Saponins containing extracts prepared from Hesperaloe, which are useful in the treatment of non-human animals.

Abstract

This invention provides a method for enhancing the immune response to an antigen in non-human animals. [Solution] A water-soluble extract from a non-woody plant of the genus Hesperaloe contains at least one saponin. Saponins extracted from Hesperaloe biomass may include 25(27)-dehydrofucleastatin, 5(6),25(27)-disdehydroyuccaloyside C, 5(6)-disdehydroyuccaloyside C, flucleastatin, and yuccaloyside. One embodiment of this application involves the administration of saponins containing a Hesperaloe extract. Another embodiment provides the administration of a Hesperaloe-derived immunomodulator containing one or more saponins for the prevention, treatment, and control of one or more conditions in non-human animals. Preferably, a Hesperaloe extract containing one or more saponins may be orally administered to poultry for the prevention and treatment of coccidiosis.

Description

Background Art

[0001] Plants produce a wide variety of types of organic compounds, most of which appear not to be directly involved in their growth and development. These substances have traditionally been called secondary metabolites or plant natural products and are often distributed among limited taxonomic groups within the plant kingdom. The functions of secondary metabolites are mostly unknown, but many compounds have been associated with useful traits in plants, such as protection against herbivores and protection against microbial infections, as attractants for pollen vectors and seed-dispersing animals, and as compounds (allelochemicals) that affect competition between plant species. Interest in plant natural products is increasing because these products often have a wide range of uses in various types of industries, such as the pharmaceutical industry, the cosmetics industry, the food industry, and the detergent industry.

[0002] A particular group of plant secondary metabolites of interest is saponins. Saponins are glycosylated compounds classified as either triterpenoids, steroids, or steroid-derived glycoalkaloids. Saponins consist of one or two sugar moieties (mono- and bisdesmosides, respectively) attached to an aglycon. Saponins can be hydrolyzed to sapogenins and sugar moieties by acid hydrolysis or enzymatic methods. Saponins are generally water-soluble high-molecular-weight compounds with molecular weights in the range of 600 to over 2,000 daltons.

[0003] The asymmetric distribution of their hydrophobic (aglycon) and hydrophilic (sugar) moieties imparts amphiphilic characteristics to these compounds, and they are mainly involved in their surfactant-like properties. By their ability to lower surface tension, saponins are potentially well-suited for use in the cosmetics industry and in the detergent industry.

[0004] Saponins also possess the ability to form insoluble complexes with cholesterol, which makes some of them suitable for use in the pharmaceutical industry as cholesterol-lowering agents. Other saponins are associated with the formation of immune-stimulating complexes that are useful in vaccine strategies.

[0005] Currently, a major limitation to the widespread use of saponins is the fact that commercially available saponins are relatively expensive. This cost is largely due to the limited number of plant extracts that contain substantial amounts of saponins. Commercially available plant extracts containing saponins currently include Saponaria officinalis, Quillaja bark and stems, Castanea sativa seeds, and extracts from various Yucca species.

[0006] Plant extracts containing saponins have thus become a matter of general interest across a wide range of industries. Consequently, there is a growing need in the art for alternative sources of saponin extracts, which are preferably inexpensive, easy to obtain, and preferably have a relatively high saponin content. [Overview of the project]

[0007] The present invention relates to compositions, methods, and kits for the administration of plant-derived immunomodulators. The compositions may be useful for sensitizing the innate and adaptive immune systems of non-human subjects, and thus can be used to treat infections or as adjuvants in immunization. Preferably, the plant-derived immunomodulators of the present invention are plant extracts containing saponins. Administration of these immunomodulators to subjects significantly enhances the cell-mediated immune system and significantly enhances antibody production in the subjects.

[0008] Accordingly, in one embodiment, the present invention provides a method for treating a non-human animal that has or is at risk of infection by administering to the animal a plant-derived immunomodulator in an amount sufficient to treat the infection. In a particularly preferred embodiment, the immunomodulator comprises at least one saponin extracted from a non-woody plant of the genus Hesperaloe.

[0009] In other embodiments, the present invention provides a method for administering a plant-derived immunomodulator to a non-human animal, wherein the plant-derived immunomodulator enhances the host's adaptive immune system and protects the non-human animal from disease caused by infectious pathogens. Preferably, administration of a plant-derived immunomodulator containing one or more saponins, prepared from non-woody plants of the genus Hesperaloe, to a subject may increase phagocytic activity in CD4 T helper (Th) cells, particularly Th1 and Th17 cells, in the subject being treated.

[0010] In further embodiments, the present invention provides the administration of plant-derived immunomodulators containing one or more saponins, preferably prepared from non-woody plants of the genus Hesperaloe, for the prevention, treatment, and control of one or more conditions in non-human animals, particularly birds, and more specifically poultry. For example, a saponin-containing composition derived from Hesperaloe may be administered to non-human animals to reduce ammonia and odor in the environment, provide a hypocholesterolemia effect, reduce inflammation, promote weight gain, and improve feed conversion efficiency. In a particularly preferred embodiment, a Hesperaloe extract containing one or more saponins may be orally administered to poultry for the prevention and treatment of coccidiosis.

[0011] In further embodiments, the present invention provides a method for enhancing the immune response to an antigen in non-human animals, comprising administering a saponin containing hesperaloe extract to a non-human animal in an amount sufficient to enhance the immune response in the non-human animal. In a particularly preferred embodiment, the saponin containing hesperaloe extract is administered to poultry, resulting in an increase in phagocytic activity in CD4 T helper (Th) cells, particularly Th1 and Th17 cells, and providing protection against Aemeria infection.

[0012] In other embodiments, the present invention provides an immunological composition useful for inducing antibody production against an antigen in a non-human animal, comprising an immunogenically effective amount of an antigen and a saponin composition extracted from a non-woody plant of the genus Hesperaloe, wherein the amount of saponin in the extract is sufficient to enhance the immune response of the non-human animal to the antigen. In a particularly preferred embodiment, the saponin comprising the extract of the present invention is administered in an Emelia vaccine to poultry that need it to increase the immune response, reduce lesion scores, and reduce oocyst shedding resulting from coccidiosis. [Brief explanation of the drawing]

[0013] [Figure 1] Figures 1A and 1B illustrate triterpenoid saponins and steroid saponins, respectively. [Figure 2A] Figures 2A-C illustrate various novel saponins extracted from non-woody plants of the genus Hesperaloe according to the present invention, including 25(27)-dehydrofucleastatin (Figure 2A), 5(6),25(27)-disdehydroyuccaloyside C (Figure 2B), and 5(6)-disdehydroyuccaloyside C (Figure 2C). [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 3] Figure 3 is a graph plotting the germinal center counts of the cecal tonsils for each member of treatment groups 1 through 6. [Figure 4] Figure 4 is a graph plotting the scores for intestinal lymphoid tissue for each of the treatment groups 1 through 6. [Figure 5] Figure 5 is a graph plotting the number of pseudoeosinophils in the mucosal intrinsic bodies of each member of treatment groups 1 to 6. [Figure 6]Figure 6 is a graph plotting the ELISA titers of each member of treatment groups 1 through 6, measured using a commercial ELISA (FlockChek® Newcastle Disease Antibody Test Kit, IDEXX) for the analysis of chicken serum.

