Patent Application
20230302079
Plants produce a vast and diverse assortment of organic compounds, the great majority of which do not appear to participate directly in their growth and development. These substances, traditionally referred to as secondary metabolites or plant natural products, often are distributed among limited taxonomic groups within the plant kingdom. The functions of secondary metabolites remain largely unknown, although a number of compounds have been associated with attributes useful to the plants e.g. protection against herbivores and protection against microbial infection, as attractants for pollinators and seed-dispersing animals, and as compounds that influence competition among plant species (allelochemicals). There is a growing interest in plant natural products, since these products often have a wide range of applications in different kinds of industries, including pharmaceutical industries, cosmetic industries, food industries, detergent industries, and the like.
A particular group of plant secondary metabolites of interest are saponins. Saponins are glycosylated compounds classified as either triterpenoids, steroids, or steroidal glycoalkaloids. Saponins consist of one or two sugar moieties which are coupled to the aglycon (mono- and bisdesmosides, respectively). 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 ranging from 600 to more than 2,000 daltons.
The asymmetric distribution of their hydrophobic (aglycone) and hydrophilic (sugar) moieties confers an amphipathic character to these compounds which are largely responsible for their detergent-like properties. The ability of lowering surface tension make saponins potentially well suited for use in the cosmetic and in the detergent industries.
Saponins also have the ability of forming 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 formation of immunostimulating complexes that are useful in vaccine strategies.
Currently, a major limitation to the broad exploitation of saponins is the fact that commercially available saponins are relatively expensive. The expenses is due in large part to the limited number of plant extracts having significant amounts of saponins. Currently, commercially available plant extracts containing saponins include Saponaria officinalis, Quillaia bark and stem, Castanea sativa seeds, and extracts of various Yucca species.
Plant extracts containing saponins are thus of general interest within a wide range of different industries. There is therefore a growing need in the art for alternative sources of saponin extracts and these plant sources should preferably be cheap, easy to obtain, and preferably the saponin content should be relatively high.
The present invention relates to compositions, methods, and kits for the administration of plant-derived immunomodulators. The compositions can be useful for sensitizing the innate and adaptive immune system of a non-human subject and thus can be used to treat an infection or as an adjuvant in an immunization. Preferably the plant-derived immunomodulators of the present invention are plant extracts containing saponins. The administration of these immunomodulators to a subject significantly enhance cell mediate immune system and enhance antibody production in the subject.
Accordingly, in one embodiment, the present invention provides a method of treating a non-human animal having, or at risk of, an infection by administering to the animal a plant-derived immunomodulator, in an amount sufficient to treat the infection. In particularly preferred embodiment the immunomodulator comprises at least one saponin extracted from non-woody plants of the genus Hesperaloe.
In other embodiments, the present invention provides a method of administering to a non-human animal a plant-derived immunomodulator wherein administration of the plant-derived immunomodulator enhances the host's adaptive immune system and protects the non-human animal against a disease caused by an infectious agent. The administration of plant-derived immunomodulator containing one or more saponins, which are preferably prepared from non-woody plants of the genus Hesperaloe, to a subject may increase the phagocytic activity in CD4 T helper (Th) cells, particularly Th1 and Th17 cells, in the subject being treated.
In yet other embodiments, the present invention provides administration of a plant-derived immunomodulator containing one or more saponins, which are 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, especially birds and more particularly poultry. For example, saponin containing compositions derived from Hesperaloe may be administered to non-human animals to reduce environmental ammonia and odor, to provide a hypocholesterolemic effect, reduce inflammation, promote weight gain, and improve feed conversion efficiency. In a particularly preferred embodiment, a Hesperaloe extract comprising one or more saponins may be administered orally to poultry for the prevention and treatment of coccidiosis.
In still other embodiments, the present invention provides a method of enhancing an immune response to an antigen in a non-human animal comprising administration of a saponin containing Hesperaloe extract to a non-human animal in an amount sufficient to enhance the immune response of the non-human animal. In a particularly preferred embodiment, the saponin containing Hesperaloe extract is administered to poultry and results in the increase the phagocytic activity in CD4 T helper (Th) cells, particularly Th1 and Th17 cells, and provides for protection against Eimeria infections.
In other embodiments, the present invention provides an immunological composition useful for inducing the production of antibodies to an antigen in a non-human animal comprising an immunogenically effective amount of an antigen and a saponin composition extracted from a non-woody plants of the genus Hesperaloe, wherein the amount of saponin in the extract is present in an amount sufficient to enhance the immune response of the non-human animal to the antigen. In particularly preferred embodiments saponin containing extracts of the present invention are administered with an Eimeria vaccine to poultry in need thereof to increase the immune response, lower lesion scores and reduced oocyst shedding resulting from coccidiosis.
As used herein the term “biomass” generally refers to whole plants and plant organs (i.e., leaves, stems, flowers, roots, etc.) of the genus Hesperaloe such as H. funifera, H. parviflora, H. nocturna, H. chiangii, H. tenuifolia, H. engelmannii, and H. malacophylla. In particularly preferred instances saponin containing compositions of the present invention may be prepared from biomass consisting essentially of the above ground portion of the plant and more particularly the portion of the plant above the crown and still more preferable the leaves of the plant.
