TECHNICAL FIELD

The present disclosure relates to an animal feed preservative or animal feed comprising one or more antimicrobial agents, wherein the one or more antimicrobial agents are selected from: one or more berberine alkaloids; one or more ursane-like triterpenoids; one or more phenylpropanoids; one or more monoterpenoids; or mixtures thereof. In particular, the present disclosure relates to a safe animal feed preservative or animal feed comprising the aforementioned antimicrobial agents.

BACKGROUND

Antimicrobial agents may be used as preservatives to avoid the spoilage of food. Examples of antimicrobial preservatives in common use include sodium benzoate, vitamin C and sodium nitrites and nitrates. Preservatives are typically selected on the basis that they are harmless to animals and humans. However, sodium benzoate and vitamin C mixtures and sodium nitrites and nitrates have all been identified as increasing cancer risk. A mixture of sodium benzoate and vitamin C can give rise to benzene in soft drinks [1]. Sodium nitrites and nitrates, which are typically found in cured meats, are classified as probably carcinogenic by WHO as they can be metabolised to release nitrosamines [2].

Some naturally-occurring compounds have the potential to act as antimicrobial preservatives when included in feed. Fewer naturally-occurring compounds have the potential to contribute to general health of food-producing animals and to promote their growth. Such compounds may be classified as phytogenic compounds. While numerous compounds have been identified as having antimicrobial activity, it cannot be predicted what value these compounds may offer as phytogenic ingredients in feed based on the limited and contradictory in vivo data publically available [3].

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims. SUMMARY

The present disclosure is based on the finding that certain natural products with antimicrobial activity can be used as preservatives in animal feed. Further, the animal feed preservatives are safe in that they show no adverse effects in animal subjects and pose an insignificant risk of disease, particularly cancer. Moreover, the animal feed preservatives disclosed herein are stable. In addition, the natural products of the present disclosure show potential as phytogenic compounds i.e. compounds that are natural growth promoters. In this regard, the natural products have been found to have a positive effect on animal growth performance including a reduction in feed conversion ratio (FCR) and to improve the gastrointestinal health of animal subjects. Thus, the natural compounds may provide an alternative to the use of antibiotics in food production helping obviate concerns such as the spread of untreatable diseases and the development of antimicrobial resistance and “superbugs” associated with antibiotics.

Thus, the present disclosure relates to an animal feed preservative comprising one or more antimicrobial agents, wherein the one or more antimicrobial agents are selected from: one or more berberine alkaloids; one or more ursane-like triterpenoids; one or more phenylpropanoids; or mixtures thereof.

The present disclosure also relates to an animal feed preservative comprising one or more antimicrobial agents, wherein the one or more antimicrobial agents are selected from: one or more berberine alkaloids; one or more ursane-like triterpenoids; one or more phenylpropanoids; or mixtures thereof, and wherein the feed preservative is safe.

The present disclosure also relates to an animal feed comprising a feed preservative as described herein. The present disclosure also relates to use of one or more antimicrobial agents as an animal feed preservative, wherein the one or more antimicrobial agents are selected from: one or more berberine alkaloids; one or more ursane-like triterpenoids; one or more phenylpropanoids; or mixtures thereof.

The present disclosure also relates to use of one or more antimicrobial agents in the preparation of an animal feed preservative, wherein the one or more antimicrobial agents are selected from: one or more berberine alkaloids; one or more ursane-like triterpenoids; one or more phenylpropanoids; or mixtures thereof.

The present disclosure also relates to use of one or more antimicrobial agents in the preparation of an animal feed, wherein the one or more antimicrobial agents are selected from: one or more berberine alkaloids; one or more ursane-like triterpenoids; one or more phenylpropanoids; or mixtures thereof.

The present disclosure also relates to use of an animal feed preservative as described herein in the preparation of an animal feed.

DEFINITIONS

Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Thus, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. For example, reference to “an animal” includes populations of a plurality of animals.

Throughout this specification, various aspects and components of the invention can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub- ranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 5, 5.5 and 6, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

Herein, the term "about" when used in connection with a measurable numerical value refers to the specified value of the variable and to all values that are within the experimental error of the specified value or within +/- 10 % of the specified value whichever is greater.

As used herein the term “acceptable excipient” refers to a solid or liquid filler, carrier, diluent or encapsulating substance that may be safely used in administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers or excipients may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water. Excipients are discussed, for example, in Remington [Reference 4].

As used herein the term “acceptable salt” refers to salts which are toxicologically safe for systemic administration. Acceptable salts include acceptable acidic/anionic or basic/cationic salts [5 to 7]. Acceptable salts of the acidic or basic compounds of the invention can be made by conventional procedures (such as reacting a free acid with the desired salt-forming base or reacting a free base with the desired salt-forming acid). Acceptable salts of acidic compounds include salts with cations and may be selected from alkali or alkaline earth metal salts, including, sodium, lithium, potassium, calcium, magnesium and the like, as well as non-toxic ammonium, quaternary ammonium (such as the berberine quaternary ammonium cation), and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolamine and the like, and salts with organic bases. Suitable organic bases include N-methyl-D-glucamine, arginine, benzathine, diolamine, olamine, procaine and tromethamine.

Acceptable salts of basic compounds include salts with anions and may be selected from organic or inorganic acids. Suitable anions include acetate, acylsulfates, acylsulfonates, adipate, ascorbate, benzoate, besylate, bromide, camsylate, caprate, caproate, caprylate, chloride, citrate, docusate, edisylate, estolate, formate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hyclate, hydrobromide, hydrochloride, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mesylate, methylbromide, methylsulfate, napsylate, nitrate, octanoate, oleate, pamoate, phosphate, polygalacturonate, salicylate, stearate, succinate, sulfate, sulfonate, sulfosalicylate, tannate, tartrate, terephthalate, tosylate, triethiodide and the like.

Berberine is a positively charged quaternary ammonium cation. Acceptable salts of beberine include without limitation chloride, hemisulfate and iodide salts.

The present disclosure also contemplates ursane-like terpenoid salts as acceptable salts. For example, an acceptable salt of berberine is berberine ursolate and, vice versa , an acceptable salt of ursolic acid is berberine ursolate, where berberine is the cation and ursolate is the anion. It will be recognised that berberine ursolate may display a combination of the biological activity possessed by the berberine ammonium cation and the biological activity possessed by the ursolate counter anion.

The present invention also contemplates phenylpropanoid salts as acceptable salts. An example of a phenylpropanoid salt is a phenolate salt of honokiol (systematic name: 2-(4-hydroxy-3-prop- 2-enyl-phenyl)-4-prop-2-enyl-phenol). For example, an acceptable salt of berberine is the honkiol salt of berberine and, vice versa, an acceptable salt of honokiol is the berberine salt of honokiol, where berberine is the cation and honokiol phenlolate is the anion. It will be recognised that the honokiol phenolate salt of berberine may display a combination of the biological activity possessed by the berberine cation and the biological activity possessed by the honokiol phenolate counter anion. The present disclosure also contemplates salts of essential oils or components of essential oils such as the salts of monoterpenoid compounds. An example of a monterpenoid compound is thymol. Thus, the present disclosure contemplates salts of thymol as acceptable salts. For example, an acceptable salt of thymol is the berberine ammonium cation salt of thymol and, vice versa , an acceptable salt of berberine is the thymol phenolate salt of berberine. It will be recognised that such a salt may display a combination of the biological activity possessed by the berberine ammonium cation and the biological activity possessed by the thymol phenolate counter anion.

As used herein “acceptable solvent” is a solvent which for the purpose of the disclosure may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, ethanol and acetic acid, glycerol, liquid polyethylene glycols and mixtures thereof. A particular solvent is water. The term “solvate” refers to a complex of variable stoichiometry formed by a solute (for example, a berberine alkaloid) and a solvent. In particular, the solvent used is an “acceptable solvent” as defined herein. When water is the solvent, the molecule is referred to as a hydrate.

The term "administering" as used herein is to be construed broadly and includes administering a feed preservative or animal feed as described herein to an animal subject. The term encompasses the normal consumption of food and water by the animal subject and oral administration (including buccal or sublingual). The term “administering” as used herein also encompasses administration by nasal administration.

As used herein the term "antimicrobial activity" is defined herein as an activity that kills or inhibits the growth of microorganisms including, but not limited to bacteria, viruses, parasites, and fungi. It would be recognised that a substance which displays antimicrobial activity may be used as a preservative to avoid the spoilage of food.

It will be understood that a reference to the term “antimicrobial”, “antimicrobial agent”, “antimicrobial compound” and the like herein encompasses, the antimicrobial/antimicrobial agent/antimicrobial compound and, where permitted, all derivatives, isomeric forms, racemates, amorphous forms, crystalline forms, solvates, acceptable salts, solvates of said salts, and prodrugs thereof in isolation from one another as well as mixtures.

As used herein, the term “berberine alkaloid(s)” refers to berberine and related compounds and derivatives thereof which share similar structures and characteristics to berberine and are suitable for the feed preservatives/animal feeds/uses of the disclosure. As described herein berberine is an isoquinoline quaternary alkaloid and plant natural product with antimicrobial activity. Berberine alkaloids include, but are not limited to protoberberine alkaloids. Non-limiting examples of berberine alkaloids are: beberine, berberrubine, coreximine, tetrahydropalmatine, jatrorrhizine, 13-hydroxyberberine chloride, coralyne chloride, 7,8-dihydro-13-methylberberine, fibrauretin (palmatine), 13-benzylberberine and acceptable salts thereof. Berberine alkaloids can exist in different isomers or different isomeric forms, for example, various tautomers or tautomeric forms. It will be understood that the term “berberine alkaloid(s)” encompasses different isomeric forms in isolation from each other as well as combinations.

Berberine alkaloids can also exist in various amorphous forms and crystalline forms (i.e. polymorphs). It will be also understood that the term “berberine alkaloid(s)” encompasses different amorphous and crystalline forms in isolation from each other as well as combinations.