[0014] definition As used herein, the term “biomass” generally refers to the whole plant and plant organs (i.e., leaves, stems, flowers, roots, etc.) of the Hesperaloe genus, such as H. funifera, H. parviflora, H. nocturna, H. ciangui, H. tenuifolia, H. engelmannii, and H. malachophylla. In particularly preferred examples, the saponins comprising the compositions of the present invention may be prepared from biomass essentially consisting of the above-ground parts of the plant, more specifically the parts above the crown of the plant, and even more preferably the leaves of the plant.

[0015] As used herein, the term “bagasse” generally refers to biomass subjected to an extraction process, such as continuous solvent extraction or grinding, the resulting solid biomass having less water-soluble solids than the biomass from which it originates. In certain embodiments, bagasse is prepared by subjecting biomass to high pressure, which can be achieved by passing the biomass through one or more pairs of opposing rolls, a mechanical press, a screw press, and by direct water pressure and other processes for applying pressure to the biomass to remove interstitial and intracellular fluids therefrom.

[0016] As used herein, the term “grinding” generally refers to the application of sufficient pressure to push the intercellular and intracellular fluids from the biomass.

[0017] As used herein, the term “saccharide” is used interchangeably with the terms “polysaccharide,” “oligosaccharide,” and “sugar,” the definitions of which are well known to those skilled in the art of sugar chemistry. It should be noted that saccharides may be in the form of mono, oligo, and / or polysaccharides. Preferably, saccharides are water-soluble and do not contain cellulose, hemicellulose, or mono, oligo, and / or polysaccharides bound to other compounds, such as glycosides (arabinose, glucose, galactose, xylose, and glucuronic acid) that bind to triterpenoids to form saponins.

[0018] As used herein, the term “saponin” generally refers to a glycoside containing a sugar component referred to as a glycan and a non-sugar component referred to as an aglycone. Depending on the structure of the aglycone, saponins may be classified as triterpenoid saponins, as illustrated in Figure 1A, or as steroidal saponins, as illustrated in Figure 1B. The aglycone portion of a saponin may be either a pentacyclic triterpenoid or a tetracyclic triterpenoid, both of which contain 30 carbon atoms. Whether steroidal or triterpenoid, saponins may be mono, bi, or tridesmodic. Monodesmodic saponins have a single saccharide, usually attached to C-3. Videsmodic saponins have two saccharides, often one attached via an ether linkage at CC-3 and the other attached via an ester linkage at CC-28, or via an ether linkage at C-20 (pentacyclic and tetracyclic triterpene saponins, respectively), or via an ether linkage at C-26 (floster saponin). In certain cases, Hesperaloe biomass may contain at least about 5 wt% total saponins, based on the oven-dry weight of the biomass, e.g., about 5–15 wt%, e.g., about 8–12 wt%, etc. Total saponins can be determined as described in the Test Methods section below.

[0019] As used herein, the term "water-soluble solids" generally refers to the dry matter remaining after the extract has been centrifuged, filtered, and all water has been evaporated. The procedure for measuring the water-soluble solids of the biomass extract of the present invention is described in detail in the section on Test Methods below. Water-soluble solids can be expressed on a percentage basis relative to the mass of the dry biomass.

[0020] As used herein, the term "water-insoluble solids" generally refers to the fraction of the extract that is removed by centrifugation and filtration during the course of measuring water-soluble solids, as described in the section on Test Methods below.

[0021] As used herein, "enhancing the immune response" refers to an increase in phagocytic activity in CD4 T helper (Th) cells, particularly Th1 and Th17 cells, in a subject treated with a saponin-containing composition derived from Yucca schidigera, as described herein, compared to the same subject before treatment.

[0022] As used herein, a "pharmaceutical composition" is a composition containing at least one saponin extracted from Yucca schidigera and formulated with one or more pharmaceutical-grade excipients in a manner that meets the requirements of the government agencies that regulate the manufacture and sale of pharmaceuticals, as part of a therapeutic regimen for the treatment or prevention of disease in non-human animals. The pharmaceutical composition can be formulated, for example, for oral administration in unit dosage forms (e.g., tablets, capsules, caplets, gelcaps, or syrups); for topical administration (e.g., as creams, gels, lotions, or ointments); for intravenous administration (e.g., as a sterile solution free of microparticulate emboli and in a solvent system appropriate for intravenous use); or for any other formulation described herein.

[0023] As used herein, the terms "subject" and "non-human animal" refer to any vertebrate animal including, but not limited to, cows, chickens, turkeys, ducks, quail, geese, pigs, and sheep.

BEST MODE FOR CARRYING OUT THE INVENTION

[0024] The present invention relates to novel pharmaceutical, nutritional supplement, and food ingredient compositions comprising at least one component selected from extracts, fractions, active compounds, and phytochemicals or mixtures thereof derived from non-woody plants of the genus Hesperaloe, including, for example, H. funifera, H. parviflora, H. nocturna, H. ciangui, H. tenuifolia, H. engelmannii, and H. malachophylla, and optionally comprising one or more pharmaceutically and nutritionally acceptable phytochemical active substances, diluents, excipients, carriers, and active substances or mixtures thereof. In a particularly preferred embodiment, the present invention provides a method for increasing phagocytic activity in CD4 T helper (Th) cells, particularly Th1 and Th17 cells, in non-human animals by administering a Hesperaloe-derived immunomodulator containing one or more saponins to non-human animals.

[0025] The compositions of the present invention are particularly well suited for the treatment of non-human animals, including, for example, cattle, poultry, pigs, sheep, and horses. For example, the methods and compositions of the present invention can be used for the treatment of cattle, chickens, turkeys, ducks, quail, geese, pigs, and sheep. In particularly preferred embodiments, the methods and compositions of the present invention can be used for the treatment of poultry, more specifically, for the prevention and treatment of coccidiosis and / or necrotizing enterocolitis.

[0026] The hesperaloe-derived immunomodulator of the present invention may contain at least 5 wt% saponins based on the oven-dry weight of the composition, as measured by the total saponin assay shown in the Test Methods section below. In certain embodiments, the saponin-containing composition used according to the present invention contains 10 wt% saponins, more preferably at least about 10 wt% saponins, even more preferably, at least about 15 wt% saponins, such as about 15 to about 25 wt% saponins, such as about 5 to about 30 wt% saponins. The effect of the composition is considered to be related to the total amount of saponins present. Thus, those skilled in the art will understand that if a particular amount of saponins is desired, this can be achieved by varying the volume of a particular concentration of the composition administered, varying the concentration of a particular volume of the composition, or both.