As used herein the term “bagasse” generally refers to biomass that has been subjected to an extraction process such as, for example, pressing or milling, so that the resulting biomass solids have less water soluble solids than the biomass from which it is derived. In certain embodiments bagasse is prepared by subjecting biomass to high pressure, which may be achieved by passing the biomass through one or more pairs of opposed rolls, a mechanical press, a screw press as well as by direct hydraulic pressure and other processes to apply pressure to the biomass and remove intercellular and intracellular liquid therefrom.
As used herein the term “milling” generally refers to the application of sufficient pressure to force the intercellular and intracellular liquid from the biomass.
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 carbohydrate chemistry. It should be noted that the saccharides can be in the form of mono-, oligo- and/or polysaccharides. Preferably saccharides are water soluble and do not include cellulose, hemicellulose or mono-, oligo- and/or polysaccharides bound to other compounds, such as glycosides (arabinose, glucose, galactose, xylose, and glucuronic acid) bound to a triterpenoid to form a saponin.
As used herein the term “saponin” generally refers to glycosides comprising a sugar component referred to as a glycone and a non-sugar component referred to as an aglycone. Depending on the structure of the aglycone the saponin may be classified as a triterpenoid saponin, illustrated in
As used herein the term “water soluble solids” generally refers to dry matter which remains after the extract has been centrifuged, filtered and all water is evaporated. The procedure for measuring water soluble solids of a biomass extract of the present invention is described in detail in the Test Methods section below. Water soluble solids may be expressed on a percentage basis relative to the mass of bone dry biomass.
As used herein the term “water insoluble solids” generally refer to the fraction of extract that is removed by centrifugation and filtration in the course of measuring water soluble solids, as described in the Test Methods section below.
As used herein, “enhancing immune response” refers to an increase the phagocytic activity in in CD4 T helper (Th) cells, particularly Th1 and Th17 cells, in a subject being treated with a saponin containing composition derived from Hesperaloe, as described herein in comparison to the same subject prior to being treated.
As used herein, “pharmaceutical composition” refers to a composition at least one saponin extracted from Hesperaloe and formulated with one or more pharmaceutical-grade excipients in a manner that conforms with the requirements of a governmental agency regulating the manufacture and sale of pharmaceuticals as part of a therapeutic regimen for the treatment or prevention of disease in a non-human animal. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or any other formulation described herein.
As used herein the terms “subject” and “non-human animal” refer to any vertebrate animal including, without limitation cattle, chickens, turkeys, ducks, quail, geese, pigs, and sheep.
The present invention relates to novel pharmaceutical, dietary supplements and food ingredient compositions comprising at least one component selected from the extract(s), fraction(s), active compound(s) and phytochemical(s) or mixtures thereof derived from non-woody plants of the genus Hesperaloe including, for example, H. funifera, H. parviflora, H. nocturna, H. chiangii, H. tenuifolia, H. engelmannii, and H. malacophylla, optionally containing one or more of pharmaceutically and dietetically acceptable phytochemical actives, diluents, vehicles, carriers and actives or mixtures thereof. In a particularly preferred embodiment, the present invention provides a method of increasing the phagocytic activity in CD4 T helper (Th) cells, particularly Th1 and Th17 cells, in a non-human animal by administering to the non-human animal a Hesperaloe derived immunomodulator containing one or more saponins.
The compositions of the present invention are particularly well suited for the treatment of non-human-animals including, for example, bovine, fowl, porcine, ovine, and equine species. By way of example, the methods and compositions of the invention can be used for the treatment of cattle, chickens, turkeys, ducks, quail, geese, pigs, and sheep. In a particularly preferred embodiment, the methods and compositions of the present invention can be used for the treatment of poultry and more particularly for the prevention and treatment of coccidiosis and/or necrotic enteritis.
The Hesperaloe derived immunomodulator of the present invention may comprise at least 5 wt %, based upon the bone dry weight of the composition, saponins as measured by the total saponin assay set forth in the Test Methods section below. In a particular embodiment, the saponin containing composition used in accordance with the invention comprises at 10 wt % saponin, more preferably at least about 10 wt % saponin and still more preferably at least about 15 wt % saponin, such as from about 5 to about 30 wt % saponin, such as from about 15 to about 25 wt %. It is believed that the effects of the composition are related to the total amount of saponins present. Thus, one of skill in the art will appreciate that if a certain amount of saponins is desired it can be achieved either through varying the volume of a certain concentration composition administered, varying the concentration of a certain volume of a composition, or both.
Saponins useful in the present invention may also be extracted from non-woody plants of the genus Hesperaloe. Hesperaloe derived saponins generally have steroidal saponins. Saponins derived from Hesperaloe may have at least one of the following aglycones or genins: kammogenin, manogenin, gentrogenin, hecogenin, tigogenin, sarsapogenin, chlorogenin and gitogenin or their corresponding isomer or oxidized or reduced forms with at least one of the following glycosidic moieties (in the form of acid or salt): glucose, xylose, rhamnose, arabinose, or galactose. In other embodiments the steroidal saponins may comprise agamenoside, agaveside, agavoside, magueyside, agavasaponi, cantalasaponin, sisalsaponin, gabrittonoside, dongnoside or amolonin, or other steroidal saponins.