As used herein, the term “berberine alkaloid(s)” encompasses acceptable salts, solvates, solvates of said salts or pro-drugs thereof.

Thus it will be understood that reference to a “berberine alkaloid(s)” encompasses, where permitted, all derivatives, isomeric forms, racemates, amorphous or crystalline forms, solvates, acceptable salts, solvates of said salts, and prodrugs thereof.

The terms “berberine” and “IRP001” and are used interchangeably herein. As used herein, “IRP001 chloride” or “IRP001 C1” denotes the chloride salt of berberine; and “IRP001 sulfate” refers to the hemisulfate salt of berberine. Thus, it would be appreciated that the terms “IRP001 sulfate”, “berberine sulfate”, “IRP001 hemisulfate, and “berberine hemisulfate” are equivalent herein. The molecular structures of berberine quaternary ammonium cation, and the chloride and hemisulfate salts are shown in Figure 1. As used herein “IRP002” refers to ursolic acid in protonated acid form; “IRP003” refers to piceid; “IRP004” refers to honokiol; and “IRP005” refers to baicalin. The term "composition" as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

As used herein, the term “derivative(s)” encompasses compounds that are generated from a parent compound. Herein, a reference to derivatives also includes a reference to metabolites. Derivatives may result from e.g. functionalization, substitution, redox manipulation, unsaturation and/or ring incorporation of the parent compound.

Monoterpenoids are hydrocarbon type compounds comprising one terpene unit; diterpenoids are hydrocarbon type compounds composed of two terpene units; triterpenoids are hydrocarbon type compounds composed of three terpene units, where each terpene unit has the molecular formula of C ₁₀ H ₁₆ . A terpene unit, itself, consists of two isoprene CH ₂ =C(CH ₃ )-CH=CH ₂ (C ₅ H ₈ ) units. Thus, a diterpenoid is a diterpene and a triterpenoid is a triterpene. It will be recognised that the terms: “monoterpenoid” and “monoterpene”; “diterpenoids” and “diterpene”; and “triterpenoid” and “triterpene” have come to be used interchangeably in the art.

The present disclosure is particularly directed to “ursane like triterpenoid(s)”. As used herein, the term “ursane like triterpenoid(s)” refers to ursane, lupane, oleanane, hopane, gammacerane, taraxastane, dammarane, lanostane and cucurbitane triterpenoids, which share similar characteristics and similar bioactivities and are suitable for the animal feed preservatives/animal feeds/uses of the invention.

Triterpenoids can exist in different isomers or different isomeric forms, for example, various tautomers or tautomeric forms. It will be understood that the term “triterpenoid(s)” encompasses different isomeric forms of triterpenoids in isolation from each other as well as combinations. It will be understood that the term “ursane like triterpenoid(s)” encompasses different isomeric forms of ursane, lupane, oleanane, hopane, gammacerane, taraxastane, dammarane, lanostane and cucurbitane triterpenoids in isolation from each other as well as mixtures.

Triterpenoids can also exist in various amorphous forms and crystalline forms (i.e. polymorphs). It will be understood that the term “triterpenoid(s)” encompasses different amorphous and crystalline forms in isolation from each other as well as combinations. It will be also understood that the term “ursane like triterpenoid(s)” encompasses different amorphous and crystalline forms in isolation from each other as well as mixtures.

As used herein, the term “triterpenoid(s)” encompasses acceptable salts, solvates, solvates of said salts or pro-drugs thereof. As used herein, the term “ursane like triterpenoid(s)” encompasses acceptable salts, solvates, solvates of said salts and pro-drugs thereof.

Triterpenoids can be found in their free form (sapogenins or aglycones) or bound to glycosides (saponins). Thus, the term “triterpenoid(s)”, as used herein, encompasses sapogenin or saponin forms. It will also be understood that the term “ursane-like triterpenoid(s)/ursane-hke triterpene(s)”, as used herein, encompasses sapogenin or saponin forms.

Thus it will be understood that reference to a “triterpenoid(s)” encompasses, where permitted, all derivatives, isomeric forms, racemates, amorphous or crystalline forms, solvates, acceptable salts, solvates of said salts, and prodrugs thereof including sapogenin forms and saponin forms.

Thus it will also be understood that reference to an “ursane-like triterpenoid(s)” encompasses, where permitted, all isomeric forms, racemates, amorphous or crystalline forms, solvates, acceptable salts, solvates of said salts, and prodrugs thereof including sapogenin forms and saponin forms.

It will also be understood that reference to “an ursolic acid” encompasses, where permitted, all isomeric forms, racemates, amorphous or crystalline forms, solvates, acceptable salts, solvates of said salts, and prodrugs thereof including sapogenin forms and saponin forms.

Phenylpropanoids are aromatic type compounds comprising a C ₆ C ₃ , i.e., phenylpropane/n- propylbenzene unit. Several phenylpropanoid skeletons can be constructed from the phenylpropane unit. Tables 1 to 8 depict phenylpropanoid skeletons with a selection of phenylpropanoid compounds.

Table 1 depicts the phenylpropane unit (1) and cinnamic acid as an example of a compound that is derived from a single phenylpropane unit. A neolignan skeleton (2) and honokiol are shown in Table 2. The dibenzylbutane skeleton (3) and diphenyl propane skeleton (4) are shown in Table 3 and Table 4 with example compounds. The stilbene skeleton (5) and resveratrol/piceid aglycone are shown in Table 5. A stilbenoid diphenyl ethane skeleton (6) and hexestrol are shown in Table 6. The diphenyl methane skeleton (7) and a representative compound are shown in Table 7. The present disclosure is also directed to compounds in which other ring structures have been incorporated into phenylpropanoid skeletons. For example, the present disclosure contemplates compounds in which a ring structure has been incorporated into a neolignan biphenyl type skeleton. For illustration, effusol, acerogenin E, a member of the acerogenin class of compounds, and syringaresinol are shown in Table 8. Table 1 Phenylpropane skeleton Table 2 Neolignan skeleton

Table 3 Dibenzylbutane skeleton Table 4 Diphenyl propane skeleton

Table 5 Stilbene skeleton

Table 6 Stilbenoid diphenyl ethane skeleton Table 7 Diphenyl methane skeleton

Table 8 Phenylpropanoid skeletons incorporating other ring structures

The present disclosure is particularly directed to “honokiol-like” or “piceid-like” phenylpropanoid compounds. As used herein, the terms: “honokiol-like phenylpropanoids”, “honokiol-like phenylpropanoid compounds”, “honokiol-like compounds”; and “piceid-like phenylpropanoids”, “piceid-like phenylpropanoid compounds”, “piceid-like compounds” and the like refer to compounds that share similar characteristics and similar bioactivities to honokiol and piceid respectively and are suitable for the animal feed preservatives/animal feeds/uses of the invention.

Phenylpropanoids can exist in different isomers or different isomeric forms, for example, various tautomers or tautomeric forms. It will be understood that the term “phenylpropanoid(s)” encompasses different isomeric forms of phenylpropanoids in isolation from each other as well as combinations.

Phenylpropanoids can also exist in various amorphous forms and crystalline forms (i.e. polymorphs). It will be understood that the term “phenylpropanoid(s)” encompasses different amorphous and crystalline forms in isolation from each other as well as combinations.

As used herein, the term “phenylpropanoid(s)” encompasses acceptable salts, solvates, solvates of said salts and pro-drugs thereof. Phenylpropanoids can be found in their free form (sapogenins or aglycones) or bound to glycosides (saponins). Thus, the term “phenylpropanoid(s)”, as used herein, encompasses sapogenin or saponin forms. Thus it will be understood that reference to a “phenylpropanoid(s)” encompasses, where permitted, all derivatives, isomeric forms, racemates, amorphous or crystalline forms, solvates, acceptable salts, solvates of said salts, and prodrugs thereof including sapogenin forms and saponin forms.

Terpenoids, including triterpenoids, and phenylpropanoids can be obtained by isolation from natural plant sources, through modification of biosynthetic pathways, through chemical synthesis and/or chemical derivatisation. Terpenoid, including triterpenoid, and phenylpropanoid derivatives can be generated from functionalisation, substitution, redox manipulation, i.e., oxidation or reduction, and unsaturation of a terpene or phenylpropane compound. In addition to functionalisation, substitution, redox manipulation and unsaturation, derivatisation may also include the incorporation of other various sized ring structures into the terpene or phenylpropane (hereinafter “ring incorporation”). This ring incorporation can be seen in Table 8 above with respect to the phenylpropanoid compounds effusol, acerogenin E and syringaresinol. The present disclosure encompasses terpenoid and phenylpropanoid derivatives that are generated from functionalisation, substitution, redox manipulation, unsaturation and ring incorporation. Herein, a reference to derivatives also includes a reference to metabolites.

The term “animal feed”, as used herein, refers to any compound, preparation, or mixture suitable for, or intended for consumption/intake by an animal.

The term “animal” or “animal subject”, as used herein, refers to a human or a non-human animal. Non-limiting examples of non-human animals are aquatic animals, mammals and birds.

The term “aquatic animal(s)”, as used herein, refers to fish including but not limited to finfish and shellfish. Examples of finfish are barramundi, bass, bream, carp, catfish, cod, crappie, drum, eel, goby, goldfish, grouper, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pangus, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish. Shellfish include but not limited to a crustacean ( e.g . crabs, crayfish, lobsters, prawns and shrimp) and a mollusc (e.g. clams, mussels, oysters, scallops and winkles).

The term “mammal(s), as used herein, refers to a human or a non-human mammal. Non- limiting examples of non-human mammals are horses, camels, rabbits, dogs, cats, goats, sheep, primates, rabbits, rodents, cattle or pigs (swine). Sheep include, for example, rams, ewes and lambs. Rodents include but are not limited to guinea pigs, mice and rats. Cattle include but are not limited to beef cattle, dairy cattle, bulls, cows and young calves. Pigs include, for example, boars, piglets, growing pigs, sows and weaners.