[0027] Saponins useful in the present invention may also be extracted from non-woody plants of the genus Hesperaloe. Saponins derived from Hesperaloe generally contain steroidal saponins. Saponins derived from Hesperaloe may contain at least one of the following aglycones or genins: cammogenin, manogenin, gentrogenin, hecogenin, tigogenin, sarsapogenin, chlorogenin, and dytogenin or their corresponding isomers or oxidized or reduced forms (in acid or salt form) accompanied by at least one of the following glycoside moieties: glucose, xylose, rhamnose, arabinose, or galactose. In other embodiments, steroidal saponins may include agamenoside, agaveside, agaboside, magueside, agabasaponi, cantharasaponin, sisarsaponin, gabritonoside, dongnoside or amoronin, or other steroidal saponins.

[0028] The extract may be recovered from non-woody plants of the genus Hesperaloe by extracting biomass, particularly leaves, more specifically leaves on the crown of the plant, and at least one solvent is selected from the group consisting of water, methanol, ethanol, butanol, isopropanol, and mixtures thereof. For example, in one embodiment, this process involves contacting the biomass with an extract solution containing water to separate the water-soluble fraction from the insoluble biomass fraction. In other embodiments, the extract solution may contain, in addition to water, a surfactant, a solvent, and optionally, an extract-supported juice. The extract-supported juice can be obtained, for example, from an earlier extraction step or an earlier grinding step.

[0029] Simple aqueous extraction of Hesperaloe biomass may yield a crude aqueous extract containing saccharides, polysaccharides, inorganic salts, saponins, and sapogens. The crude extract may also be produced by extracting the biomass using methanol as a solvent, or using a mixture of methanol and water, which may have been previously extracted with acetone or diethyl ether to remove lipids and pigments. In other examples, the biomass may be extracted with a 4:1 ethanol-water solvent, followed by defatting of the extract with a nonpolar solvent, such as hexane. In certain examples, the defatted extract may be subjected to further processing to isolate certain water-soluble components, such as saponins, which may be purified from the defatted extract by mixing with butanol and separating the butanol phase to obtain a mixture of saponins substantially free of proteins and free saccharides and polysaccharides.

[0030] Hot water extracts can also be used. For example, in one embodiment, the water-soluble solid may be extracted from Hesperaloe biomass, particularly leaves, by extracting the biomass with hot water ethanol or isopropanol (75-95% by weight alcohol). The aqueous alcohol extract may then be filtered and concentrated, and lipid-soluble substances may be removed by mixing the extract with a non-polar solvent, such as hexane. A substantially pure saponin composition may then be prepared by further extracting the defatted extract with a polar solvent, such as butanol.

[0031] For the purpose of preparing the compositions of the present invention and for use in this method, a simple aqueous extract may be preferred, but other extraction methods are within the scope of the present invention. In a particularly preferred embodiment, the hesperaloe biomass may be equally diced and pressed and extracted with an aqueous solvent to remove water-soluble extracts, such as inorganic salts, saccharides, polysaccharides, organic acids, and saponins. The water-soluble extract may be collected and concentrated by techniques well known in the art, such as evaporation, spray drying, or drum drying. The extract may be concentrated until it has a solid content of about 20 to about 100% by weight, for example, about 20 to about 95% by weight, or for example, about 20 to about 80% by weight.

[0032] In a particularly preferred embodiment, the water-soluble extract is concentrated by spray drying by supplying the extract solution to a spraying device. Suitable spraying devices include, but are not limited to, rotary wheel sprayers, pressure nozzle sprayers, and two-phase fluid nozzle sprayers. Rotary wheel sprayers, pressure nozzle sprayers, and two-phase fluid nozzle sprayers are known to those skilled in the art and include sprayers in spray dryers that are commercially available from various sources, such as GEA Process Engineering.

[0033] As described in more detail below, the biomass may be crushed and the bagasse and water-soluble solids separated using rolls, screws, and other forms of presses. In certain preferred embodiments, the biomass is passed between one or more nip-like sections of opposing, counter-rotating rolls to maximize the mechanical removal of the juice. The bagasse can then be brought into contact with the juice in a subsequent crushing step, as described more thoroughly below. In certain examples, the biomass may be purified by being cut equally before crushing. Cutting and purification may be carried out using methods well known in the art. In particularly preferred embodiments, the biomass is purified to remove fragments, such as dust, without using water or other solvents. While it may be preferable to cut the biomass equally before extraction, in certain embodiments it is useful not to crush, pulp, shred or macerate the biomass before it is crushed. While such physical processing steps may be advantageous in that they expose more biomass surface to the extract solution, they can break down plant cell walls, excessively shorten fiber length, and create an excessive amount of fine particles. It is generally desirable to avoid negatively affecting the bagasse during the extraction stage in this manner. In this manner, the extraction method of the present invention typically yields bagasse fibers that can be further processed, for example, by pulping, and yields pulp fibers suitable for the manufacture of paper products.

[0034] In other embodiments, water-soluble solids can be recovered from biomass by diffusion. In diffusion, the biomass is brought into contact with a liquid to extract the liquid components. Typically, the biomass is prepared by first cutting, without shearing or grinding, to minimize damage to the fibers and avoid the generation of excessive amounts of fine particles. The prepared biomass is then repeatedly washed, usually with a solvent, to extract the liquids contained within the biomass. The solvent can be any of the aforementioned solvents. Exemplary treatment solvents include water, especially hot water, such as water heated to a temperature of about 40 to about 90°C. The solvent can be recycled and reused so that the solvent used for the first extraction is reused as a solvent for extracting subsequent prepared biomass.

[0035] Various types of diffusers are known in the art and can be adapted for use in the biomass described herein. Suitable diffusers include ring diffusers, tower diffusers, or drum diffusers. Exemplary diffusion systems are discussed, for example, in U.S. Patents 4,182,632, 4,751,060, 5,885,539, and 6,193,805, the contents of which are incorporated herein by reference in a manner consistent with this disclosure. Numerous other diffusion methods and devices for diffusion methods are known and can be adapted for use in the methods described herein. One such diffuser is the Crown Model III Percolation Extractor, a continuous loop, counterflow, shallow-bed diffuser commercially available from Crown Iron Works (Blaine, Minnesota).

[0036] The biomass may be extracted, either cleaved or uncleaved, by any suitable extraction method as discussed above. In a particularly preferred embodiment, the solvent used for extraction is water. Those skilled in the art will recognize that the ratio of extraction solvent to biomass will vary based on the solvent, the amount of biomass to be extracted, and the extraction procedure. In a particular preferred embodiment, the extraction solvent is water, and the ratio of extraction solvent to biomass is about 1:5 to about 1:100, for example, about 1:5 to about 1:50, more preferably about 1:5 to about 1:20, based on kilograms of oven-dried biomass per liter of extraction solvent.

