preferable and a temperature in the range of from 0.142 Industrially, multiple extractions can be performed about 20 to about 30° C. being most preferable. using a counter-current multiple stage solid-liquid extraction US 2007/01965.17 A1 Aug. 23, 2007 equipment. Alternatively, if the filtration after the first of aquatic fresh water mollusks, the inventive product may extraction is performed in a conventional plate and frame be added at a solids concentration from about 10 to about 40 filter equipment, fresh water can be used to wash residual ppm, most preferably from about 20 to about 35 ppm, in the Saponins. volume of water to be treated. Depending on the solid 0143 Concentration: the extract is optionally concen concentration of the final product (g Solids/L), the amount of trated by heating at atmospheric or reduced pressure to final product to be added may vary. obtain a final solids concentration in the range of 200 to 500 Determination of Saponins Concentration and Extent of g solids/L, most preferably 200 to 350 g solids/L. Alkaline Treatment 0144 Alkaline Treatment: the liquid extract is treated 0153. A preferred method of estimating the saponin con with a basic reagent to modify the pH of the solution. Any tent utilizes reverse phase HPLC based on the method convenient base may be utilized; sodium hydroxide is pre developed for Ouillaia Saponaria Molina triterpenic ferred. This basic reagent is added directly to the extract in saponins (San Martin and Briones, 2000). This method can a powder or liquid form. also used to assess the extent of the alkaline treatment, since the more hydrophobic saponin derivatives elute at longer 0145 The concentration of the basic agent in the reaction retention times in the chromatogram. In RP-HPLC chro mixture may vary from about 0.1 N to about 1 N. In matograms, the non-treated quinoa saponins elute in the particular, if the end product is to be a liquid product, early part of the chromatogram. After alkaline treatment, preferably in the range of from 0.3 N to 0.8 N, most these peaks weaken or disappear, and new peaks are formed preferably in the range of from about 0.4 N to about 0.6 N. at longer retention times (more hydrophobic compounds). 0146 The pH of the solution is raised to a pH in the range The extent of the reaction can be monitored, by following of from about 8 to about 13, preferably to a range of from the area of the remaining hydrophilic peaks. Complete about 10 to about 13, most preferably in the range of from reaction corresponds to almost complete disappearance of about 12 to about 13. the hydrophilic peaks. 0147 The alkaline aqueous solution is heated attempera Effect of Alkaline Treatment ture in the range of from about 50 to about 100° C. 0154 Previous papers describe that exposure of quinoa preferably to a temperature in the range of from about 70 to saponins (mainly bidesmosidic) to basic conditions causes a about 100° C., most preferably to a temperature in the range release of the glucose in carbon 28, resulting in a monodes of from about 90 to about 100° C. mosidic saponin. 0148 Heating of the basic aqueous solution generally continues for a period of from about 0.5 to about 3 hours, 0.155 Surprisingly, we have now discovered that the preferably for a period of from about 1 to about 3 hours, conversion of bidesmosidic saponins to the monodesmosidic most preferably for a period of from about 1.5-2.5 hours. reported in the literature appear to occur to completion only This alkaline treatment converts the present Saponins to in the presence of pure Saponins. In the presence of multiple more hydrophobic saponins. other components such as are present in crude aqueous saponin extracts obtained from natural sources, exposure to 0149. In particular, where the end product is to be a liquid alkaline conditions results in a complex alteration of many product, the alkaline treatment parameters are shown in of the salts, phenols, proteins, starch, fat and saponins Table 5. present. It is the combination of the alteration in the structure of these multiple components that yields the Synergistic TABLE 5 effect demonstrated by the present product. Alkaline treatment parameters for liquid product 0156 RP-HPLC analysis revealed that the saponin origi nal present shows peaks in the hydrophilic part of the Dry Most chromatogram (short retention times). After alkaline treat Product Suitable Preferred Preferred ment, the Saponin peaks shift from short retention times, to Base used NaOH NaOH NaOH longer retention times. Almost all original saponin peaks O. 1-1 N O3-08 N O4 0.6 N disappear with 2 hours of exposure to alkaline conditions. pH 8-13 10-13 12-13 Temp ( C.) SO-100 70-100 90-100 0157 Samples of the original quinoa husks and the Time (hrs) OS-3 1-3 1.5-2.5 alkaline treated material may also be analyzed using LC/MS. This method allows the identification of saponin structures based on the molecular mass of each Saponin. 