Extractives may be recovered from non-woody plants of the genus Hesperaloe by extracting biomass, particularly the leaves and more particularly the leaves above the crown of the plant, with at least one solvent selected from the group consisting of water, methanol, ethanol, butanol, and isopropanol, and mixtures thereof. For example, in one embodiment, the process comprises contacting biomass with an extractant solution comprising water and separating the water soluble fraction from the insoluble biomass fraction. In other embodiments the extractant solution may comprise, in addition to water, a surfactant, a solvent and optionally extract-bearing juice. The extract-bearing juice can come from, for example, an earlier extraction step or an earlier milling step.
A simple water extraction of Hesperaloe biomass may yield a crude aqueous extract comprising saccharides, polysaccharides, inorganic salts, saponins and sapogenins. A crude extract may also be produced using methanol as a solvent, or a mixture of methanol and water, to extract biomass, which may have been previously extracted with acetone or diethyl ether to remove lipids and pigments. In other instances, the biomass may be extracted with a 4:1 ethanol-water solvent, followed by subsequent defatting of the extract with a non-polar solvent such as hexane. In certain instances, the defatted extract may be subjected to further treatment to isolate specific water soluble components, such as saponins, which may be purified from the defatted extract by mixing with butanol and separating the butanol phase to yield a mixture of saponins that are substantially free from proteins and free saccharides and polysaccharides.
Hot aqueous extractants can also be used. For example, in one embodiment water soluble solids may be extracted from Hesperaloe biomass, particularly the leaves, by extracting the biomass with hot aqueous ethanol or isopropanol (75 to 95% by weight alcohol). The aqueous alcohol extraction fluid may then be filtered and concentrated, and the fat-soluble material may be removed by mixing the extraction fluid with a non-polar solvent such as hexane. A substantially pure saponin composition may then be prepared by further extracting defatted extract with a polar solvent such as butanol.
For the purpose of preparing the compositions of the present invention, and for use in the present method, a simple aqueous extract may be preferred, although other extraction methods are within the scope of the present invention. In a particularly preferred embodiment, Hesperaloe biomass may be cut to size, 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 extracts are collected and may be concentrated by techniques well known in the art such as, for example, evaporation, spray-drying, drum drying and the like. The extract may be concentrated until it has a solids content of about 20 to about 100% solids by weight, such as from about 20 to about 95% solids by weight, such as from about 20 to about 80% solids by weight.
In a particularly preferred embodiment water soluble extracts are concentrated by spray drying by feeding the extract solution to atomizing equipment. Suitable atomizing equipment includes, but is not limited to, a rotary wheel atomizer, a pressure nozzle atomizer, and a dual fluid nozzle atomizer. Rotary wheel, pressure nozzle and dual fluid nozzle atomizers are known to those of ordinary skill in the art and include those in spray dryers commercially available from a variety of sources, such as GEA Process Engineering.
As will be described in more detail below, the biomass may be milled to separate the bagasse and water soluble solids using a roll, screw, and other forms of presses. In certain preferred embodiments biomass is passed between one or more nips of opposed counter-rotating rolls to maximize the mechanical removal of juice. The bagasse can then be contacted with the juice in a subsequent milling step, as will be described more fully below. In certain instances, the biomass may be cut to size and cleaned prior to milling. Cutting and cleaning may be carried out using well known methods in the art. In a particularly preferred embodiment, the biomass is cleaned to remove debris such as dirt without the use of water or other solvents. While it may be preferable to cut the biomass to size prior to extraction, in certain embodiments it may be useful not to grind, pulp, shred or macerate the biomass before it is milled. While such physical processing steps can be advantageous in that they expose more of the biomass surface to the extractant solution, they can break the plant cell walls, excessively shorten fiber length, and create an excessive amount of fines. It is generally desirable to avoid negatively affecting the bagasse in this manner during the extraction phase. In this manner, the extraction method of the present invention typically yields bagasse fiber that may be further processed, such as by pulping, to yield pulp fibers well suited for the manufacture of paper products.
In other embodiments the water soluble solids may be recovered from biomass by diffusion. In diffusion, the biomass brought into contact with the liquid to extract the liquid components. Usually, the biomass is prepared by first cutting, but not shearing or crushing so as to minimize the damage to fibers and avoid the creation of an excessive amount of fines. The prepared biomass is then washed repeatedly, usually using a solvent, to extract the liquid contained in the biomass. The solvent can be any of the foregoing solvents. An exemplary treatment solvent is water, particularly hot water such as water heated to a temperature from about 40 to about 90° C. The solvent can be circulated and reused so that the solvent used for a first extraction is reused as a solvent to extract subsequent prepared biomass.
Various types of diffusers are known in the art and can be adapted for use with biomass as described herein. Suitable diffusers include a ring diffuser, a tower diffuser, or a drum diffuser. Exemplary diffusion systems are discussed, for example, in U.S. Pat. Nos. 4,182,632, 4,751,060, 5,885,539 and 6,193,805 the contents of which are hereby incorporated in a manner consistent with the present disclosure. Numerous other diffusion methods and devices for the diffusion method are known and can be adapted for use in the methods described herein. One such diffuser is the continuous-loop, counter-current, shallow-bed Crown Model Ill Percolation Extractor, commercially available from Crown Iron Works, Blaine, MN.