[0037] The pH of the extraction solvent can be between approximately pH 5.0 and 8.0, for example, between approximately pH 6.0 and 8.0 and between approximately pH 6.5 and 7.5. In certain embodiments, the extraction solvent is water having a pH between approximately pH 6.5 and 7.5. In embodiments where the extraction includes impregnation with crude juice, the impregnation solution may have a pH of approximately 4.0 to 5.0.

[0038] Extraction may be carried out at temperatures between approximately 25 and 90°C, for example, between approximately 30 and 80°C, between approximately 35 and 75°C, between approximately 40 and 70°C, between approximately 45 and 65°C, or between approximately 50 and 60°C.

[0039] In embodiments where the extraction method is a batch extraction method, the extraction time may be in the range of approximately 0.25 to approximately 24 hours, for example, approximately 0.5 to approximately 2 hours, approximately 1 to approximately 8 hours, or approximately 1 to approximately 6 hours.

[0040] In embodiments where the extraction method is a continuous method, the extraction time may be in the range of approximately 0.25 to approximately 5 hours, for example, approximately 0.5 to approximately 3 hours.

[0041] After extraction, the water-insoluble biomass material may be separated from the water-soluble solids by filtration to provide a filtrate containing inorganic salts, saccharides, polysaccharides, organic acids, and saponins (referred to herein as “first filtrate”). Separation can be achieved by any suitable means, but is not limited to gravity filtration, plate-frame filter press, cross-flow filter, screen filter, Nutsche filter, belt filter, ceramic filter, membrane filter, microfilter, nanofilter, ultrafilter, or centrifugation. Various filtration aids, such as diatomaceous earth, bentonite, and zeolite, may also be used in this method.

[0042] After separation, the pH of the first filtrate may be adjusted to remove additional impurities. In one embodiment, the pH of the first filtrate can be adjusted to between approximately 8.5 and approximately 10.0 by treatment with a base, such as calcium oxide or calcium hydroxide (approximately 1.0% of the volume of the filtrate), with slow stirring.

[0043] In a particularly preferred embodiment, water-soluble solids are removed from the biomass, especially Hesperaloe leaves, before pulping by a series of mills, such as two, three, four, five, six, or seven mills arranged in tandem, which may involve impregnation and / or depissing. Generally, the biomass treatment according to the present invention removes at least about 25% of water-soluble solids, more preferably at least about 50%, even more preferably at least about 75%, for example, about 25-98%, for example, about 50-90%, for example, about 75-90%, etc., from the biomass.

[0044] The amount of water-soluble solids recovered from biomass may vary depending on the extraction efficiency, but in certain cases, about 100 to about 400 grams of water-soluble solids, e.g., about 120 to about 350 grams / kilogram, or e.g., about 150 to about 300 grams / kilogram, may be extracted per kilogram of oven-dry biomass. Of the extracted water-soluble solids, total saponins may contain about 5 to about 40 wt%, e.g., about 10 to about 30 wt%, based on the oven-dry weight of the water-soluble solids. In certain cases, the amount of total saponins that can be extracted from biomass may range from about 10 to about 400 grams / kilogram of oven-dry biomass, e.g., about 20 to about 300 grams, e.g., about 25 to about 200 grams, e.g., about 10 to about 100 grams. In certain cases, the amount of material removed from the biomass during the extraction process (oven-dry grams per kilogram of oven-dry biomass) may range as shown in Table 1 below. [Table 1]

[0045] In addition to saponins, the water-soluble solid may contain saccharides, proteins, lipids, and inorganic salts. For example, in certain cases, the water-soluble solid may contain at least about 1 wt% of saccharides, based on the oven-dry weight of the water-soluble solid, such as about 1 to about 15 wt%, or for example, about 2 to about 10 wt%. The saccharides may include monosaccharides and oligosaccharides. In other cases, the water-soluble solid may contain at least about 15 wt% of inorganic salts, based on the oven-dry weight of the water-soluble solid, such as about 15 to about 30 wt%.

[0046] Generally, grinding is carried out by adding an aqueous solvent, such as water, having a pH in the range of about 5 to 9, for example, about 6 to 7 to 8. The water-soluble solid is generally recovered from the grinding process as a crude extract and may be subjected to further processing to recover specific compounds, such as saccharides, polysaccharides, organic acids, and saponins.

[0047] Suspended solids, also referred to herein as water-insoluble fractions, may be removed from the crude extract by known processes including, for example, clarification, filtration, centrifugation, or a combination thereof. The amount of water-insoluble solids in the extract (dry grams per kilogram of dry biomass) may range from about 1.0 to about 30 grams and may include hydrophobic substances, such as waxes.

[0048] After removing suspended solids, the clarified juice may be used directly, concentrated, or subjected to further processing to isolate one or more water-soluble solids, such as saccharides, polysaccharides, organic acids, saponins, and sapononins. In another example, the clarified juice may be further purified to remove saccharides, polysaccharides, and organic acids to obtain a composition containing saponins.

[0049] The juice obtained as a result of the extraction method described above can be subjected to further extraction to obtain saponins in the form of a crude saponin extract, or in a substantially purified form containing saponins at a concentration of about 30 to about 90% by weight. The extraction method may include mixing the juice extracted from non-woody plants of the genus Hesperaloe with a water-immiscible polar solvent. Suitable water-immiscible polar solvents include, for example, alcohols having 4 to 6 carbon atoms, such as butyl, amyl, hexyl, and cyclohexyl alcohols. Extraction of the juice using a water-immiscible polar solvent generally removes impurities remaining in the aqueous phase, such as proteins, sugars, and organic acids, and transfers the saponins to the solvent phase.

[0050] The solvent phase containing the saponin may be subjected to further treatment to separate the saponin from the alcohol phase. This can be achieved in various ways, for example, by dehydrating the solvent extract by cooling, or by adding an organic solvent that is miscible with the alcohol solvent but insoluble in the saponin. Suitable precipitation solvents include, for example, diethyl ether, petroleum ether, acetone, and chloroform.

[0051] In a particularly preferred embodiment, the saponin is separated from the alcohol by flash evaporation. Flash evaporation is a known technique in preparative chemistry for the rapid removal of volatile components from liquid mixtures. The volatile liquid is removed from the solution by rapid conversion to the vapor phase, by forming a thin film of the solution over a large surface area under reduced pressure, often with an increasing temperature of the solution, which is above ambient temperature but below the boiling point of the solution at atmospheric pressure. The actual thickness of the film and the area to which it is applied are chosen to provide optimal evaporation and ease of use, however, the evaporation can be substantially instantaneous (hence the name "flash" evaporation). Flash evaporation avoids prolonged use at high temperatures that can decompose the intended product and has the ability to remove almost all of the alcohol component (therefore, the remaining solution is suitable for a preferred implementation of spray drying used in the next step). The alcohol may be recovered from this step and reused in this extraction method.