0150 Neutralization: once the alkaline treatment is fin ished, the extract is cooled to room temperature and con 0158. The LC chromatogram has confirmed that after centrated acid is added to bring the extract to a pH in the alkaline treatment, more hydrophobic saponin derivatives range of from about 3 to about 7.5, preferably in the range are formed of from about 4 to about 7.5, most preferably to a pH in the 0159. The MS analysis of the saponins in the raw mate range of from about 6.5 to about 7.5. rial showed a combination of bidesmosidic, but also some 0151. This extract is packaged and is ready for sale. In monodesmosidic saponins, confirming existing literature this phase, excipients or preservatives can also be added to reports. The main Saponins are based on structures of improve its stability. oleanolic acid, phytolaccagenic acid and hederagenin. 0152 Mollusk Control: This composition is utilized to 0.160 However the analysis of the product surprisingly control fresh water snails or other mollusks. For the control does not show lower molecular weight monodesmosidic US 2007/01965.17 A1 Aug. 23, 2007

saponins as would have been expected. On the contrary, the (Lindeboom, 2005), and filtered with the aid of 5 g/l of MS shows that the product contains a complex mixture of diatomaceous earth and Whatman #2. The extract was then high molecular weight compounds. These compounds are dial filtered with 8 volumes of distilled water, using 10 derivatives of the original Saponins. Instead of obtaining kDalton ultrafiltration membranes (Amicon, USA) to Smaller compounds, it appears that some Sugars and also remove low molecular weight non-Saponin impurities. unknown compounds attached to the original Saponins. Saponins are retained by the UF membranes due to their capacity to form micelles at concentrations above the critical 0161 During the alkaline treatment step a portion of the micelle concentration. Since the proximal analysis showed saponins are modified. The degree of modification may be the presence of 20% w/w non-Saponin compounds (protein, measured by any convenient method. fat, ash), the Saponin content of the standard was estimated 0162 The following non-limiting examples further illus at approximately 80% w/w. trate the invention but do not define the full scope of the 0170 Product yield of 1.1 kg of dry product was invention obtained. The saponin content of the raw material, as well the extent of the alkaline reaction were estimated using EXAMPLES RP-HPLC analysis. FIG. 4 shows RP-HPLC chromatograms Example 1 of an aqueous extract of the raw material (FIG. 4a) and the purified sample used as standard (FIG. 4b). Both chromato grams are similar, except that for the standard the UV Preparation of Powder Type Product and Saponin absorbing impurities that elute in the early part of the Analysis chromatogram have been reduced (e.g. elution time<4 min) 0163. One kg of husks of quinoa real (origin: Bolivia) due to diafiltration. Similar RP-HPLC chromatograms con were contacted in a tank with 3 L of water. The mixture was taining three main peaks (peaks A, B and C) have been stirred for 30 min at room temperature. Then 80 g of NaOH reported for quinoa saponins (Muir et al. 2002). The proxi were dissolved in 2 L of water and added to the mixture. The mal analysis of the standard revealed the presence of 20% final soda concentration was 0.4 N (80 g of soda in 5 L of W/w non-Saponin compounds (ash, protein, fat). This was water, molecular weight of NaOH: 40 g/gmol. This mixture probably due to the entrapment of impurities in the Saponin was stirred at 95°C. for 2 hours. After completion of the micelles and the partial loss of Saponins through the mem reaction, the mixture was cooled at room temperature and brane. These observations agree well with previous reports neutralized with HCl (final pH 7.0).The mixture was air that indicate that quinoa samples treated with lower molecu dried for 16 hours at 70° C. (see FIG. 3). lar weight cut-off UF membranes (e.g. 1000 kDalton) can achieve a higher Saponin content, e.g. 85-90% w/w Saponins 0164. The saponin content in the raw material was esti (Muir et al. 2002). To estimate the saponin content of the raw mated using reverse phase HPLC based on the method material, 100 g quinoa husks were mixed with 1 L of water developed for Ouillaja Saponaria Molina triterpenic for 2 hours. An average of 55 g soluble solids/L was saponins (San Martin and Briones, 2000), using a Waters obtained, with a saponin content of