The biomass, cut or uncut, may be extracted by any suitable extraction process as discussed above. In a particularly preferred embodiment, the solvent used for extraction comprises water. One of skill in the art will recognize 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 certain preferred embodiments, the extraction solvent is water and the ratio of extraction solvent to biomass, on the basis of liters of extraction solvent to kilogram of bone-dry biomass, is from about 1:5 to about 1:100, such as from about 1:5 to about 1:50 and more preferably from about 1:5 to about 1:20.
The pH of the extraction solvent can be between about pH 5.0 and 8.0, such as, for example, between about pH 6.0 and about pH 8.0, between about pH 6.5 and about pH 7.5. In a particular embodiment, the extraction solvent is water having a pH between about pH 6.5 and about pH 7.5. In those embodiments where extraction includes imbibition with a crude juice, the imbibition fluid may have a pH from about 4.0 to about 5.0.
The extraction may be carried out at temperatures between about 25 and about 90° C., such as, for example, between about 30 and about 80° C., between about 35 and about 75° C., between about 40 and about 70° C., between about 45 and about 65° C. or between about 50 and about 60° C.
In embodiments where the extraction process is a batch extraction process, the duration of extraction may range from about 0.25 to about 24 hours, such as, for example, from about 0.5 to about 2 hours, from about 1 to about 8 hours, or from about 1 to about 6 hours.
In embodiments where the extraction process is a continuous process, the duration of extraction may range from about 0.25 to about 5 hours, such as, for example, from about 0.5 to about 3 hours.
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 the “first filtrate”). Separation can be achieved by any suitable means including, but not limited to, gravity filtration, a plate-and-frame filter press, cross flow filters, screen filters, Nutsche filters, belt filters, ceramic filters, membrane filters, microfilters, nanofilters, ultrafilters or centrifugation. Optionally various filtration aids such as diatomaceous earth, bentonite, zeolite, and the like, may also be used in this process.
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 about 8.5 and about 10.0 by treatment with a base, such as, for example, calcium oxide or hydroxide (about 1.0% from the volume of filtrate) with slow agitation.
In a particularly preferred embodiment water soluble solids are removed from biomass, particularly Hesperaloe leaves, prior to pulping by a series of mills, such as two, three, four, five, six or seven mills arranged in tandem, optionally with imbibition and/or depithing. Generally, processing biomass according to the present invention removes at least about 25% of the water soluble solids from the biomass, more preferably at least about 50%, still more preferably at least about 75%, such as from about 25 to about 98%, such as from about 50 to about 90%, such as from about 75 to about 90%.
The amount of water soluble solids recovered from biomass may vary depending on the extraction efficiency, however, in certain instances from about 100 to about 400 grams of water soluble solids may be extracted per kilogram of bone dry biomass, such as from about 120 to about 350 grams per kilogram, such as from about 150 to about 300 grams per kilogram. Of the extracted water soluble solids, the total saponins may comprise from about 5 to about 40 wt %, such as from about 10 to about 30 wt %, based upon the bone dry weight of the water soluble solids. In certain instances the amount of total saponins that may be extracted from biomass may range from about 10 to about 400 grams per bone dry kilogram of biomass, such as from about 20 to about 300 grams, such as from about 25 to about 200 grams, such as from about 10 to about 100 grams. In certain instances, the amounts of materials (on bone dry grams per kilogram of bone dry biomass) removed from the biomass during the extraction process may range as set forth in Table 1, below.
In addition to saponins, the water soluble solids may comprise saccharides, proteins, lipids, and inorganic salts. For example, in certain instances, the water soluble solids may comprise from at least about 1 wt %, based upon the bone dry weight of water soluble solids, saccharides, such as from about 1 to about 15 wt %, such as from about 2 to about 10 wt %. The saccharides may comprise monosaccharides and oligosaccharides. In other instances, the water soluble solids may comprise from at least about 15 wt %, based upon the bone dry weight of water soluble solids, inorganic salts, such as from about 15 to about 30 wt %.
Generally milling is carried out with the addition of an aqueous solvent, such as water, having a pH ranging from about 5 to about 9, such as from about 6 to about 7 to about 8. The water soluble solids are generally recovered from the milling process as a crude extract and may be subjected to further processing to recover specific compounds, such as saccharides, polysaccharides, organic acids and saponins.
The suspended solids, also referred to herein as the water insoluble fraction, may optionally be removed from the crude extract by well-known processes including, for example, clarification, filtration, centrifugation, or a combination thereof. The amount of water insoluble solids in the extract (on bone dry grams per kilogram of bone dry biomass) may range from about 1.0 to about 30 grams and may comprise hydrophobic substances such as waxes and the like.
After removal of 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 sapogenins. In other instances, the clarified juice may be further purified to remove saccharides, polysaccharides, and organic acids to yield composition comprising saponins.
The juice resulting from the foregoing extraction process may be subjected to further extraction to obtain saponin in the form of a crude saponin extract or its substantially purified form comprising saponins at a concentration from about 30 to about 90% in weight. The extraction method may comprise mixing 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 from 4 to 6 carbon atoms, such as butyl, amyl, hexyl and cyclohexyl alcohols. Extraction of the juice with a water-immiscible polar solvent generally removes impurities such as proteins, carbohydrates, and organic acids, which remain in the aqueous phase, the saponin being transferred to the solvent phase.