[0052] The saponin content of the alcohol extract can be further increased by passing it over an ultrafiltration membrane without significant change to or loss of the saponin composition. This concentrated saponin fraction, with a saponin content in the range of 85-90%, can then be further purified in liquid form or reduced to a dry state. Individual saponins can be recovered by a combination of reversed-phase solid-phase extraction and preparative reversed-phase HPLC. Alternatively, the alcohol extract containing saponins can be directly fractionated by a combination of reversed-phase solid-phase extraction and preparative reversed-phase HPLC.

[0053] In further embodiments, the saponin may be purified from a juice prepared according to the present invention, comprising the step of mixing the juice with a salt and a solvent to form a first solution. The solvent may include one or more solvents selected from acetic acid, acetone, acetonitrile, benzene, 1-butanol, 2-butanol, 2-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethane, diethylene glycol, diethyl ether, diglym, 1,2-dimethoxyethane, dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexamethylphosphoramide, hexamethylphosphate triamide, hexane, methanol, methyl-t-butyl ether, methylene chloride, N-methyl-2-pyrrolidinone, pentane, perchloroethylene, petroleum ether, 1-propanol, 2-propanol, pyridine, tetrahydrofuran, toluene, triethylamine, trifluorotoluene, water, xylene, or any combination thereof. In some embodiments, the solvent is water. The salt may be selected from alkali metal salts, alkaline earth salts, transition metal salts, ammonium salts, or combinations thereof. In certain preferred embodiments, the salt added to the plant extract to form the solution is an alkaline earth metal salt. In particularly preferred embodiments, the salt is calcium chloride (CaCl2), magnesium chloride (MgCl2), or a mixture thereof.

[0054] The pH of the first solution is generally adjusted to about 6.0 to about 9.0, for example, about 6.0 to about 8.0, or for example, about 6.0 to about 7.0. At least one phosphoric acid may then be added to the first solution to form an ion-polysaccharide complex precipitate. Useful phosphoric acids include, for example, sodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), sodium phosphate (Na3PO4), or sodium bishydrogen phosphate (Na2H2PO7).

[0055] The precipitated ion-polysaccharide complex may be removed by filtration to obtain a second solution, which may be further purified to produce an extract of purified saponins. Optionally, the extract may be concentrated by any filtration technique known in the prior art. Preferably, the concentration of the purified saponin extract is carried out by nanofiltration, ultrafiltration, and diafiltration, or any combination thereof. In some embodiments, the saponin extract is substantially free of proteins. In some embodiments, the saponin extract is substantially free of polysaccharides. In some embodiments, the saponin extract is substantially free of phenolic compounds.

[0056] The total amount of saponins that can be extracted from Hesperaloe biomass according to the present invention may range from about 10 to about 100 grams per kilogram of oven-dried biomass, for example, about 20 to about 80 grams, or for example, about 25 to about 75 grams. The saponins may be provided as part of a crude juice, as part of a dried water-soluble solid composition, as a partially purified composition, or as a substantially pure composition containing a mixture of saponins.

[0057] In certain embodiments, saponins extracted from Hesperaloe biomass include 25(27)-dehydrofucleastatin (Figure 2A), 5(6),25(27)-disdehydroyucca leuside (Figure 2B), 5(6)-disdehydroyucca leuside (Figure 2C), flucleastatin, and yucca leuside.

[0058] A useful composition in the present invention can be prepared by mixing an aqueous extract from Hesperaloe biomass with one or more polyhydroxy alcohols, including glycerol, propylene glycol, polyalkylene glycol, such as polyethylene glycol and polypropylene glycol, and polyglycerol. Preferred polyhydroxy alcohols have fewer than about eight carbon atoms. Glycerol and propylene glycol are particularly preferred polyhydroxy alcohols.

[0059] The composition may also contain saccharides, which may be present in the aqueous extract or added after extraction during formulation. Useful saccharides in the compositions of the present invention include monosaccharides, such as glucose; disaccharides, such as sucrose; and polysaccharides, such as starch.

[0060] In further embodiments, compositions according to embodiments of the present invention may include various other additives known in the art so as to be beneficial for the maintenance and welfare of non-human animals. For example, compositions may include components such as vitamin E, vitamin A propionic acid, vitamin A palmitate, vitamin B1, vitamin B2, vitamin B6, vitamin B12, D-activated animal sterols (a source of vitamin D3), yeast components, dried egg solids, dried casein, and dried whey.

[0061] The saponin-containing composition of the present invention may be in liquid or dry form. For example, a saponin containing hesperaloe extract may be dried into a powder form. In this form, the saponin-containing composition may be administered to animals as a pill or bolus, or mixed with other components, such as feed. For example, a dried powder formulation of the saponin-containing composition may be added to feed via a microcomponent machine, or added to a feed mixing track and thoroughly mixed to ensure even distribution in the feed. The saponin containing hesperaloe extract may also be in liquid form with a carrier liquid, such as water. In this form, the saponin-containing composition may be administered to animals as a liquid medicine.

[0062] The saponin-containing composition of the present invention may be administered to non-human animals requiring it as part of a feeding regimen, either as a single dose or as multiple doses. For example, a non-human animal may receive an initial dose, followed by a smaller maintenance dose. A non-human animal may receive multiple doses of the saponin-containing composition in a single day, or multiple doses over multiple days.

[0063] In certain embodiments, the compositions of the present invention may be useful as immunomodulators or adjuvants. In certain embodiments, saponins comprising compositions derived from Hesperaloe may be administered to non-human animals in need to induce an adaptive immune response. In particularly preferred embodiments, administration of saponins comprising extracts of the present invention results in an increase in phagocytic activity in CD4 T helper (Th) cells, particularly Th1 and Th17 cells, in a subject. In this form, extracts of the present invention may be administered as a pharmaceutical composition without the addition of antigens to enhance the immune response of the subject.

[0064] In other embodiments, the Hesperaloe extract of the present invention may be administered to non-human animals as an antigen to enhance the immune response of a target. Suitable antigens include microbial pathogens, bacteria, viruses, proteins, glycoproteins, lipoproteins, peptides, glycopeptides, lipopeptides, toxoids, sugars, and tumor-specific antigens. A mixture of two or more antigens may be used. In certain preferred embodiments, the composition of the present invention may be administered in a vaccine intended for the prevention of coccidiosis in non-human animals, particularly poultry, characterized in that the coccidia is selected from the group consisting of Aemeria, Isospora, Toxoplasma, Vesnoitia, and Neospora. Thus, the present invention provides an adjuvant system that is particularly advantageous in inducing active immunity against an antigen in non-human animals, for example, when preparing and using vaccines and other immunostimulatory compositions to treat or prevent diseases.