Birds include, for example, poultry such as chickens, ducks, geese, turkeys, quail, guinea fowl, pigeons (including squabs) and birds of prey (including hawks, eagles, kites, falcons, vultures, harriers, ospreys, and owls). Chickens include, for example, broiler chickens (broilers), broiler breeders, chicks, fryers, roosters and layer hens (layers).

The term “animal subject”, as used herein, encompasses companion animals (such as cats, dogs) and food-producing animals (as defined herein) and aquarium and zoo animals.

Further, pseudo-ruminant animals include, for example, horses, rabbits and guinea pigs.

Ruminant animals include, for example, animals such as cattle, sheep, goats and deer. Monogastric animals include but not limited to pigs, cats, dogs, rats and mice.

As used herein, the term “food-producing animal” refers to an animal that is farmed for the production of food for consumption by another animal, for example, a human. It would be understood that the term “food-producing animal” includes, for example, a chicken or pig.

It will be understood that the term “isomer” refers to structural or constitutional isomers, tautomers, regioisomers, geometric isomers, or stereoisomers including enantiomers or diastereisomers. Further, a racemate will be understood to comprise an equimolar mixture of a pair of enantiomers.

It will be understood that the term “prodrug” refers to an inactive form of a compound which is transformed in vivo to the active form. Suitable prodrugs include esters, phosphonate esters etc , of the active form of the compound. Discussion of pro-drugs may be found in [8] to [10]. Further discussion of ursane-like terpenoid bio availability and pro-drugs may be found in [11].

As used herein, a “safe” residue level of an antimicrobial agent is one that poses an insignificant risk of disease, particularly cancer. More specifically, an insignificant risk of cancer is defined as a 1 in 1 million increase in risk.

As used herein, the term “no residue” refers to any residue remaining in the edible tissues of food-producing animals that is so low that it presents an insignificant risk of cancer to consumers. As used herein, the term “substituted” means that the corresponding radical, group or moiety has one or more substituents, or has one or more substituents present. Accordingly, the term "unsubstituted" means that the corresponding radical, group or moiety has no substituents. The term "optionally substituted”, as used herein, means that the corresponding radical, group or moiety is “substituted” or “unsubstituted”.

Where a radical has a plurality of substituents, and a selection of various substituents is specified, the substituents are selected independently of one another and do not need to be identical. When a radical, group or moiety is a substituted group, at least one hydrogen atom on the radical, group of moiety is replaced with a substituent. In the case of an oxo substituent (=0) two hydrogen atoms are replaced. In this regard, substituents may include: alkyl, alkene, alkyne, halogen, nitro, cyano, hydroxy, sulfonic, thiol, ether, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclo, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialkylamino) alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (heterocyclo)alkyl, (cycloalkylamino)alkyl, (C ₁ -C ₄ haloalkoxy)alkyl, (hetero aryl) alkyl, or oxo, heterocycle, -OR ^x , -NR ^X R ^Y , -NR ^x C(=O)R ^y - NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SO _q R ^x and -SO _q NR ^x R ^y , wherein q is 0, 1 or 2, R ^x and R ^y are the same or different and independently selected from hydrogen, alkyl, oxaalkyl, cycloalkyl, cyclooxaalkyl, alkenyl, oxaalkenyl, alkadienyl, oxaalkadienyl, aryl or heterocyclo, and each of said alkyl, oxaalkyl, cycloalkyl, cyclooxaalkyl, alkenyl, oxaalkenyl, alkadienyl, oxaalkadienyl, aryl or heterocyclo substituents may be further substituted with, for example, one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , heterocyclo, -NR ^x R ^y , - NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y ,-C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y .

As used herein, "alkyl" means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon. The alkyl group may contain from 1 to 25 carbon atoms. The term “C ₁ - ₁₀ alkyl” means an alkyl group containing from 1 to 10 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls, “cycloalkyls”, include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term “C _1-10 cycloalkyl” means a cycloalkyl group containing from 1 to 10 carbon atoms. An “alkyl” radical may be substituted or unsubstituted. The term "oxaalkyl" means an alkyl group, as defined above, containing an oxygen atom i.e., the alkyl group contains the species -O-. The term “C _1-10 oxaalkyl” means an alkyl group containing from 1 to 10 carbon atoms and also containing an oxygen atom i.e., the C _1-10 alkyl group contains the species -O-. The term “C _1-10 oxacycloalkyl” means a cycloalkyl group containing from 1 to 10 carbon atoms and also containing an oxygen atom within the ring i.e., the oxacycloalkyl group is a heterocycle that contains the species -O- within the ring. An “oxaalkyl” or “oxacycloalkyl” radical may be substituted or unsubstituted.

The term "alkenyl" means an alkyl, as defined above, containing one double bond between adjacent carbon atoms. As used herein, the alkenyl group may contain from 1 to 25 carbon atoms. The term “C _1-10 alkenyl” means an alkenyl group containing from 1 to 10 carbon atoms, where C ₁ alkenyl denotes a double bond between a methylene substituent and the carbon atom bearing said methylene substituent. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2- butenyl, and the like. Unsaturated cyclic alkenyls, “cycloalkenyls”, include cyclopentenyl and cyclohexenyl, and the like. The term “C _1-10 cycloalkenyl” means a cycloalkenyl group containing from 1 to 10 carbon atoms. An “alkenyl” or “cycloalkenyl” radical may be substituted or unsubstituted.

The term "oxaalkenyl" means an alkenyl group, as defined above, containing an oxygen atom i.e., the alkenyl group contains the species -O-. As used herein, the term “C _1-10 oxaalkenyl” means an alkenyl group, as defined above, containing from 1 to 10 carbon atoms and also containing an oxygen atom i.e., the alkenyl group contains the species -O-. The term “C ₁ - ₁₀ oxacycloalkenyl” means a cycloalkenyl group containing from 1 to 10 carbon atoms and also containing an oxygen atom withn the ring i.e., the cycloalkenyl group is a hetereocycle that contains the species -O- within the ring. An “oxaalkenyl” or “cyclooxaalkenyl” radical may be substituted or unsubstituted.

Further, the term "alkadienyl" means an alkyl group, as defined above, containing two double bonds where an individual double bond is between adjacent carbon atoms. As used herein, the alkadienyl group may contain from 1 to 25 carbon atoms. The term “C _3-10 alkadienyl” means an alkadienyl group containing from 3 to 10 carbon atoms and two double bonds. The term “C ₃ alkadienyl” denotes a group where there is a double bond between a propylene substituent and the carbon atom bearing said propylene substituent. Alkadienyls include cis and/or trans isomers i.e an alkadienyl group may include: cis:cis:, trans:trans; cis:trans, or trans:cis double bonds. An “alkadienyl” radial may be substituted or unsubstituted.

As used herein, the term “cycloalkadienyl” means a cycloalkyl group, as defined above, containing two double bonds where one double bond is between a first pair of adjacent carbon atoms and the other double bond is between a second pair of adjacent carbon atoms as in the structure -C=C-C=C-. The term “cycloalkadienyl”, as used herein, also means a cycloalkyl group, as defined above, wherein one carbon atom has two double bonds with each of its two adjacent carbon atoms as in the structure -C=C=C-C-. A “cycloalkadienyl” radial may be substituted or unsubstituted.

As used herein, the term “oxaalkadienyl” means an alkadienyl group, as defined above, which contains an oxygen atom, i.e., the alkadienyl group contains the species -O-. The term “C ₁ - ₁₀ oxaalkadienyl” means an oxaalkadienyl group containing from 1 to 10 carbon atoms and also containing an oxygen atom, i.e., the oxaalkadienyl group contains the species -O-. An “oxaalkadienyl” radical may be substituted or unsubstituted.

As used herein, the term “cyclooxaalkadienyl” means a cycloalkadienyl group, as defined above, which contains an oxygen atom, i.e., the cycloalkadienyl group contains the species -O-. The term “C _1-10 cyclooxaalkadienyl” means a cyclooxaalkadienyl group containing from 1 to 10 carbon atoms and also containing an oxygen atom, i.e., the cyclooxaalkadienyl group contains the species -O-. A “cyclooxaalkadienyl” radial may be substituted or unsubstituted.

The term "alkynyl", as used herein, means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. The alkynyl group may contain from 2 to 25 carbon atoms. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and thelike. An “alkynyl” radical may be substituted or unsubstituted.

The term "aryl" as used herein refers to a mono- or polycyclic aromatic hydrocarbon systems. The aryl systems may have 3 to 22 carbon atoms, which can be optionally substituted. The term "aryl" also includes systems in which the aromatic cycle is part of a bi- or polycyclic saturated, partially unsaturated and/or aromatic system, such as where the aromatic cycle is fused to an aryl, cycloalkyl, heteroaryl or heterocyclo group as defined herein via any desired and possible ring member of the aryl radical. Bonding can be affected via any possible ring member of the aryl radical. Non-limiting examples of suitable aryl radicals are phenyl, biphenyl, naphthyl, 1- naphthyl, 2-naphthyl, binaphthyl, 1,2, 3, 4-tetrahydro naphthyl, acenaphthyl, anthracenyl, azulenyl, benzfluoryl, benzphenanthryl, chrysyl, indanyl, indenyl, fluoryl, fluorenyl, picenyl and pyrenyl. An “aryl” radical may be substituted or unsubstituted.

As used herein, the term "heteroaryl" refers to an unsaturated aromatic hydrogen radical having at least one heteroatom. The heteroaryl group may have, for example, one, two, three, four, five or six rings, which may be fused or bicyclic. In certain embodiments, "heteroaryl" refers to an aromatic monocyclic ring system containing five members of which at least one member is a N, O or S atom and which optionally contains one, two or three additional N atoms; an aromatic monocyclic ring having six members of which one, two or three members are a N atom, an aromatic bicyclic or fused ring having nine members of which at least one member is a N, O or S atom and which optionally contains one, two or three additional N atoms; or an aromatic bicyclic ring having ten members of which one, two or three members are a N atom. By way of non- limiting example, suitable heteroaryl groups include furanyl, pyridyl, phthalimido, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, pyronyl, pyrazinyl, tetrazolyl, thionaphthyl, benzofuranyl, isobenzofuryl, indolyl, oxyindolyl, isoindolyl, indazolyl, indolinyl, azaindolyl, isoindazolyl, coumarinyl, isocoumarinyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxadinyl, chromenyl, chromanyl, isochromanyl, carbolinyl, thiazolyl, isoxazolyl, isoxazolonyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, benzodioxepinyl and pyridazyl. A “heteroaryl” radical may be substituted or unsubstituted.