The solvent phase containing the saponin may be subjected to further treatment to separate the saponin from the alcohol phase. This can be accomplished in various ways including, for example, by cooling, by dehydrating the solvent extract, or by adding an organic solvent which is miscible with the alcohol solvent but in which the saponin is insoluble. Suitable precipitating solvents include, for example, diethyl ether, petroleum ether, acetone, and chloroform.
In a particularly preferred embodiments, the saponin is separated from the alcohol by flash evaporation. Flash evaporation is a technique known in preparative chemistry for the rapid removal of a volatile component from a liquid mixture. The volatile liquid is removed from solution by rapid conversion to a vapor phase by creating a thin film of the solution over a large surface area under reduced pressure often accompanied by an increase of temperature of the solution above ambient but less than the boiling point of the solution at atmospheric pressure. The actual thickness of the film and the area over which it is applied is chosen to provide optimum evaporation and ease of use, but evaporation may be substantially instantaneous (hence the name “flash” evaporation). Flash evaporation avoids the prolonged use of high temperatures that may degrade the intended product and has the ability to remove almost all of the alcohol component (which makes the remaining solution suitable for the preferred practice of spray drying employed in the next step. The alcohol may be recovered from this step and reused in the extraction process.
The saponin content of the alcohol extract can be further increased by passage over an ultrafiltration membrane without significant alteration to or loss of the saponin composition. This concentrated saponin fraction where the saponin content is in the range of 85-90%, can then be further purified in a liquid state or reduced to a dry state. Individual saponins may be recovered by a combination of reversed-phase solid phase extraction and preparative reversed-phase HPLC. Alternatively, the alcohol extract containing saponins can be fractionated directly by a combination of reversed-phase solid phase extraction and preparative reversed-phase HPLC.
In still other embodiments saponins may be purified from juice prepared according to the present invention comprises the steps of mixing the juice with a salt and a solvent to form a first solution. The solvent may comprise 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, diglyme, 1,2-dimethoxyethane, dimethylformamide, dimethylsulfoxide, 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexamethylphosphoramide, hexamethylphosphorous 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 of the forgoing. In some embodiments the solvent is water. The salt may be selected from an alkali metal salt, an alkaline earth salt, a transition metal salt, an ammonium salt, or combinations of the forgoing. In certain preferred embodiment 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.
The pH of the first solution is generally adjusted to a pH from about 6.0 to about 9.0, such as from about 6.0 to about 8.0, such as from about 6.0 to about 7.0. At least one phosphate may then be added to the first solution to form an ion-polysaccharides complex precipitate. Useful phosphates include, for example, sodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), sodium phosphate (Na3PO4), or sodium hydrogen bisphosphate (Na2H2PO7).
The precipitated ion-polysaccharides complex may be removed by filtration to yield a second solution, which may be further clarified to produce an extract of purified saponins. Optionally, the extract can be concentrated by any filtration technique known in the prior art. Preferably, the concentration of the extract of purified saponins is carried out by nanofiltration, ultrafiltration and diafiltration, or any combination of these techniques. 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.
The total amount of saponins that may be extracted from Hesperaloe biomass according to the present invention may range from about 10 to about 100 grams per bone dry kilogram of biomass, such as from about 20 to about 80 grams, such as from about 25 to about 75 grams. The saponins may be provided as part of a crude juice, as part of a dried water soluble solids compositions, as a partially purified compositions or as a substantially pure composition comprising a mixture of saponins.
In certain embodiments saponins extracted from Hesperaloe biomass may comprise 25(27)-dehydrofucreastatin (
Compositions useful in the present invention may be prepared by blending 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 less than about eight carbon atoms. Glycerol and propylene glycol are particularly preferred polyhydroxy alcohols.
The composition may also comprise saccharides, which may be present in the aqueous extract or may be added after extraction during formulation. Saccharides useful in compositions of the present invention include monosaccharides such as glucose, disaccharides such as sucrose and polysaccharides such as starch.
In still other embodiments compositions in accordance with embodiments of the invention can include various other additives known in the art to have benefits for the maintenance and well-being of non-human animals. By way of example, compositions can also include components such as Vitamin E, Vitamin A Propionate, Vitamin A Palmitate, Vitamin B1, Vitamin B2, Vitamin B6, Vitamin B12, D-Activated Animal Sterol (source of Vitamin D3), yeast components, dried egg solids, dried casein, and dried whey.
Saponin containing compositions of the present invention may be in liquid or dry forms. By way of example, a saponin containing Hesperaloe extract may be dried into a powder form. In this form, the saponin containing composition may be administered to an animal as a pill or bolus or mixed in with other components such as a feed ration. For example, dry powder formulations of saponin containing compositions may be added to the feed ration via a micro-ingredient machine or added to a feed mix truck and mixed thoroughly to assure even distribution in the feed. Saponin containing Hesperaloe extract may also be in liquid form with an amount of a carrier liquid such as water. In this form, the saponin containing composition may be administered to an animal as a liquid drench.
Saponin containing compositions of the present invention may be administered to non-human animals in need there of as a single dose, as multiple doses as part of a feeding regiment. For example, a non-human animal may receive an initial dose and then receive subsequent maintenance doses in lesser amounts. A non-human animal may receive multiple doses of a saponin containing composition in one day or may receive multiple doses over multiple days.