[0065] In a particularly preferred embodiment, the saponin-containing extract may be administered with the Aemeria vaccine to poultry that require it to increase the immune response, reduce the lesion score, and lessen oocyst shedding due to coccidiosis. The immunogenic composition of the present invention may be delivered orally or subcutaneously in dose volumes appropriate to increase the immune response, such as dose levels of less than about 50 μg, e.g., less than about 40 μg, e.g., less than about 30 μg, e.g., about 1 to about 50 μg, e.g., about 5 to about 30 μg.

[0066] The compositions of the present invention exhibit an adjuvant effect when administered over a wide range of doses and ratios to the antigen being administered. In one embodiment, the saponin is administered at an adjuvant ratio of 3.0 or less, preferably 1.0 or less, to the antigen (w / w), based on the weight of the saponin.

[0067] Saponins extracted from non-woody plants of the genus Hesperaloe according to the present invention may be used as adjuvants in crude or purified form and may be mixed with other non-saponin adjuvants to achieve enhancement of the immune response to an antigen. Such non-saponin adjuvants useful in the present invention include oil adjuvants (e.g., Freund complete and imperfect), liposomes, mineral salts (e.g., AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), silica, alum, Al(OH)3, Ca3(PO4)2, kaolin, and carbon), polynucleotides (e.g., polyIC acid and polyAU acid), and certain natural substances (e.g., wax D from Mycobacterium tubercurosis, as well as substances found in members of the genera Corynebacterium parhum, Bordetella pertasis, and Blue Cera).

[0068] Testing method water soluble solid The total biomass water-soluble solids may be determined using an Accelerated Solvent Extraction system (ASE), such as Dionex® ASE® 350 (Thermo Fisher Scientific, Waltham, Massachusetts). Approximately 10 grams of recovered biomass are dried in an oven, typically at 125°C for 4 hours, to a constant weight. After drying, 1.5–2.0 grams of oven-dried biomass are accurately weighed, and the weight (W) is measured. bThe amount was recorded to the nearest 0.001 gram. Using water as the solvent, the biomass was extracted using the conditions shown in the table below. The biomass-to-solvent ratio was generally 21:1, and five consecutive water extraction cycles were performed. At the end of each extraction cycle, the liquid phase was collected and dried under vacuum at approximately 40°C to obtain the dried material (W i Record the weight of the water-soluble solid (W) to the nearest 0.001g. e ) for each extraction cycle (W i It is calculated by summing the weights of the solids recovered from the ). The total water-soluble solids as a percentage of the oven-dried biomass are then determined using the following equation: Water-soluble solids (wt%) = W e / W b *100. [Table 2]

[0069] The total water-soluble solids in the biomass extract can be determined by taking a suitable aliquot, typically about 10–50 ml, and transferring it to a clean, dry centrifuge tube. Centrifuge the tube at 7000 rpm for 20 minutes. Calculate the weight of the extract (W1). Transfer the supernatant aliquot to a clean, pre-weighed beaker (D0) and weigh it. Weigh the beaker and sample to the nearest 0.001 g and record the weight (D2). Place the beaker containing the sample in a hot air oven at 140°C overnight to dry. Remove the beaker from the oven, dry it, cool it to room temperature, and then weigh it to the nearest 0.001 gram (D1). Determine the weight percentage of soluble solids based on the weight of the extract using the following formula.

number

[0070] Total saponins Total saponins were measured as generally described in Makkar, Harinder PS, Sidhuraju, P., Becker, Klaus (2007) Plant Secondary Metabolites, chapter 17, pp93-100. A standard saponin solution was prepared by weighing 10 mg of diosgenin (MilliporeSigma > 93%), dissolving it in 16 mL of methanol, and adding 4 mL of distilled water. The solution was thoroughly mixed to obtain a 0.5 mg / mL diosgenin solution in 80% methanol solvent. Calibration curves were prepared by transferring various amounts of standards (0, 10, 20, 40, 60, 80, and 100 μL) into 13 mm glass test tubes using standards. A solution of 80% aqueous methanol was added to a total volume of 100 μL.

[0071] Before testing, biomass extract samples were diluted with water to a total solids content of approximately 0.5 wt% to ensure that the absorbance results were within the range of the saponin standard calibration curve. The diluted extract (20 μL) was pipetted into 13 mm glass test tubes, and the volume was increased to 100 μL with 80 μL of methanol. Each sample was tested in three different ways.

[0072] To each sample, 100 μL of vanillin reagent (prepared by dissolving 800 mg of vanillin in 10 mL of 99.5% ethanol (analytical grade)) was added, followed by 1.0 mL of 72% (v / v) sulfuric acid (prepared by adding 72 mL of sulfuric acid (analytical grade, 95%, w / w) to 28 mL of distilled water). The solutions were thoroughly mixed and heated at 60°C for 10 minutes. The samples were then cooled in an ice bath, and 1 mL of the solution was transferred to each cuvette, where the absorbance at 544 nm was read. The total mass of saponins in the samples may be calculated based on a standard absorbance curve as follows: Saponin (μg) = [slope] × measured absorbance - [intercept]

[0073] Total saponins Total saponins were measured as generally described in Makkar, Harinder PS, Sidhuraju, P., Becker, Klaus (2007) Plant Secondary Metabolites, chapter 17, pp93-100. A standard saponin solution was prepared by weighing 10 mg of diosgenin (MilliporeSigma > 93%), dissolving it in 16 mL of methanol, and adding 4 mL of distilled water. The solution was thoroughly mixed to obtain a 0.5 mg / mL diosgenin solution in 80% methanol solvent. Calibration curves were prepared by transferring various amounts of standards (0, 10, 20, 40, 60, 80, and 100 μL) into 13 mm glass test tubes using standards. A solution of 80% aqueous methanol was added to a total volume of 100 μL.

[0074] Before testing, biomass extract samples were diluted with water to a total solids content of approximately 0.5 wt% to ensure that the absorbance results were within the range of the saponin standard calibration curve. The diluted extract (20 μL) was pipetted into 13 mm glass test tubes, and the volume was increased to 100 μL with 80 μL of methanol. Each sample was tested in three different ways.