approximately 52-53% 600 equipment and UV detection at 210 nm. This analysis W/w. This did not change significantly with longer mixing was also used to assess the extent of the alkaline treatment. time. Based on this, the Saponin content of the quinoa husks Aqueous extracts for analysis were prepared by mixing 100 used in laboratory experiments was estimated at 29% w/w. g of husks or product with 1 L of water for 2 h at room temperature and agitation, followed by filtration with What 0171 FIG. 5 shows the chromatogram of aqueous man # 2. The samples were injected at approximately 30 g extracts of the raw material after treatment with NaOH for soluble solids/L; the exact value was determined through 2 hour. The chromatogram shows that the three major peaks drying at 70° C. until constant weight. The injection volume present in the untreated samples disappeared almost com was 20 uI. In all chromatograms, Saponins were considered pletely and new peaks formed at longer elution times (more to elute after 4 min, since non-Saponin hydrophilic com hydrophobic compounds). With shorter reaction times, a pounds (e.g. polyphenols) elute in the early part of the fraction of the three major original saponin peaks remained, organic solvent gradient. Saponin content was estimated by indicating incomplete reaction (chromatograms not shown). comparing area of Saponins in the sample to that of the standard containing 80% w/w saponins (see below): Example 3 0165 % saponins (w/w): (A sample/A standard)x (Soluble solids standard/Soluble solids sample)x80% Effect of Products on Golden Apple Snails and Fish 0172 Product toxicology in snails was assessed with 0166 The overall saponin content was thus estimated as: golden apple snails (Pomacea canaliculata) from Argen 0167 % saponin (w/w): % saponin (w/w) in soluble tinean origin. Quinoa husks and the powder type product SolidsxTSS described in Example 1 was used. For this purpose, 20-35 mm adult Snails were used. The Snails were maintained in 0168 Total soluble solids (TSS) were estimated as the 25x20x15 cm. glass aquaria with lids. Each aquarium was concentration of soluble solids in the filtrate (in g/L) times filled with 2.5 L of tap water, previously treated with a the total volume of water added (in L). chlorine neutralizing product. The Snails were kept at about 0169. The standard was prepared by mixing 100 g of 22°C. using electrical heaters (3 Watt resistance). The pH husks with 1 L of water at ambient temperature for 2 h. The was about 7.5. Before starting the tests, the snails were extract was filtered through Whatman #2 filtering paper. The acclimatized to lab conditions for a week, receiving filtrate (pH 5.7) was acidulated with concentrated HCl to pH aquarium fish feed flakes every 12 h. Four snails were placed 3.5, to obtain the isoelectric precipitation of quinoa proteins in each aquarium and each experiment was carried out in US 2007/01965.17 A1 Aug. 23, 2007 triplicate. Snail mortality was assessed by stimuli with a a) contacting a saponin containing plant material with stainless steal needle and detection or lack of movement. water for a time sufficient to extract a portion of the After 24h, Snails classified as dead were placed in a separate Saponins from the plant material, container with fresh water and checked for recovery. Only those snails that did not recover within additional 24 h in b) adding a basic agent to alkaline modify the Saponins fresh water were reported dead. present in the solution from Step a), 0173 Product toxicology in fish was assessed in 2-3 g c) reducing the pH of the solution from step b) to no goldfish (Carassius auratus) using the products and the greater than 7.5, and aquarium system previously described, except air pumps d) drying the mixture of step c). were added. This time 5 liters of water per aquarium were 2. The composition of claim 1 where used at a temperature 16-18°C. Four fish were used in each step a) comprises contacting the Saponin containing plant aquarium and each experiment was carried out in triplicate. material with water in a ratio of 1 part by weight of In some experiments 7-8 g tilapias (Oreochromis sp.) were plant material with from 1 to 10 parts by weight of used at 21-22 C. Every 24 h during 96 h, the number of water at a temperature from 20° to 90° C., from 0.1 to dead fish per aquarium was recorded. 