In certain embodiments, the compositions of the present invention, may be useful as an immunomodulator or adjuvant. In certain embodiments saponin containing compositions derived from Hesperaloe may be administered to a non-human animal in need thereof to elicit an adaptive immune response. In a particularly preferred embodiment administration of the saponin containing extracts of the present invention causes an increase in the phagocytic activity in CD4 T helper (Th) cells, particularly Th1 and Th17 cells, in the subject. In this manner the extracts of the present invention may be administered as a pharmaceutical composition without the addition of an antigen to enhance the immune response of the subject.
In other embodiments the Hesperaloe extracts of the present invention may be administered to non-human animals with an antigen to enhance the immune response of the subject. Suitable antigens include microbial pathogens, bacteria, viruses, proteins, glycoproteins lipoproteins, peptides, glycopeptides, lipopeptides, toxoids, carbohydrates, and tumor-specific antigens. Mixtures of two or more antigens may be employed. In certain preferred embodiments the compositions of the present invention may be administered with a vaccine intended for the prevention of coccidiosis in non-human animals, in particular poultry, characterized in that the coccidia are chosen from the group consisting of Eimeria, Isospora, Toxoplasma, Besnoitia, and Neospora. Thus, the present invention provides an adjuvant system that is particularly advantageous in making and using vaccine and other immunostimulant compositions to treat or prevent diseases, such inducing active immunity towards antigens in non-human animals.
In a particularly preferred embodiment of the saponin containing extracts may be administered with an Eimeria vaccine to poultry in need thereof to increase the immune response, lower lesion scores and reduced oocyst shedding resulting from coccidiosis. The immunogenic composition of the present invention may be delivered orally or subcutaneously in a dose volume suitable for increasing an immune response, such as a does level of less than about 50 μg, such as less than about 40 μg, such as less than about 30 μg, such as from about 1 to about 50 μg, such as from about 5 to about 30 μg.
The compositions of the present invention exhibit adjuvant effects when administered over a wide range of dosages and a wide range of ratios to the antigen being administered. In one embodiment, the saponin is administered in a ratio of adjuvant, based upon the weight of saponin, to antigen (w/w) of 3.0 or less, preferably 1.0 or less.
Saponins extracted from non-woody plant of the genus Hesperaloe according to the present invention may be used as adjuvants in crude or purified forms and may be admixed with other non-saponin adjuvants to achieve the enhancement of the immune response to an antigen. Such non-saponin adjuvants useful with the present invention are oil adjuvants (for example, Freund's Complete and Incomplete), liposomes, mineral salts (for example, AlK(SO4)2, AlNa(SO4)2, AlNH4 (SO4), silica, alum, Al(OH)3, Ca3 (PO4)2, kaolin, and carbon), polynucleotides (for example, poly IC and poly AU acids), and certain natural substances (for example, wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella).
Total biomass water soluble solids may be determined using an Accelerated Solvent Extraction system (ASE) such as a Dionex™ ASE™ 350 (Thermo Fisher Scientific, Waltham, MA). Approximately 10 grams of harvested biomass is dried to a constant weight in an oven, typically 4 hours at 125° C. After drying 1.5-2.0 grams of the bone dry biomass is accurately weighed and the weight (Wb) recorded to the nearest 0.001 gram. Using water as the solvent, biomass is extracted using the conditions set forth in the table below. The ratio of biomass to solvent is generally 21:1 and five consecutive water extraction cycles are performed. At the end of each extraction cycle, the liquid phase is collected, dried under vacuum at approximately 40° C. and the weight of the dried material (Wi) is recorded to the nearest 0.001 g. The total weight of water soluble solids (We) is calculated by summing the weight of solids recovered from each extraction cycle (Wi). Total water soluble solids as a percentage of bone dry biomass is then determined using the following equation: Water Soluble Solids (wt %)=We/Wb*100.
The total water soluble solids in biomass extract may be determined by withdrawing an appropriate aliquot, typically about 10-50 ml, transferring to clean, dry, centrifuge tube. The tube is centrifuged at 7000 rpm for 20 minutes. The weight of extract (W1) is calculated. An aliquot of the supernatant is then transferred to clean, pre-weighed beaker (D0), and weighed. The beaker and sample are then weighed to the nearest 0.001 g and the weight (D2) recorded. The beaker containing the sample is then placed at 140° C. in a hot air oven for overnight drying. The beaker is removed from the oven and desiccated to cool to room temperature then weighed to the nearest 0.001 gram (D1). The weight percentage of soluble solids, based upon the weight of the extract, is determined using the formula below:
D1=mass of empty beaker+dried soluble solids, D0=mass of empty beaker, D2=mass of biomass extract and empty beaker.
Total saponins were measured generally as described in Makkar, Harinder P. S., Sidhuraju, P., Becker, Klaus (2007) Plant Secondary Metabolites, chapter 17, pp 93-100. A standard saponin solution was prepared by weighing 10 mg of diosgenin (MilliporeSigma>93%), dissolving in 16 mL of methanol and adding 4 mL of distilled water. The solution was mixed thoroughly to yield a 0.5 mg/mL diosgenin solution in 80% methanol solvent. The standard was used to produce a calibration curve by transferring various amounts of the standard (0, 10, 20, 40, 60, 80, and 100 μL) into 13-mm glass test tubes. A solution of 80% aqueous methanol was added to a total volume of 100 μL.