[0075] To each sample, 100 μL of vanillin reagent (prepared by dissolving 800 mg of vanillin in 10 mL of 99.5% ethanol (analytical grade)) was added, followed by 1.0 mL of 72% (v / v) sulfuric acid (prepared by adding 72 mL of sulfuric acid (analytical grade, 95%, w / w) to 28 mL of distilled water). The solutions were thoroughly mixed and heated at 60°C for 10 minutes. The samples were then cooled in an ice bath, and 1 mL of the solution was transferred to each cuvette, where the absorbance at 544 nm was read. The total mass of saponins in the samples may be calculated based on a standard absorbance curve as follows: Saponin (μg) = [slope] × measured absorbance - [intercept] [Examples]

[0076] Example 1 A total of 150 one-day-old broiler chicks were randomly assigned to six experimental groups for a 28-day cage trial. Live coccidia were manually introduced to the birds on their 14th day of life. The treatment codes included two control codes using a basal diet with and without coccidia challenge (control+), as listed in Table 3. All remaining treatment codes were challenged with coccidia using a basal diet concentrated with the composition of the present invention at two different dosages, or a yucca extract commercially available under the trade name FOAMATION® (commercially available from Ingredion, Westchester, Illinois). FOAMATION® contained 50 wt% water-soluble solids of the composition, of which 10 wt% consisted of saponins. Bird weight gain (BW) and feed consumption were measured weekly for each pen. Feed equivalent ratio (FCR) is the ratio between kilograms of feed consumed and kilograms of weight gain. A lower FCR value indicates better feed. [Table 3]

[0077] The extract of this invention was prepared by harvesting mature Hesperaloe funifera leaves from the crown, cutting the leaves into sections ranging from approximately 0.50 to 8.0 cm, and pressing the cut biomass using a tandem press. The biomass was pressed three times, the crude juice was collected, passed through a 25 mm filter, and heated to concentrate the extract to a 29% solid. The water-soluble solid contained 21 wt% total saponins based on the oven-dry weight of the water-soluble solid.

[0078] At the end of the 28-day clinical trial, the challenged control (control+) group showed a reduction in feed consumption of approximately 140 g / chicken and a reduction in weight gain of approximately 160 g / chicken compared to the unchallenged control. These reductions, however, were not observed in chickens fed the feed containing the composition of the present invention, as illustrated in Table 4 below. [Table 4]

[0079] Example 2 A total of 512 one-day-old broiler chicks were randomly assigned to eight experimental groups, with eight cages and eight birds per cage for each group during a 21-day trial. Live coccidia were manually introduced into the young birds at 14 days of age. Bird weight gain (WG), feed conversion rate (FCR), lesion score, and oocyst count were measured. Treatment codes included basal diets without coccidia challenge (control) and with challenge (control+), as listed in Table 5. The remaining treatment codes were coccidia-challenged codes using basal diets with Coban (Elanco Animal Health, commercially available from Greenfield, Indiana), Micro-Aid (DPI Global, commercially available from Porterville, California), and two different inventive samples at two different dosages. The basal diets met the National Research Council's minimum requirements for poultry. [Table 5]

[0080] Sample 1 of the present invention was prepared by harvesting mature Hesperaloe funifera leaves from the crown, cutting the leaves into fragments ranging from approximately 0.50 to approximately 8.0 cm, and pressing the cut biomass using a tandem press. The biomass was pressed three times, the crude juice was collected, passed through a 25 mm filter, and heated to concentrate the extract to 29% solid. Sample 2 of the present invention was prepared by harvesting mature Hesperaloe funifera leaves from the crown, cutting the leaves into fragments ranging from approximately 0.50 to approximately 8.0 cm, and pressing the cut biomass once using a tandem press. The collected juice was then heated to obtain an extract having 14% solid. All processed materials were prepared by mixing each additive with the basal feed at the specified loading level in a mixer.

[0081] Chickens fed the composition of the present invention showed weight gain, improved feed conversion rates, reduced lesion scores, and lower oocysts, as summarized in Table 6 below. In many cases, the improvements were comparable to or better than those observed in chickens fed Coban or Micro-Aid Green. The composition of the present invention is effective even at relatively low doses of saponin. [Table 6]

[0082] The composition of the present invention is particularly useful in reducing or preventing coccidiosis. The lesion score, a score between 0 and 4 (where 0 indicates a normal intestinal appearance, while 4 indicates a severely damaged intestine), is a means of evaluating the development of coccidiosis through intestinal injury in chickens. Chickens fed the composition of the present invention for three weeks showed improved lesion scores (23-27%) compared to unchallenged controls. By reducing the number of infections and protecting the digestive system of chickens, the chickens were able to digest better, absorb nutrients better, and grow at a faster rate.

[0083] Example 3 Ross x Ross male broiler chicks, totaling 210 days from hatching, were obtained from Aviagen Hatchery, Blairsville, Georgia. At birth, the birds received routine vaccinations (HVTSB1). The birds were randomly assigned to six experimental groups for a 28-day cage trial. The treatment groups included the first group (no vaccine or hesperaloe extract), the group (hesperaloe extract alone), the group (Newcastle disease virus (Lasota strain) and hesperaloe extract), and the group (inactivated Newcastle disease virus (Lasota strain)), as listed in Table 7. [Table 7]

[0084] Birds treated with Newcastle disease virus received an oil-type emulsion Newcastle disease virus (Lasota strain) vaccine administered at 0.10 ml SQ via the posterior neck on day 0. Hesperaloe extract was substantially prepared as described in Example 2 by pressing the cut biomass once using a tandem press, and the collected juice was then heated to obtain an extract having 14% solids.

[0085] Each group of 35 broiler chicks was housed in a 13.4' × 15.7' chamber. The isolation chamber environment was controlled by an independent HEPA filtration system and heat pump unit, with a single heat lamp providing supplemental heat during egg rearing. The birds were raised at ambient humidity and provided with a lighting program according to major breeder recommendations. Upon placement, each pen contained approximately 4 inches of fresh pine shavings. The litter was not changed during the trial course. Each compartment included a tube feeder and a bell waterer, resulting in a ratio of 35 birds / feeder and waterer.

[0086] All diets contained 113.5 g / ton of amprolium to prevent coccidiosis, but no other concomitant drug treatments were used during the study. Starter foods were weighed and fed from DOT 0 to DOT 28. The feed formulations consisted of non-medicated commercial broiler starter and producer diets, formulated with commonly used U.S. feeds representing topical formulations, which were calculated by analysis to meet or exceed NRC standards. No antibiotics were added to any of the feeds.

[0087] In DOT 28, 10 birds were euthanized per procedure, and organ specimens (large Peyer's patches in the duodenum, cecal tonsils, half of the bursa of Fabricius, and 0.5 cm sections of the jejunum) were collected and placed in individual vials. The tissue samples were fixed in 10% buffered formalin and embedded in paraffin wax. Sections of the paraffin-embedded tissue (approximately 5 microns) were stained with Mayer's hematoxylin and eosin (H&E).

[0088] The lymphoid follicular region and lymphoid follicular cortex were measured as described in Muniz, et al, Brazilian J Poult Sci 8, 217-220, 2006. Five follicles, with complete presentation of anatomical features, were measured sac-wise. Measurements were performed using ImageJ software 1.37, a Java-based image processing software with free hand tools and line-to-area functionality, developed by the National Institutes of Health and available free of charge on the internet. Pixel-to-micrometer measurements were performed using AmScope MR400 calibration slides.