As used herein the term, "heterocyclo" refers to a saturated or partially unsaturated ring having at least three members of which at least one member is a heteroatom such as N, O or S and which optionally contains one additional O atom or additional N atom; a saturated or partially unsaturated ring having four members of which at least one member is a N, O or S atom and which optionally contains one additional O atom or one or two additional N atoms; a saturated or partially unsaturated ring having five members of which at least one member is a N, O or S atom and which optionally contains one additional O atom or one, two or three additional N atoms; a saturated or partially unsaturated ring having six members of which one, two or three members are an N, O or S atom and which optionally contains one additional O atom or one, two or three additional N atoms; saturated or partially unsaturated ring having seven members of which one, two or three members are an N, O or S atom and which optionally contains one additional O atom or one, two or three additional N atoms; a saturated or partially unsaturated ring having eight members of which one, two or three members are an N, O or S atom and which optionally contains one additional O atom or one, two or three additional N atoms; a saturated or partially unsaturated bicyclic ring having nine members of which at least one member is a N, O or S atom and which optionally contains one, two or three additional N atoms; or a saturated or partially unsaturated bicyclic ring having ten members of which one, two or three members are an N, O or S atom and which optionally contains one additional O atom or one, two or three additional N atoms. By way of non-limiting example, suitable heterocyclo groups include pyrrolinyl, pyrrolidinyl, dioxolanyl, tetrahydrofuranyl, morpholinyl, imidazolinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, thiopyranyl, tetrahydrothiopyranyl and piperazinyl. A “heterocyclo” radical may be substituted or unsubstituted.

As used herein, the term “halo” refers to a halogen. In particular, the term “halo” refers to any one of fluorine, chlorine, bromine and iodine.

As used herein, the term “heteroatom” refers to an atom other than carbon or hydrogen. Examples of a heteroatom are N, O, S and P and Si. In particular, the term “heteroatom” refers to any one of N, O and S.

As used herein, the term "alkaryl" refers to an aryl group with an alkyl substituent. Binding is through the aryl group. The alkyl and aryl moieties of such a group are as defined herein. Non- limiting examples of alkaryl include tolyl, xylyl, butylphenyl, mesityl, ethyltolyl, methylindanyl, methylnaphthyl, methyltetrahydronaphthyl, ethylnaphthyl, dimethylnaphthyl, propylnaphthyl, butylnaphthyl, methylfluoryl and methylchrysyl. An “alkaryl” group may be substituted or unsubstituted.

As used herein, the term "aralkyl" refers to an alkyl group with an aryl substituent. Binding is through the alkyl group. The aryl and alkyl moieties of such a group are as defined herein. Non- limiting examples of aralkyl include benzyl, methylbenzyl, ethylbenzyl, dimethylbenzyl, diethylbenzyl, methylethylbenzyl, methoxybenzyl, chlorobenzyl, dichlorobenzyl, trichlorobenzyl, phenethyl, phenylpropyl, diphenylpropyl, phenylbutyl, biphenylmethyl, fluorobenzyl, difluorobenzyl, trifluorobenzyl, phenyltolylmethyl, trifluoromethylbenzyl, bis(trifluoromethyl)benzyl, propylbenzyl, tolylmethyl, fluorophenethyl, fluorenylmethyl, methoxyphenethyl, dimethoxybenzyl dichlorophenethyl, phenylethylbenzyl, isopropylbenzyl, diphenylmethyl, propylbenzyl, butylbenzyl, dimethylethylbenzyl, phenylpentyl, tetramethylbenzyl, phenylhexyl, dipropylbenzyl, triethylbenzyl, cyclohexylbenzyl, naphthylmethyl, diphenylethyl, triphenylmethyl and hexamethylbenzyl. An “aralkyl” group may be substituted or unsubstituted.

As used herein, the term "heterocycloalkyl" refers to an alkyl group with a heterocyclo substituent. Binding is through the alkyl group. The heterocyclo and alkyl moieties of such a group are to be understood with regard to the definitions of heterocyclo and alkyl provided herein. By way of non-limiting example, suitable heterocyclolalkyl groups include methyl, ethyl, propyl, butyl, pentyl and hexyl substituted with one or more of the heterocyclo groups including pyrrolidinyl, tetrahydrofuranyl, morpholinyl, piperidinyl and piperazinyl. The heterocycloalkyl may be substituted or unsubstituted.

The term "arylamino" refers to an amine group with an aryl substituent. Binding is through the amine group. Such groups have the number of carbon atoms as indicated. The aryl moiety of such a group may be substituted as defined herein, with regard to the definition of aryl. By way of non-limiting example, suitable arylamino groups include phenylamino, biphenylamino, methylphenylamino, methoxyphenylamino, tolylamino and chlorophenylamino. The arylamino may be substituted or unsubstituted.

As used herein, the term “alkoxy or “alkoxyl” refers to the group alkyl as defined above which contains at least one O atom, where the at least one oxgen atom is at the position where the alkoxy group is attached to the remainder of the organic compound. By way of non-limiting example, suitable alkoxy groups include, for example, methoxy (-O-CH ₃ ), ethoxy (-O-CH ₂ - CH ₃ ), propoxy (-O-CH ₂ -CH ₂ -CH ₃ (straight chain alkyl) or -O-CH-(CH ₃ ) ₂ (branched chain alkyl)) and -O-CH ₂ -CH ₂ -O-CH ₃ . The alkoxy may be substituted or unsubstituted. In this regard, the term “haloalkoxy”, as used herein, refers to “alkoxy” substituted with one or more halo i.e. one or more of F, Cl, Br or I. An example of “haloalkoxy” is -OCF ₃ .

As used herein, the term “aryloxy” refers to the group aryl as defined above which contains at least one O atom, where the at least one oxgen atom is at the position where the aryloxy group is attached to the remainder of the organic compound. By way of non-limiting example, suitable aryloxy groups include, for example, phenoxy, tolyloxy and xylyoxy. The aryloxy may be substituted or unsubstituted.

Other “compound” group definitions will be readily understandable by the skilled person based on the previous definitions of their constituent parts, and the usual conventions of nomenclature. With respect to structural notation it would be understood that refers to either a single bond or a double bond. Thus, the structural motif: refers to aromatic, unsaturated or saturated systems including: which are used interchangeably to denote aromatic systems; or and the like which are used to denote di-unsaturated systems; or and the like which are used to denote mono-unsaturated systems; or which is used to denote a fully saturated system.

For illustration, the structural formula below encompasses: the molecule piperitone , wherein b = 1; R ⁵ is absent and is a double bond; a = 2 and R ⁴ is independently C ₁ alkyl and C ₁ alkyl; and also encompasses the molecule beta-phellandrene wherein b = 0; a = 1; and R ⁴ is independently C ₁ alkenyl and C ₁ alkyl.

In addition, it would be understood that a wavy line when drawn disposed from an asymmetric centre in a molecule refers to the case where the stereochemistry at that asymmetric centre is undefined.

BRIEF DESCRIPTION OF DRAWINGS Figure 1 depicts the molecular structure of berberine quaternary ammonium cation, berberine chloride and berberine hemisulfate.

Figure 2 depicts the molecular structures and names of representative berberine alkaloids of the disclosure including 13 -hydroxyberberine, 13-benzylberberine, 7,8-dihydro-13-methylberberine, berberrubine, fibrauretin (palmatine), palmatine chloride, tetrahydropalmatine, coralyne, coreximine, jatrorrhizine.

Figure 3 depicts a general formula for pentacyclic triterpene skeletons (ring E is present).

Whilst rings A, B and C are six-membered, rings D and E have variable size. For example, rings D and E may be three-, four-, five-, six-, seven- or eight-membered rings. The general formula heads a number of representative pentacyclic triterpene skeletons: ursane, lupane, oleanane, hopane, gammacerane and taraxastane.

Figure 4 depicts a general formula for tetracyclic triterpene skeletons (ring E is absent). Whilst rings A, B and C are six-membered, ring D has variable size. For example, ring D may be three- , four-, five-, six-, seven- or eight- membered. The general formula heads a number of representative tetracyclic triterpene skeletons: dammarane, lanostane and cucurbitane.

Figure 5 depicts the molecular structure of ursane with numbering and a selection of ursane like triterpenoids: neoilexonol, regelin, β-boswellic acid, urmiensolide, alstoprenylene, asiatic acid, corosolic acid and ursolic acid. It would be understood that the depicted ursane structure is optionally unsaturated with an optional double bond between the C12 and C13 positions.

Figure 6 depicts molecular structures of ursolic acid (A: without stereochemistry; B: with stereochemistry) .

Figure 7 depicts the molecular structure of lupane and a selection of lupane type triterpenoids: lupanol, lupeol acetate, 3-oxolupenal, betulonic acid and betulinic acid derivatives and bevirimat.

Figure 8 depicts the molecular structure of oleanane and a selection of oleanane type triterpenoids: oleanolic acid, erythrodiol, β-amyrin, maslinic acid, α-boswellic acid, myricadiol, mupinensisone, miliacin, enoxolone, and lucyin A. It would be understood that the depicted oleanane structure is optionally unsaturated with an optional double bond between the C12 and C13 positions; an optional double bond between the C14 and C15 positions; and/or an optional double bond between the C18 and C19 positions.