3 hours, 0174 Table 6 shows the results of the experiments for the step b)comprises adding sufficient basic agent to adjust original quinoa husks and the powder type product tested the pH to a pH in the range of 8 to 13 while heating for against GAS and fish. 0.5 to 3 hours at a temperature from 50 to 1000 C, and 0175. In the case of untreated quinoa husks, no dead step c) comprises cooling the mixture of step b) to snails or fish were observed even at the highest concentra ambient temperature and reducing the pH to a pH in the tion tested (e.g. 121 ppm of product). However, the alkali range of 3 to 7.5. treated husks killed 100% of the snails at about 33 ppm. At 3. The composition of claim 1 where the saponin con this concentration, goldfish and tilapia did not die in 96 taining plant material is at least one part of a quinoa plant. hours. In fact, no dead fish were observed even at the highest 4. The composition of claim 3 where the plant material is dose tested, e.g. 54 ppm product. quinea husk. 0176) These results indicate that the composition based 5. A liquid molluscicidal composition comprising alkaline on alkaline treated husks is lethal for GAS at lower doses modified plant Saponins, obtained by a process comprising than for fish, making it possible to use it safely in rice fields. Another interesting observation is that the snails closed their a) contacting a saponin containing plant material with operculum and ceased all movement almost immediately water for a time sufficient to extract a portion of the after the addition of 33 ppm of product. This probably allows Saponins from the plant material, for the protecetion of germinating seeds and young rice b) removing soluble proteins from the extract, seedlings, reducing crop establishment losses in direct seeded rice. Tests with husks treated with alkali for less than c) separating insoluble materials from the liquid extract 2 h, or with lower NAOH or temperature conditions, were from step b) and recovering the extract, significantly less lethal to GAS (results not shown). d) optionally concentrating the extract recovered in c), e) adding a basic agent to alkaline modify the saponins TABLE 6 present in the extract from step d), Putinoa Saponin toxicology in Snails f) reducing the pH of the extract from step e) to no greater and fish under laboratory conditions than 7.5, and Snails Goldfish Tilapia fish g) optionally adding excipients and/or preservatives. % % % % % % 6. The composition of claim 5 where ppm dead dead dead dead dead dead step a) comprises contacting the plant material with water at a ratio of 1 part by weight plant material to from 1 part to 15 parts by weight of water at a temperature from 20° to 100° C., for a period of from 0.1 hour to 3 hours, step b) comprises removing soluble proteins from the extract by a method selected from adding acid in sufficient quantity to bring the pH to 3.5 to 4 to precipitate the proteins or by adding bentonite to the Solution in the range of 1-5 g/L to adsorb the proteins, step d) comprises concentrating the extract obtained in c) to a solids content of 200 to 500 g/L, step e) comprises contacting the extracts of step d) with a sufficient quantity of a base to adjust the pH to a pH in 1. A particulate molluscicidal composition containing the range of from 8 to 13 while simultaneously heating alkaline modified plant Saponins obtained by a process to a temperature in the range of from 50 to 100° C. to comprising: alkaline modify the extracted Saponins, and US 2007/01965.17 A1 Aug. 23, 2007

step f) comprises reducing the pH to a pH in the range of part to 15 parts by weight of water at a temperature from 3 to 7.5. from 20° to 100° C., for a period of from 0.1 hour to 3 7. The composition of claim 5 where the saponin con hours, taining plant material is at least one part of a quinoa plant. step b) comprises removing soluble proteins from the 8. The composition of claim 7 where the plant material is extract by a method selected from adding acid in quinoa husk. sufficient quantity to bring the pH to 3.5 to 4 to 9. The composition of claim 5 where the insoluble mate precipitate the proteins or by adding bentonite to the rials from step c) are re-extracted at least one additional Solution in the range of 1-5 g/L to adsorb the proteins, time, Subject them again to step c) and the extracts are combined prior to step d). step d) comprises concentrating the extract obtained in c) 10. A process for producing a particulate molluscicidal to a solids content of 200 to 500 g/L, composition containing alkaline modified plant Saponins step e) comprises contacting the extracts of step d) with a comprising: sufficient quantity of a base to adjust the pH to a pH in a) contacting a saponin containing plant material with the range of from 8 to 13 while simultaneously heating water for a time sufficient to extract a portion of the to a temperature in the range of from 50 to 100° C. to Saponins from the plant material, alkaline modify the extracted Saponins, and step f) comprises reducing the pH to a pH in the range of b) adding a basic agent to alkaline modify the saponins from 3 to 7.5. present in the solution from Step a), 16. The process of claim 15 where the Saponin containing c) reducing the pH of the solution from step b) to no plant material is at least one part of a quinoa plant. greater than 7.5, and 17. The process of claim 16 where the plant material is quinoa husk. d) drying the mixture of step c). 