Prior to testing samples of biomass extract were adjusted to about 0.5 wt % total solids by dilution with water to ensure absorbency result fell along the saponin standard calibration curve range. Samples of diluted extract (20-μL) were pipetted into 13-mm glass test tubes and the volume was brought up to 100 μL with 80 μL methanol. Each sample was tested in triplicate.
To each sample 100 μL of vanillin reagent (prepared by dissolving 800 mg of vanillin in 10 mL of 99.5% ethanol (analytical grade)) and then 1.0 mL of 72% (v/v) sulfuric acid (72% (v/v) sulfuric acid prepared by adding 72 mL of sulfuric acid (analytical grade, 95%, w/w) to 28 mL of distilled water) were added. Solutions were mixed well and heated at 60° C. for 10 minutes. Samples were then cooled in an ice bath and 1 mL of solution was transferred into respective cuvette and absorbance at 544 nm was read. The total mass of saponins in the sample may be calculated based upon the standard absorbency curve as follows:
Saponin(μg)=[Slope]×Measured Absorbency−[Intercept]
Total saponins were measured generally as described in Makkar, Harinder P. S., Sidhuraju, P., Becker, Klaus (2007) Plant Secondary Metabolites, chapter 17, pp 93-100. A standard saponin solution was prepared by weighing 10 mg of diosgenin (MilliporeSigma>93%), dissolving in 16 mL of methanol and adding 4 mL of distilled water. The solution was mixed thoroughly to yield a 0.5 mg/mL diosgenin solution in 80% methanol solvent. The standard was used to produce a calibration curve by transferring various amounts of the standard (0, 10, 20, 40, 60, 80, and 100 μL) into 13-mm glass test tubes. A solution of 80% aqueous methanol was added to a total volume of 100 μL.
Prior to testing samples of biomass extract were adjusted to about 0.5 wt % total solids by dilution with water to ensure absorbency result fell along the saponin standard calibration curve range. Samples of diluted extract (20-μL) were pipetted into 13-mm glass test tubes and the volume was brought up to 100 μL with 80 μL methanol. Each sample was tested in triplicate.
To each sample 100 μL of vanillin reagent (prepared by dissolving 800 mg of vanillin in 10 mL of 99.5% ethanol (analytical grade)) and then 1.0 mL of 72% (v/v) sulfuric acid (72% (v/v) sulfuric acid prepared by adding 72 mL of sulfuric acid (analytical grade, 95%, w/w) to 28 mL of distilled water) were added. Solutions were mixed well and heated at 60° C. for 10 minutes. Samples were then cooled in an ice bath and 1 mL of solution was transferred into respective cuvette and absorbance at 544 nm was read. The total mass of saponins in the sample may be calculated based upon the standard absorbency curve as follows:
Saponin(μg)=[Slope]×Measured Absorbency−[Intercept]
A total of 150 one-day-old broiler chicks were randomly distributed to six experimental groups in a 28-day cage study. Live coccidia were manually introduced to the birds at their 14th day of age. The treatment codes, listed in Table 3, included two control codes using basal diet with (Control+) and without (Control) coccidian challenges. Remaining treatment codes were all challenged with coccidian using base diet enriched with an inventive composition at two different dosages or a Yucca extract marketed under the tradename FOAMATION™ (commercially available from Ingredion, Westchester, IL). FOAMATION™ comprised 50% by weight of the composition water soluble solids, of which saponins comprised 10 wt %. Bird body weight gain (BW) and feed consumption for each pen were measured on a weekly basis. Feed conversion rate (FCR) is the ratio between kilograms of feed consumed and kilograms of body weight gain. The lower FCR value indicates a better feed.
The inventive extract was prepared by forage harvesting mature Hesperaloe funifera leaves above the crown, cutting the leaves into pieces ranging from about 0.50 to about 8.0 cm and pressing the cut biomass using a tandem press. The biomass was pressed three times and the crude juice was collected and passed through 25 mm filter and heated to concentrate the extract to 29% solid. The water soluble solids comprised 21 wt % total saponins, based upon the bone dry weight of water soluble solids.
At the end of 28-day trial, the challenged control (Control+) group decreased feed consumption by about 140 g/bird and body weight gain by was reduced by about 160 g/bird compared to the control without challenge. These decreases, however, were not observed in chickens administered feeds comprising the inventive composition, as illustrated in Table 4, below.
A total of 512 one-day-old broiler chicks were randomly distributed to 8 experimental groups, 8 cages for each group and 8 birds per cage in a 21-day study. Live coccidian was manually introduced to the young birds at 14th day of age. Bird weight gain (WG), feed conversion rate (FCR), lesion score and oocyst counts were measured. The treatment codes, listed in Table 5, included basal diet with no coccidian challenge (control) and with challenge (Control+).Remaining treatment codes were coccidian challenged codes using base diet with Coban (commercially available from Elanco Animal Health, Greenfield, IN), Micro-Aid (commercially available from DPI Global, Porterville, CA) and two different inventive samples at two different dosages. Base diet met the minimum National Research Council requirements for poultry.