Figure 9 depicts the molecular structure of hopane and a selection of hopane type triterpenoids: carandinol, capillirol B, capillirone, and cylindrin. It would be understood that the depicted hopane structure is optionally unsaturated with an optional double bond between the C9 and C11 positions.

Figure 10 depicts the molecular structure of gammacerane and a selection of gammacerane type triterpenoids based on tetrahymanol and tetrahymanone derivatives.

Figure 11 depicts the molecular structure of taraxastane and a representative taraxastane type triterpenenoid (Ilexpublesnin F). It would be understood that the depicted oleanane structure is optionally unsaturated with an optional double bond between the C12 and C13 positions and/or an optional double bond between the C18 and C19 positions.

Figure 12 depicts the molecular structure of dammarane and a selection of dammarane type triterpenenoids: ixorene, azadirahemiacetal, polystanin E, and mauritic acid. It would be understood that the depicted dammarne structure is optionally unsaturated with an optional double bond between the C12 and C13 positions and/or an optional double bond between the C14 and C15 positions.

Figure 13 depicts the molecular structure of lanostane and a selection of lanostane type triterpenenoids: cycloartenol, ganoderol A, eburicol, and suberosol. It would be understood that the depicted oleanane structure is optionally unsaturated with an optional double bond between the C7 and C8 positions; an optional double bond between the C8 and C9 positions; and/or an optional double bond between the C9 and C11 positions.

Figure 14 depicts the molecular structure of curcubitane and a selection of curcubitan type triterpenenoids: curcurbitacin A, B, C, D, E, I, J, K, L, O, P and Q, 11-deoxycucurbitacin I, 23,24-dihydrocucurbitacin B. It would be understood that the depicted curcubitane structure is optionally unsaturated with an optional double bond between the C1 and C2 positions; an optional double bond between the C23 and C24 positions.

Figure 15 depicts the molecular structures and names of representative antimicrobial compounds/preserving agents of the disclosure including matrine, oxymatrine, arecoline, acrecoline hydrobromide, baicalin, baicalein anemonin, andrographolide.

Figure 16 depicts the molecular structures and names of representative compounds of the invention based on a neolignan skeleton including honokiol, isohonokiol, magnolignan, magnolol.

Figure 17 depicts the molecular structures and names of representative compounds of the invention based on a stilbene skeleton including ethylstilbestrol, rhapontin aglycone, astringin aglycone, resveratrol/piceid aglycone and piceid.

Figure 18 depicts the molecular structures and names of representative compounds of the invention based on a diphenylethane skeleton including dienestrol and hexestrol.

Figure 19 depicts the molecular structures of related monoterpenes which are essential oils or components of essential oils. Thymol, p-cymene, m-cymene, o-cymene, carvacrol, limonene, beta-phellandrene, piperitone and terpinolene are listed. Figure 20 depicts calibration curved to quantitate piceid (A); berberine (B); and ursolic acid (C) in Batch 1 of Example 5.

Figure 21 depicts calibration curved to quantitate piceid (A); berberine (B); and ursolic acid (C) in Batch 1 of Example 5.

Figure 22 depicts a process flow chart for manufacture of berberine chloride by liquid extraction of bark from Phellodendron chinense. Raw material is dry bark of Phellodendron chinense which conforms to in-house QC Standards as assessed by an examination step; step i) washing, drying and crushing; step ii) extracting with hot water and ethanol; step iii) concentrating and decolouring by active carbon; step iv) crystallising and dissolving in ethanol to re-crystallise; step v) vacuum desiccator; step vi) mixing; step vii) testing.

Figures 23A and 23B depict a process flow chart for manufacture of piceid by extraction from the dry root of Polygonum cuspidatum.

Figure 23A. Raw material is dry root of Polygonum cuspidatum which conforms to in-house working standards. Step i) washing, drying and crushing; step ii) extracting with ethanol; step iii) evaporating solvent, dissolving in ethanol and filtering; step iv) acidifying with sulphuric acid, evaporating ethanol and filtering; step v) dissolving in water, neutralising with sodium hydroxide and centrifuging; step vi) dissolving in water by stirring, adding enzyme with stirring; step vii) centrifuging and extracting with ethanol and evaporating ethanol; step viii) settling for crystallising and centrifuging; step ix) extracting with ethanol, adding active kieselgur, heating to boiling with stirring, centrifuging and evaporating ethanol; step x) extracting with ethanol, adding active carbon, heating to boiling with stirring, centrifuging and evaporating ethanol.

Figure 23B. Step xi) cooling to room temperature, settling, centrifuging and crystallising; step xii) drying oven; step xiii) pulverising, sieving and packing; step xiv) testing.

Specific embodiments of the disclosure are described below. It will be appreciated that these embodiments are illustrative and not restrictive.

DETAILED DESCRIPTION OF INVENTION

The present disclosure contemplates an animal feed preservative comprising at least one antimicrobial agent. Examples of antimicrobial agents are berberine alkaloids; triterpenoids, such as ursane-like triterpenoids; phenylpropanoids, such as honokiol-like phenylpropanoids, piceid-like phenylpropanoids; and monoterpenoids. Thus, the present disclosure relates to an animal feed preservative comprising one or more antimicrobial agents, wherein the one or more antimicrobial agents are selected from: one or more berberine alkaloids; one or more ursane-like triterpenoids; and/or one or more phenylpropanoids.

The present disclosure also relates to an animal feed preservative comprising one or more antimicrobial agents, wherein the one or more antimicrobial agents are selected from: one or more berberine alkaloids; one or more ursane-like triterpenoids; and/or one or more phenylpropanoids; and wherein the feed preservative is safe.

Berberine alkaloids

Berberine

Berberine is an isoquinoline quaternary alkaloid with antimicrobial activity. It is found present in and can be extracted from many plants: Hydrastis canadensis (Goldenseal), B. vulgaris (barberry), Coptis chinensis (Chinese goldenthread), Rhizoma coptidis, Phellodendri chinensis cortex, and other herbs. Example 6 describes the manufacture of berberine chloride by extraction from the dry bark of Phellodendron chinense as a raw material. This plant, for example, has been widely used in traditional Chinese medicine over hundreds of years.

According to the Chinese Pharmacopoeia, the berberine content of Rhizoma coptidis, Phellodendri chinensis and Phellodendron amurense and Berberidis radix are 5.5%, 3.0%, 0.6% and 0.6% respectively. Rhizoma coptidis (Huanglian in Chinese) belongs to family Ranunculaceae and contains three main Coptis species: Coptis chinensis (Weilian in Chinese), Coptis deltoidea (Yalian in Chinese), and Coptis teeta (Yunlian in Chinese). Rhizoma coptidis is harvested in autumn and sliced after the removing the fibrous roots. Those with bright yellow sections and very bitter taste are considered of good quality. The bitter taste of berberine (and other berberine alkaloids as disclosed herein) makes taste-masking/palatability an important issue to consider when formulating berberine alkaloids for administration to animal subjects.

Berberine (systematic name: 5,6-dihydro-9,10-dimethoxybenzo[g]-1,3-benzodioxolo[5,6- α]quinolizinium; CAS [2086-83-1] is an odourless, yellow crystalline powder and is described in The Merck Index [12A]. Berberine is a quaternary ammonium cation with molecular formula of C ₂₀ H ₁₈ NO ₄ ⁺ and molecular weight of 336.36. The chloride dihydrate salt (molecular formula: C ₂₀ H ₁₈ CINO ₄ .2H ₂ O; molecular weight 407.81; CAS [633-65-8]) is slightly soluble in cold water, but freely soluble in boiling water. It is practically insoluble in cold ethanol. Anhydrous berberine chloride has the molecular formula of C ₂₀ H ₁₈ CINO ₄ ; molecular weight of 371.81. The hemisulfate salt is soluble in about 30 parts water, slightly soluble in ethanol. Figure 1 depicts the molecular structure of the berberine ammonium cation, berberine chloride salt, and berberine hemisulfate salt.

As used herein, the term “berberine alkaloid(s)” refers to berberine and related compounds which share similar structures and characteristics to berberine and are suitable for the feed preservatives/animal feeds/uses of the disclosure.

In one example of the feed preservatives described herein, the one or more berberine alklaoids are one or more protoberberines. In one example, the one or more berberine alkaloids are selected from: berberine, 13-benzylberberine, 13 -hydroxyberberine, 7,8-dihydro-13- methylberberine, berberrubine, fibrauretin (palmatine), tetrahydropalmatine, coralyne, coreximine, jatrorrhizine or an acceptable salt thereof.

The molecular structures of 13-benzylberberine, 13-hydroxyberberine, 7,8-dihydro- 13- methylberberine, berberrubine, fibrauretin (palmatine), palmatine chloride (X = C1), tetrahydropalmatine, coralyne, coreximine and jatrorrhizine are shown in Figure 2.

Fibrauretin (Palmatine)

Fibrauretin or palmatine is an example of a berberine alkaloid. It is a bitter tasting compound extracted from Fibauera recisa Pierre. According to the Chinese Pharmacopoeia, Fibrauera recisa Pierre consists of no less than 2.0% fibrauretin. Another source is Coptidis rhizoma, the rhizome of Coptis chinensis Franch, Coptis deltoidea and Coptis teeta Wall. Coptidiz rhizoma consists of no less than 1.5% fibrauretin. Palmatine chloride is a yellow solid, which is soluble in hot water, sparingly soluble in water, and slightly soluble in ethanol. Its melting point is 196-198 °C. Its molecular formula is C ₂₁ H ₂₂ NO ₄ Cl with a molecular weight of 387.86. The molecular structure of the palmatine quaternary ammonium cation and palmatine chloride (X = C1) are set out in Figure 2. In one example of the feed preservatives described herein, the one or more berberine alkaloids is berberine or an acceptable salt thereof. In one example, the acceptable salt is selected from berberine sulfate or berberine chloride. In one example, the acceptable salt is berberine sulfate. In another example, the acceptable salt is berberine chloride.