[0089] The area of ​​the cecal tonsils and Peyer's patches was measured in the same manner as the lymphoid follicle area, and germinal centers were counted manually. The germinal center counts of the cecal tonsils for each of the treatment groups 1 to 6 are shown in Figure 3.

[0090] Gut-associated lymphoid tissue (GALT) proliferation (hyperplasia) was scored on a scale of 0 to 5: 0 (not apparent), 1 (minimal presence), 2 (mild), 3 (moderate), 4 (noticeable), and 5 (severe). GALT proliferation occurred locally, locally widespread, and diffusely, but was within the normal range; however, the degree of hyperplasia varied. GALT scores for each of the treatment groups 1 to 6 are shown in Figure 4.

[0091] Intestinal heterophylls appeared as clusters of heterophylls in the lamina propria. The total number of clusters was counted and recorded for each jejunal section. The total number of clusters in the lamina propria for each of the treatment groups 1 to 6 is shown in Figure 5.

[0092] During DOT 28, blood samples were collected from 10 birds per treatment, serum samples were gathered, and evaluated using a commercially available enzyme-linked immunosorbent assay (ELISA) Newcastle disease antibody test (FlockChek®, IDEXX, commercially available from Maine, USA). The resulting ELISA titers for each group are shown in Figure 6 (treatment groups 1 to 6, shown from left to right in the graph).

Claims

1. A method for enhancing the immune response in a non-human animal, comprising the step of administering an immunogenically effective amount of an extract from a non-woody plant of the genus Hesperaloe containing at least one saponin to the non-human animal.

2. The method according to claim 1, wherein the extract further comprises a saccharide, a protein, and a lipid.

3. The method according to claim 1, wherein the extract is substantially free of saccharides, proteins, and lipids.

4. The method according to claim 1, wherein the at least one saponin comprises cammogenin, manogenin, gentrogenin, hecogenin, tygogenin, sarsapogenin, chlorogenin, or dytogenin, and at least one glycoside moiety selected from glucose, xylose, rhamnose, arabinose, and galactose.

5. The method according to claim 1, wherein the at least one saponin is 25(27)-dehydrofucreastatin, 5(6),25(27)-disdehydroyuccaloyside, 5(6)-disdehydroyuccaloyside, flucreastatin, or yuccaloyside.

6. The method according to claim 1, wherein the extract comprises a mixture of 25(27)-dehydrofucleastatin, 5(6),25(27)-disdehydroyucca leuside, 5(6)-disdehydroyucca leuside, flucleastatin, and yucca leuside.

7. The method according to claim 1, wherein the non-woody plant of the genus Hesperaloe is Hesperaloe funifera, Hesperaloe nocturna, Hesperaloe parviflora, or Hesperaloe thianguii.

8. The method according to claim 1, wherein the amount of saponin in the extract is in the range of about 10 to about 25% by weight of the extract.

9. A method for treating coccidiosis and / or necrotizing enterocolitis in poultry, comprising administering to it a composition comprising at least one saponin extracted from a non-woody plant of the genus Hesperaloe.

10. The method according to claim 9, wherein the extract further comprises a saccharide, a protein, and a lipid.

11. The method according to claim 9, wherein the extract is substantially free of saccharides, proteins, and lipids.

12. The method according to claim 9, wherein the at least one saponin comprises cammogenin, manogenin, gentrogenin, hecogenin, tygogenin, sarsapogenin, chlorogenin, or dytogenin, and at least one glycoside moiety selected from glucose, xylose, rhamnose, arabinose, and galactose.

13. The method according to claim 9, wherein the at least one saponin is 25(27)-dehydrofucreastatin, 5(6),25(27)-disdehydroyucca leuside, 5(6)-disdehydroyucca leuside, flucreastatin, or yucca leuside.

14. The method according to claim 9, wherein the extract comprises a mixture of 25(27)-dehydrofucleastatin, 5(6),25(27)-disdehydroyucca leuside, 5(6)-disdehydroyucca leuside, flucleastatin, and yucca leuside.

15. The method according to claim 9, wherein the non-woody plant of the genus Hesperaloe is Hesperaloe funifera, Hesperaloe nocturna, Hesperaloe parviflora, or Hesperaloe thianguii.

16. The method according to claim 9, wherein the amount of saponin in the extract is in the range of about 10 to about 25% by weight of the extract.

17. The method according to claim 9, further comprising administering an immunogenically effective amount of antigen.

18. A method for enhancing an adaptive immune response in a non-human animal, comprising administering to the non-human animal an amount of a hesperaloe-derived immunomodulator effective in increasing the activity of at least one adaptive immune defense mechanism in the non-human animal, wherein the hesperaloe-derived immunomodulator comprises at least one saponin.

19. The method according to claim 18, wherein the hesperaloe-derived immunomodulator comprises a mixture of two or more saponins.

20. The method according to claim 18, wherein the at least one saponin comprises cammogenin, manogenin, gentrogenin, hecogenin, tygogenin, sarsapogenin, chlorogenin, or dytogenin, and at least one glycoside moiety selected from glucose, xylose, rhamnose, arabinose, and galactose.

21. The method according to claim 18, wherein the hesperaloe-derived immunomodulator comprises two or more saponins selected from 25(27)-dehydrofucleastatin, 5(6),25(27)-disdehydroyucca leuside, 5(6)-disdehydroyucca leuside, flucleastatin, and yucca leuside.

22. The method according to claim 18, wherein the hesperaloe-derived immunomodulator comprises 25(27)-dehydrofucleastatin, 5(6),25(27)-disdehydroyucca leuside, 5(6)-disdehydroyucca leuside, flucleastatin, and a mixture of yucca leuside.

23. An immunogenic or vaccine composition for coccidiosis, infectious bronchitis, infectious bursal disease, laryngotracheitis, Marek's disease, or Newcastle disease in chickens, comprising at least one parasite, microorganism, antigen, immunogen, epitope, or vaccine, and an adjuvant comprising 1 to about 50 μg of total saponins extracted from Hesperaloe.

24. The composition according to claim 23, further comprising a pharmaceutically effective carrier.

25. The composition according to claim 23, wherein at least one parasite, microorganism, antigen, immunogen, epitope, or vaccine is a coccidiosis vaccine.

26. The composition according to claim 25, wherein the coccidiosis vaccine comprises one or more strains of E. aserbrina, E. maxima, E. mitis, or E. tenera.

27. The composition according to claim 23, wherein the at least one parasite, microorganism, antigen, immunogen, epitope, or vaccine is selected from the group comprising infectious bronchitis vaccine, infectious bursal disease vaccine, laryngotracheitis vaccine, Marek's disease vaccine, and Newcastle disease vaccine.

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