Inventive sample 1 was prepared by forage harvesting mature Hesperaloe funifera leaves above the crown, cutting the leaves into pieces ranging from about 0.50 to about 8.0 cm and pressing the cut biomass using a tandem press. The biomass was pressed three times and the crude juice was collected and passed through 25 mm filter and heated to concentrate the extract to 29% solid. Inventive sample 2 was prepared by forage harvesting mature Hesperaloe funifera leaves above the crown, cutting the leaves into pieces ranging from about 0.50 to about 8.0 cm and pressing the cut biomass using a tandem press once, followed by heating of the collected juice to obtain an extract having 14% solids. All treatment materials were made by mixing each additive to the base feed at the designated loading level in a mixer.
Chickens fed the inventive composition exhibited weight gain, improved feed conversion rate, decreased lesion score and lower oocysts as summarized in Table 6, below. In many instances the improvements were the comparable to, or better, then those observed in chickens fed Coban or Micro-Aid Green. Even at relatively low dosages of saponin, the inventive compositions were effective.
Compositions of the present invention are particularly useful in reducing or preventing coccidial. Lesion score is a means of assessing coccidial development through chicken's intestinal damage on a score between 0-4 (0 indicates normal intestinal appearance while 4 indicates severe damaged intestine). Chickens fed the inventive composition, over 3 weeks, improved lesion score (23-27%), compare to the unchallenged control. By reducing the instances of infection and protecting the chickens' digestive system, the chickens were able to better digest and absorb nutrients and grow at a greater rate.
A total of 210 day-of-hatch Ross×Ross male broiler chicks were obtained from Aviagen Hatchery, Blairsville, GA. Upon birth the birds received routine vaccinations (HVTSB1). The birds were randomly distributed to six experimental groups in a 28-day cage study. The treatment groups, listed in Table 7, included a first group that received no vaccine or Hesperaloe extract, groups that received the Hesperaloe extract alone, groups that received Newcastle disease virus (Lasota strain) and Hesperaloe extract, as well as a group that received an inactivated Newcastle disease virus (Lasota strain).
Hesperaloe Extract
Birds treated with the Newcastle disease virus received an oil emulsion Newcastle disease virus (Lasota strain) vaccine administered at 0.10 ml S.Q. in back of neck on Day 0. The Hesperaloe extract was prepared substantially as described in Example 2 by pressing the cut biomass using a tandem press once, followed by heating of the collected juice to obtain an extract having 14% solids.
Each group of 35 broiler chicks were housed in a room measuring 13.4′×15.7′. The isolation room environment is controlled by independent HEPA filtration systems and heat pump units with one (1) heat lamp providing supplemental heat during brooding. Birds were raised under ambient humidity and provided a lighting program as per the primary breeder recommendations. At placement, each pen contained approximately 4 inches of fresh pine shavings. Litter was not replaced during the study course. Each division contained a tube feeder and a bell drinker resulting in a 35 bird/feeder and drinker ratio.
All diets contained 113.5 g/ton amprolium to prevent coccidosis with no other concomitant drug therapy used during the study. Starter rations were weighed and fed DOT 0 through DOT 28. Feed formulations consisted of un-medicated commercial-type broiler starter and grower diets compounded with commonly used United States feedstuffs representative of local formulations, calculated analyses to meet or exceed NRC standards. No antibiotics were added to any feed.
On DOT 28 ten birds per treatment were euthanized and organ specimens (large Peyer's Patch in duodenum, Cecal tonsil, one-half of bursa of fabricius, 0.5 cm section of jejunum) were collected and placed into individual jars. The tissue sample was fixed in 10% buffered formalin and embedded in paraffin wax. Sections (approximately 5 microns) of the paraffin-embedded tissues were stained with Mayer's hematoxylin and eosin (H&E).
Lymphoid follicle area and lymphoid follicular cortex were measured as described by Muniz, et al, Brazilian J Poult Sci 8, 217-220, 2006. Five follicles with full presentation of anatomic features were measured per bursa. Measurements were made with the free hand tool and line to area feature in ImageJ software 1.37, Java based image processing software, which was developed at the U.S. National Institutes of Health and is freely available on the internet. Pixels to micrometers were calibrated using an AmScope MR400 calibration slide.
The area of cecal tonsil and Peyer's patch were measured similarly to the measurement of lymphoid follicle area and the germinal centers were manually counted. Cecal tonsil germinal center counts for each of treatment groups 1 through 6 are illustrated in
Gut associated lymphoid tissue (GALT) expansion (hyperplasia) was scored on a scale from 0 to 5: 0 (not apparent), 1 (minimal presence), 2 (mild), 3 (moderate), 4 (marked), and 5 (severe). GALT expansion occurred as focal, locally extensive, and diffuse, within normal limits but to variable degrees of hyperplasia. GALT score for each of treatment groups 1 through 6 is illustrated in
Intestinal heterophils appeared as clusters of heterophils in the lamina propria. The total number of clusters were counted and recorded for each jejunum section. The total number of clusters in the lamina propria for each of treatment groups 1 through 6 are illustrated in
ON DOT 28 ten birds per treatment were bled and the serum samples were collected and assessed using a commercial enzyme-linked immunosorbent assay (ELISA) Newcastle disease antibody test (FlockChek®, commercially available from IDEXX, Maine, USA). The resulting ELISA titers for each of the groups is shown in
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/38274 | 6/21/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63041224 | Jun 2020 | US |