18. The process of claim 14 where the insoluble materials 11. The process of claim 10 where from Step c) are re-extracted at least one additional time, Subject them again to step c) and the extracts combined prior step a) comprises contacting the Saponin containing plant to step d). material with water in a ratio of 1 part by weight of 19. A method of controlling fresh water mollusks com plant material with from 1 to 10 parts by weight of prising applying a molluscicidal effective amount of a water at a temperature from 20° to 90° C., from 0.1 to composition of claim 1 to a body of fresh water where such 3 hours, fresh water mollusks are found. step b)comprises adding Sufficient basic agent to adjust 20. The method of claim 19 where the fresh water the pH to a pH in the range of 8 to 13 while heating for mollusks are members of the subclass Prosobranchia, family 0.5 to 3 hours at a temperature from 50 to 1000 C, and Pilidae. 21. The method of claim 20 where the fresh water step c) comprises cooling the mixture of step b) to mollusks are members of the genera Pila and Pomacea. ambient temperature and reducing the pH to a pH in the 22. The method of claim 20 where the fresh water range of 3 to 7.5. mollusks are apple Snails. 12. The process of claim 10 where the Saponin containing 23. A method according to claim 19, where the freshwater plant material is at least one part of a quinoa plant. Snails are selected from Pomacea ssp. Isidorella ssp. 13. The process of claim 12 where the plant material is Biomphalaria ssp. Lynnaea spp. quinoa husk. 24. A method according to claim 23, where the freshwater 14. A process for producing a liquid molluscicidal com Snails are of the species Pomacea canaliculata. position comprising alkaline modified plant saponins com 25. The method of claim 19 where the fresh water prising mollusks are Zebra mussels. a) contacting a saponin containing plant material with 26. A method according to claim 19, where the body of water for a time sufficient to extract a portion of the fresh water is selected from flooded plantations, rivers, Saponins from the plant material, ponds, fish farms and lakes. 27. A method according to claim 19, where the effective b) removing soluble proteins from the extract, amount is 20-35 ppm alkali treated quinoa solids in the water to be treated. c) separating insoluble materials from the liquid extract 28. A method according to claim 19 where the body of from step b) and recovering the extract, fresh water is a fish farm shrimp farm or rice paddy. d) optionally concentrating the extract recovered in c), 'e) 29. A method of controlling fresh water mollusks com adding a basic agent to alkaline modify the Saponins prising applying a molluscicidal effective amount of a present in the extract from step c), composition of claim 5 to a body of fresh water where such fresh water mollusks are found. f) reducing the pH of the extract from step d) to no greater 30. The method of claim 29 where the fresh water than 7.5, and mollusks are members of the subclass Prosobranchia, family g) optionally adding excipients and/or preservatives. Pilidae. 15. The process of claim 14 where 31. The method of claim 30 where the fresh water mollusks are members of the genera Pila and Pomacea. step a) comprises contacting the plant material with water 32. The method of claim 30 where the fresh water at a ratio of 1 part by weight plant material to from 1 mollusks are apple Snails. US 2007/01965.17 A1 Aug. 23, 2007

33. A method according to claim 29, where the freshwater 37. A method according to claim 29, wherein the effective Snails are selected from Pomacea ssp. Isidorella ssp. amount is 20-35 ppm of alkali treated quinoa solids in the Biomphalaria ssp. Lynnaea spp. water to be treated. 34. A method according to claim 33, where the freshwater 38. A method according to claim 29, where the liquid Snails are of the species Pomacea canaliculata. composition comprises from 200 to 500 g/L of alkaline 35. The method of claim 29 where the fresh water modified quinoa solids and an excipient. mollusks are Zebra mussels. 39. A method according to claim 29 where the body of 36. A method according to claim 29, where the body of fresh water is a fish farm shrimp farm or rice paddy. fresh water is selected from flooded plantations, rivers, ponds, fish farms and lakes. k k k k k