Triterpenoid compounds Triterpenoids are hydrocarbon compounds composed of three terpene units where each terpene unit has the molecular formula of C ₁₀ H ₁₆ . Thus, a triterpenoid has a molecular formula C ₃₀ H ₄₈ . Triterpenoids can be found in their free form (sapogenins or aglycones) or bound to glycosides (saponins). Triterpenoids are often bioactive and as they are naturally occurring possess desirable pharmacological properties such as low toxicity and safety. Triterpenoids include tetracyclic and pentacyclic terpenoids which are based on the general formula: shown in Figure 3 (and Figure 4). It would be understood that triterpenoids encompass compounds that are derived from the above carbon skeleton whether that be through, where permitted, substitution through the carbon atoms of rings A, B, C, D and E or functionalisation, redox manipulation, i.e. oxidation or reduction, unsaturation and/or ring incorporation of the rings A, B, C, D or E or any ring substituent. With reference to the general formula a tetracyclic triterpene is a compound where ring D is a cycloalkyl ring and where ring E is absent. A pentacyclic triterpene is a compound where ring D is a cycloalkyl ring and ring E is present as a cycloalkyl ring. Tetracyclic and pentacyclic triterpenoids include many subgroups which are defined on the basis of their carbon skeleton.

For example, whilst ursanes, lupanes, oleananes, hopanes, gammaceranes and taraxastanes are pentacyclic triterpenoids, dammaranes, lanostanes and cucurbitanes are tetracyclic triterpenoids. These pentacyclic and tetracyclic subgroups are shown in Figure 3 and Figure 4.

The subgroups are structurally related and share similar characteristics and similar bio activities. Thus, pentacyclic triterpenoids based on gammaceranes, hopanes, lupanes, oleananes skeletons can share similar characteristics and similar bioactivities to ursane pentacyclic triterpenoids as can tetracyclic triterpenoids based on dammarane, lanostanes and cucurbitanes. As used herein, the term “ursane like triterpenoid” includes ursane, lupane, oleanane, hopane, gammacerane, taraxastane, dammarane, lanostane and cucurbitane based triterpenoids which share similar characteristics and similar bio activities.

In the feed preservatives described herein, the one or more ursane-like triterpenoids are selected from: ursolic acid neoilexonol, regelin, β-boswellic acid, urmiensolide, alstoprenylene, asiatic acid, corosolic acid, uvaol, rotundic acid, lupanol, lupeol acetate, 3-oxolupenal, betulonic acid, betulinic acid, bevirimat, oleanolic acid, erythrodiol, β-amyrin, maslinic acid, α-boswellic acid, myricadiol, mupinensisone, miliacin, enoxolone, lucyin A, echinocystic acid, sumaresinolic acid, gypsogenic acid, imberic acid, carandinol, capillirol B, capillirone, cylindrin, tetrahymanol and tetrahymanone triterpenoids, Ilexpublesnin F, ixorene, azadirahemiacetal, polystanin E, mauritic acid, cycloartenol, ganoderol A, eburicol, suberosol, curcurbitacin A, B, C, D, E, I, J, K, L, O, P and Q, 11-deoxycucurbitacin I, 23,24-dihydrocucurbitacin B, colocynthin, an acceptable salt thereof and any combination thereof.

The present disclosure relates to ursane-like triterpenoids according to the general formula (I):

wherein:

E is absent or present, and when present E is C _3-8 cycloalkyl and y takes an integer value from the group consisting of: 2, 4, 6, 8, 10, 12 and 14 or E is C _3-8 cycloalkenyl and y takes y takes an integer value from the group consisting of: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12;

D is C _3-8 cycloalkyl or C _3-8 cycloalkenyl and when E is absent, x takes an integer value from the group consisting of: 2, 4, 6, 8, 10 and 12; and when E is present, x is 0 or takes an integer value selected from the group consisting of: 2, 4, 6, 8 and 10; are each independently absent or selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclo, halogen, nitro, cyano, hydroxy, sulfonic, thiol, ether, amino, haloalkyl, hydroxyalkyl, heteroaryloxy, aralkyloxy, alkylthio, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialky lamino) alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (heterocyclo)alkyl, (cycloalkylamino)alkyl, (C ₁ - C ₄ haloalkoxy)alkyl, (hetero aryl) alkyl, oxo, -OR ^x , -NR ^x R ^Y , -NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y , - C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SO _q R ^x and -SO _q NR ^x R ^y , wherein q is 0, 1 or 2, R ^x and R ^y are each independently selected from hydrogen, alkyl, oxaalkyl, alkenyl, oxaalkenyl, alkadienyl, oxaalkadienyl, aryl, heteroaryl or heterocyclo, and each of said alkyl, oxaalkyl, alkenyl, oxaalkenyl, alkadienyl, oxaalkadienyl, aryl, heteroaryl or heterocyclo substituents is optionally further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , heterocyclo, - NR ^x R ^y , -NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y ,-C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and - SONR ^x R ^y . Preferably, each of said alkyl, oxaalkyl, alkenyl, oxaalkenyl, alkadienyl, oxaalkadienyl, aryl, heteroaryl or heterocyclo substituents is further substituted with one or more of oxo, halogen, - OH, -CN, alkyl, -OR ^x , heterocyclo, -NR ^x R ^y , -NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y ,-C(=O)R ^x , -C(=O)OR ^x , - C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y .

Preferably, are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclo, halogen, nitro, cyano, hydroxy, sulfonic, thiol, ether, amino, haloalkyl, hydroxyalkyl, heteroaryloxy, aralkyloxy, alkylthio, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialky lamino) alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (heterocyclo)alkyl, (cycloalkylamino)alkyl, (C ₁ - C4haloalkoxy)alkyl, (hetero aryl) alkyl, oxo, -OR ^x , -NR ^x R ^y , -NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SO _q R ^x and -SO _q NR ^x R ^y , wherein q is 0, 1 or 2, R ^x and R ^y are each independently selected from hydrogen, alkyl, oxaalkyl, alkenyl, alkadienyl, aryl or heterocyclo, and each of said alkyl, oxaalkyl, alkenyl, alkadienyl, aryl, heteroaryl or heterocyclo substituents is optionally further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , heterocyclo, -NR ^x R ^y , -NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y ,-C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y .

Preferably, each of said alkyl, oxaalkyl, alkenyl, alkadienyl, aryl, heteroaryl or heterocyclo substituents is further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , heterocyclo, -NR ^x R ^y , -NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y ,-C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y .

Preferably, are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclo, halogen, nitro, cyano, hydroxy, sulfonic, thiol, ether, amino, haloalkyl, hydroxyalkyl, aralkyloxy, alkylthio, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialky lamino) alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (heterocyclo)alkyl, (cycloalkylamino)alkyl, (C ₁ -C ₄ haloalkoxy)alkyl, oxo, -OR ^x , -

NR ^x R ^y , -NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SO _q R ^x and - SO _q NR ^x R ^y , wherein q is 0, 1 or 2, R ^x and R ^y are each independently selected from hydrogen, alkyl, oxaalkyl, alkenyl, alkadienyl, aryl, heteroaryl or heterocyclo, and each of said alkyl, oxaalkyl, alkenyl, alkadienyl, aryl, heteroaryl or heterocyclo substituents is optionally further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , heterocyclo, -NR ^x R ^y , - NR ^x C(=O)R ^y , -NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y . Preferably, each of said alkyl, oxaalkyl, alkenyl, alkadienyl, aryl, heteroaryl or heterocyclo substituents is further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , heterocyclo, -NR ^x R ^y , -NR ^x C(=O)R ^y , -NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y .

Preferably, are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclo, halogen, nitro, cyano, hydroxy, sulfonic, thiol, ether, amino, haloalkyl, hydroxyalkyl, aralkyloxy, alkylthio, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialkylamino)alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (cycloalkylamino)alkyl, (C ₁ -C ₄ haloalkoxy)alkyl, oxo, -OR ^x , -NR ^x R ^y , - NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SO _q R ^x and -SO _q NR ^x R ^y , wherein q is 0, 1 or 2, R ^x and R ^y are each independently selected from hydrogen, alkyl, oxaalkyl, alkenyl, alkadienyl, aryl, heteroaryl or heterocyclo, and each of said alkyl, oxaalkyl, alkenyl, alkadienyl, aryl, heteroaryl or heterocyclo substituents is optionally further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , -heterocyclo, NR ^x R ^y , -NR ^x C(=O)R ^y - NR ^x SO ₂ R ^y ,-C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y .

Preferably, each of said alkyl, oxaalkyl, alkenyl, alkadienyl, aryl, heteroaryl or heterocyclo substituents is further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , heterocyclo, -NR ^x R ^y , -NR ^x C(=O)R ^y , -NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y .

Preferably, are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclo, halogen, nitro, cyano, hydroxy, sulfonic, thiol, ether, amino, haloalkyl, hydroxyalkyl, alkylthio, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialkylamino) alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (cycloalkylamino)alkyl, (C ₁ -C ₄ haloalkoxy)alkyl, oxo, -OR ^x , -NR ^x R ^y , -NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SO _q R ^x and -SO _q NR ^x R ^y , wherein q is 0, 1 or 2, R ^x and R ^y are each independently selected from hydrogen, alkyl, oxaalkyl, alkenyl, alkadienyl, aryl, heteroaryl, heterocyclo and each of said alkyl, oxaalkyl, alkenyl, alkadienyl, aryl, heteroaryl or heterocyclo substituents is optionally further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , heterocyclo, -NR ^x R ^y , -NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y ,-C(=O)R ^x , -C(=O)OR ^x , - C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y . Preferably, each of said alkyl, oxaalkyl, alkenyl, alkadienyl, aryl, heteroaryl or heterocyclo substituents is further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , heterocyclo, -NR ^x R ^y , -NR ^x C(=O)R ^y , -NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y .

Preferably, are each independently selected from hydrogen, alkyl, aryl, heteroaryl, heterocyclo, halogen, nitro, cyano, hydroxy, sulfonic, thiol, ether, amino, haloalkyl, hydroxyalkyl, alkylthio, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialkylamino)alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (cycloalkylamino)alkyl, (C ₁ -C ₄ haloalkoxy)alkyl, oxo, -OR ^x , -NR ^x R ^y , -NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SO _q R ^x and -SO _q NR ^x R ^y , wherein q is 0, 1 or 2, R ^x and R ^y are each independently selected from hydrogen, alkyl, oxaalkyl, aryl, heteroaryl or heterocyclo, and each of said alkyl, oxaalkyl, aryl, heteroaryl or heterocyclo substituents is optionally further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , heterocyclo, -NR ^x R ^y , - NR ^x C(=O)R ^y , -NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y .

Preferably, each of said alkyl, oxaalkyl, aryl, heteroaryl or heterocyclo substituents is further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , heterocyclo, -NR ^x R ^y , - NR ^x C(=O)R ^y , -NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y .

Preferably, are each independently selected from hydrogen, alkyl, aryl, heteroaryl, halogen, nitro, cyano, hydroxy, sulfonic, thiol, ether, amino, haloalkyl, hydroxyalkyl, alkylthio, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialkylamino)alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (cycloalkylamino)alkyl, (C ₁ -C ₄ haloalkoxy)alkyl, oxo, -OR ^x , -NR ^x R ^y , -NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SO _q R ^x and -SO _q NR ^x R ^y , wherein q is 0, 1 or 2, R ^x and R ^y are each independently selected from hydrogen, alkyl, oxaalkyl, aryl or heteroaryl, and each of said alkyl, oxaalkyl, aryl or heteroaryl substituents is further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , -NR ^x R ^y , -NR ^x C(=O)R ^y , -NR ^x SO ₂ R ^y ,-C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y .

Preferably, each of said alkyl, oxaalkyl, aryl or heteroaryl substituents is further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , -NR ^x R ^y , -NR ^x C(=O)R ^y , -NR ^x SO ₂ R ^y , - C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y . Preferably, are each independently selected from hydrogen, alkyl, aryl, halogen, nitro, cyano, hydroxy, sulfonic, thiol, ether, amino, haloalkyl, hydroxyalkyl, alkylthio, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl,

(dialkylamino)alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (cycloalkylamino)alkyl, (C ₁ -C ₄ haloalkoxy)alkyl, oxo, -OR ^x , -NR ^x R ^y , -NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SO _q R ^x and -SO _q NR ^x R ^y , wherein q is 0, 1 or 2, R ^x and R ^y are each independently selected from hydrogen, alkyl, oxaalkyl or aryl, and each of said alkyl, oxaalkyl or aryl substituents is further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , - NR ^x R ^y , -NR ^x C(=O)R ^y , -NR ^x SO ₂ R ^y ,-C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and - SONR ^x R ^y .

Preferably, each of said alkyl, oxaalkyl or aryl substituents is further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , -NR ^x R ^y , -NR ^x C(=O)R ^y , -NR ^x SO ₂ R ^y , -C(=O)R ^x , - C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y .

Preferably, are each independently selected from hydrogen, alkyl, halogen, nitro, cyano, hydroxy, sulfonic, thiol, ether, amino, haloalkyl, hydroxyalkyl, alkylthio, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialkylamino) alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (cycloalkylamino)alkyl, (C ₁ - C ₄ halo alkoxy ) alkyl, oxo, -OR ^x , -NR ^x R ^y , -NR ^x C(=O)R ^y -NR ^x SO ₂ R ^y , -C(=O)R ^x , -C(=O)OR ^x , - C(=O)NR ^x R ^y , -SO _q R ^x and -SO _q NR ^x R ^y , wherein q is 0, 1 or 2, R ^x and R ^y are each independently selected from hydrogen, alkyl, oxaalkyl, and each of said alkyl or oxaalkyl substituents is optionally further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , -NR ^x R ^y , -NR ^x C(=O)R ^y , -NR ^x SO ₂ R ^y ,-C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y .

Preferably, each of said alkyl or oxaalkyl substituents is further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , -NR ^x R ^y , -NR ^x C(=O)R ^y , -NR ^x SO ₂ R ^y , -C(=O)R ^x , - C(=O)OR ^x , -C(=O)NR ^x R ^y , -SOR ^x and -SONR ^x R ^y .

In one or more embodiments, E is present. In one or more embodiments, E is present and D is C ₆ cycloalkyl. In one or more embodiments, E is C ₅ cycloalkyl. In one or more embodiments, E is C ₆ cycloalkyl.

In one example, the ursane-like triterpenoid is an ursane triterpenoid. In one example, the ursane-like triterpenoid is preferably selected from the group consisting of: neoilexonol, regelin, β-boswellic acid, urmiensolide, alstoprenylene, asiatic acid, corosolic acid and ursolic acid.

In one example, the ursane-like triterpenoid is a lupane triterpenoid. In one example, the lupane triterpenoid is selected from the group consisting of: lupanol, lupeol acetate, 3-oxolupenal, betulonic acid, betulinic acid and bevirimat.

In one example, the ursane-like triterpenoid is an oleanane triterpenoid. In one example, the oleanane triterpenoid is selected from the group consisting of: oleanolic acid, erythrodiol, β- amyrin, maslinic acid, α-boswellic acid myricadiol, mupinensisone, miliacin, enoxolone, and lucyin A.

In one example, the ursane-like triterpenoid is a hopane triterpenoid. In one example, the hopane triterpenoid is selected from the group consisting of: carandinol, capillirol B, capillirone, and cylindrin.

In one example, the ursane-like triterpenoid is a gammacerane triterpenoid. In one example, the gammacerane triterpenoid is selected from tetrahymanol and tetrahymanone triterpenoids.

In one example, the ursane-like triterpenoid is a taraxastane triterpenoid. In one example, the taraxastane triterpenoid is Ilexpublesnin F.

In one embodiment, E is absent. In one example, the ursane-like triterpenoid is a dammarane triterpenoid. In one example, the dammarane triterpenoid is selected from the group consisting of: ixorene, azadirahemiacetal, polystanin E, and mauritic acid.

In one example, the ursane-like triterpenoid is a lanostane triterpenoid. In one example, the lanostane triterpenoid is selected from the group consisting of: cycloartenol, ganoderol A, eburicol, and suberosol.

Ursolic acid

Ursolic acid is a pentacyclic triterpenoid widely found in the peels of fruits, (such as apples, bilberries, cranberries and prunes), as well as in herbs (such as rosemary and thyme) and spices. Molecular structures for ursolic acid are shown in Figure 6. Formula A shows the structure of ursolic acid with no stereochemistry defined. Formula B shows the structure of naturally occurring ursolic acid with stereochemistry defined as shown in The Merck Index [12B]. The Chemical Abstracts Registry Number for naturally occurring ursolic acid as defined by Formula B is [77-52-1]. Ursolic acid is also referred to (3β)-3-Hydroxyursen-12-en-28-oic acid; urson; prunol; micromerol; and malol. The molecular formula is C ₃₀ H ₄₈ O ₃ and molecular weight is 456.70. In one example, the ursane-like triterpenoid is ursolic acid. In one example, the ursane-like triterpenoid is an acceptable salt of ursolic acid. In one example, the acceptable salt is selected from potassium ursolate or sodium ursolate. In one example, the acceptable salt is potassium ursolate. In one example, the acceptable salt is sodium ursolate.

Phenylpropanoids Phenylpropanoids are aromatic type compounds comprising a C ₆ C ₃ , i.e., phenylpropane/n- propylbenzene unit. Several phenylpropanoid skeletons can be constructed from the phenylpropane unit. Tables 1 to 8 depict phenylpropanoid skeletons with a selection of phenylpropanoid compounds.

As defined above, the present disclosure is particularly directed to “honokiol-like” or “piceid- like” phenylpropanoid compounds. As used herein, the terms: “honokiol-like phenylpropanoids”, “honokiol-like phenylpropanoid compounds”, “honokiol-like compounds”; and “piceid-like phenylpropanoids”, “piceid-like phenylpropanoid compounds”, “piceid-like compounds” and the like refer to compounds that share similar characteristics and similar bioactivities to honokiol and piceid respectively and are suitable for the animal feed preservatives/animal feeds/uses of the invention.

Honokiol-like phenylpropanoids

Honokiol-like compounds have a neolignan skeleton (as represented in Table 2). In one example of the feed preservatives described herein, the one or more phenylpropanoids is one or more honokiol-like phenylpropanoids. In one example, the one or more honokiol-like phenylpropanoids is selected from: honokiol, isohonokiol, dehydrodieugenol, diferulic acid, magnolignan, magnolol, randainol, an acceptable salt thereof and any combination thereof. In one example, the one or more honokiol-like phenylpropanoids is honokiol or an acceptable salt thereof.

Honokiol Honokiol is a phenylpropanoid compound with a neolignan skeleton ( Table 2). It is widely found in the plant kingdom, specifically in the bark of magnolia plants. Its molecular formula is C ₁₈ H ₁₈ O ₂ and molecular weight is 266.34. The molecular structure for honokiol and other phenylpropanoids with a neolignan skeleton are shown in Figure 15. As described above, honokiol-like phenylpropanoid compounds have a neolignan skeleton.

Thus, one example of the phenylpropanoid compounds presently disclosed is a compound according to Formula (II): wherein: x and y are each independently selected from the group consisting of 0, 1, 2, 3, 4, and 5 such that the sum of x and y is not greater than 5;

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, C _1-10 alkenyl, C _2-10 oxaalkenyl, C _3-10 cycloalkenyl, C _2-10 oxacycloalkenyl, C _3-10 alkadienyl, C _{3- 10} oxaalkadienyl, C _4-10 cycloalkadienyl, C _4-10 oxacycloalkadienyl, (O)Ph, C(O)CH ₂ Ph, C(O)C ₁ - 1 ₀ alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl, C(O)C _2-10 oxacycloalkyl, C(O)C _2-10 alkenyl, C(O)C _2-10 oxaalkenyl, C(O)C _3-10 cycloalkenyl, C(O)C _3-10 oxacycloalkenyl, C(O)C _4-10 alkadienyl, C(O)C _4-10 oxaalkadienyl, C(O)C _4-10 cycloalkadienyl, C(O)C _4-10 oxacycloalkadienyl;

Preferably: R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, C _1-10 alkenyl, C _2-10 oxaalkenyl, C _3-10 cycloalkenyl, C _2-10 oxacycloalkenyl, C _3-10 alkadienyl, C _{3- 10} oxaalkadienyl, (O)Ph, C(O)CH ₂ Ph, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl, C(O)C _2-10 oxacycloalkyl, C(O)C _2-10 alkenyl, C(O)C _2-10 oxaalkenyl, C(O)C _3-10 cycloalkenyl, C(O)C _3-10 oxacycloalkenyl, C(O)C _4-10 alkadienyl, C(O)C _4-10 oxaalkadienyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, C _1-10 alkenyl, C _2-10 oxaalkenyl, C _3-10 cycloalkenyl, C _2-10 oxacycloalkenyl, (O)Ph, C(O)CH ₂ Ph, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl, C(O)C _2-10 oxacycloalkyl, C(O)C _{2- 10} alkenyl, C(O)C _2-10 oxaalkenyl, C(O)C _3-10 cycloalkenyl, C(O)C _3-10 oxacycloalkenyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, C _1-10 alkenyl, C _2-10 oxaalkenyl, (O)Ph, C(O)CH ₂ Ph, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl, C(O)C ₃ - ₁₀ cycloalkyl, C(O)C _2-10 oxacycloalkyl, C(O)C _2-10 alkenyl, C(O)C _2-10 oxaalkenyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, (O)Ph, C(O)CH ₂ Ph, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl, C(O)C _{2- 10} oxacycloalkyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, C(O)C _{1- 10} alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl, C(O)C _2-10 oxacycloalkyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C(O)C _1-10 alkyl, C(O)C _{1- 10} oxaalkyl, C(O)C _3-10 cycloalkyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl;

Preferably, where R ¹ is C _1-10 alkyl, C _1-10 oxaalkyl, C(O)C _1-10 alkyl or C(O)C _1-10 oxaalkyl, R ¹ forms a ring structure with the 6-membered ring A or the 6-membered ring B. Preferably, the ring structure is derivatised. Preferably:

R ¹ is independently H, C _1-10 alkyl, C(O)C _1-10 alkyl;

Preferably, where R ¹ is C _1-10 alkyl or C(O)C _1-10 alkyl, R ¹ forms a ring structure with the 6- membered ring A or the 6-membered ring B. Preferably, the ring structure is derivatised. Piceid-like phenylpropanoids

Piceid-hke compunds have stilbenoid skeletons (as represented in Table 5 and Table 6) .In one example of the feed preservatives described herein, the one or more phenylpropanoids is one or more piceid-like phenylpropanoids. In one example, the one or more piceid-hke phenylpropanoids is selected from: piceid, ethylstilbestrol, rhapontin, astringin, resveratrol, lysidiside A, hexestrol, dienestrol, chlorophorin, 3-hydroxy-5-methoxy-6-prenylstilbene-2- carboxylic acid, isorhapontin, piceatannol, pinosylvin, pinosylvin methyl ether, 4- prenylresveratrol, pterostilbene, oxyresveratrol, an acceptable salt thereof and amy combination thereof. In one example, the one or more piceid-like phenylpropanoids is piceid.

Piceid

Piceid or polydatin (systematic name 2-[3-hydroxy-5-[(E)-2-(4- hydroxyphenyl)ethenyl]phenoxy]-6-(hydroxymethyl)oxane-3,4,5- triol); CAS [65914-17-2], is a stilbenoid glucoside ( Table 6) with molecular formula of C ₂₀ H ₂₂ O ₈ and molecular weight of 390.388. It is an off white powder and is described in The Merck Index [12C] and is a major resveratrol derivative in grape juices. It can be found in the bark of Picea sitchensis. It can also be isolated from Fallopia japonica, the Japanese knotweed (syn. Polygonum cuspidatum ). Example 7 describes the manufacture of piceid from Polygonum cuspidatum. The molecular structure for piceid and other stilbenoid phenylpropanoids are shown in Figure 17.

As described above, piceid-like phenylpropanoid compounds have a stilbenoid skeleton. Thus, one example of the phenylpropanoid compounds presently disclosed is a compound according to Formula (III):

wherein: x and y are each independently selected from the group consisting of 0, 1, 2, 3, 4, and 5 such that the sum of x and y is not greater than 5;

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, C _1-10 alkenyl, C _2-10 oxaalkenyl, C _3-10 cycloalkenyl, C _2-10 oxacycloalkenyl, C _3-10 alkadienyl, C _{3- 10} oxaalkadienyl, C4 _-10 cycloalkadienyl, C4 _-10 oxacycloalkadienyl, (O)Ph, C(O)CH ₂ Ph, C(O)C ₁ - 1 ₀ alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl, C(O)C _2-10 oxacycloalkyl, C(O)C _2-10 alkenyl, C(O)C _2-10 oxaalkenyl, C(O)C _3-10 cycloalkenyl, C(O)C _3-10 oxacycloalkenyl, C(O)C _4-10 alkadienyl, C(O)C _4-10 oxaalkadienyl, C(O)C _4-10 cycloalkadienyl, C(O)C _4-10 oxacycloalkadienyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, C _1-10 alkenyl, C _2-10 oxaalkenyl, C _3-10 cycloalkenyl, C _2-10 oxacycloalkenyl, C _3-10 alkadienyl, C _{3- 10} oxaalkadienyl, (O)Ph, C(O)CH ₂ Ph, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl, C(O)C _2-10 oxacycloalkyl, C(O)C _2-10 alkenyl, C(O)C _2-10 oxaalkenyl, C(O)C _3-10 cycloalkenyl, C(O)C _3-10 oxacycloalkenyl, C(O)C _4-10 alkadienyl, C(O)C _4-10 oxaalkadienyl.

Preferably: R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, C _1-10 alkenyl, C _2-10 oxaalkenyl, C _3-10 cycloalkenyl, C _2-10 oxacycloalkenyl, (O)Ph, C(O)CH ₂ Ph, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl, C(O)C _2-10 alkenyl, C(O)C _2-10 oxaalkenyl, C(O)C _3-10 cycloalkenyl, C(O)C _3-10 oxacycloalkenyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, C _1-10 alkenyl, C _2-10 oxaalkenyl, (O)Ph, C(O)CH ₂ Ph, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl, C(O)C ₃ - ₁₀ cycloalkyl, C(O)C _2-10 alkenyl, C(O)C _2-10 oxaalkenyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, (O)Ph, C(O)CH ₂ Ph, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, C(O)C ₁ - ₁₀ alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C(O)C _1-10 alkyl, C(O)C ₁ - ₁₀ oxaalkyl, C(O)C _3-10 cycloalkyl.

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl.

Preferably, where R ¹ is C _1-10 alkyl, C _1-10 oxaalkyl, C(O)C _1-10 alkyl or C(O)C _1-10 oxaalkyl, R ¹ forms a ring structure with the 6-membered ring A or the 6-membered ring B. Preferably, the ring structure is derivatised.

Preferably:

R ¹ is independently H, C _1-10 alkyl, C(O)C _1-10 alkyl.

Preferably, where R ¹ is C _1-10 alkyl or C(O)C _1-10 alkyl, R ¹ forms a ring structure with the 6- membered ring A or the 6-membered ring B. Preferably, the ring structure is derivatised. Preferably, the phenylpropanoid compound is a compound according to Formula (IV): wherein: x and y are each independently selected from the group consisting of 0, 1, 2, 3, 4, and 5 such that the sum of x and y is not greater than 5; m = 1 or 2;

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, C _1-10 alkenyl, C _2-10 oxaalkenyl, C _3-10 cycloalkenyl, C _2-10 oxacycloalkenyl, C _3-10 alkadienyl, C _{3- 10} oxaalkadienyl, C _4-10 cycloalkadienyl, C _4-10 oxacycloalkadienyl, (O)Ph, C(O)CH ₂ Ph, C(O)C ₁ - ₁₀ alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl, C(O)C _2-10 alkenyl, C(O)C _2-10 oxaalkenyl, C(O)C _3-10 cycloalkenyl, C(O)C _3-10 oxacycloalkenyl, C(O)C _4-10 alkadienyl, C(O)C ₄ - ₁₀ oxaalkadienyl, C(O)C _4-10 cycloalkadienyl, C(O)C _4-10 oxacycloalkadienyl;

Preferably: R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, C _1-10 alkenyl, C _2-10 oxaalkenyl, C _3-10 cycloalkenyl, C _2-10 oxacycloalkenyl, C _3-10 alkadienyl, C _{3- 10} oxaalkadienyl, (O)Ph, C(O)CH ₂ Ph, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl, C(O)C _2-10 oxacycloalkyl, C(O)C _2-10 alkenyl, C(O)C _2-10 oxaalkenyl, C(O)C _3-10 cycloalkenyl, C(O)C _3-10 oxacycloalkenyl, C(O)C _4-10 alkadienyl, C(O)C _4-10 oxaalkadienyl; Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, C _1-10 alkenyl, C _2-10 oxaalkenyl, C _3-10 cycloalkenyl, C _2-10 oxacycloalkenyl, (O)Ph, C(O)CH ₂ Ph, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl, C(O)C _2-10 oxacycloalkyl, C(O)C ₂ - ₁₀ alkenyl, C(O)C _2-10 oxaalkenyl, C(O)C _3-10 cycloalkenyl, C(O)C _3-10 oxacycloalkenyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, C _1-10 alkenyl, C _2-10 oxaalkenyl, (O)Ph, C(O)CH ₂ Ph, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl, C(O)C ₃ - ₁₀ cycloalkyl, C(O)C _2-10 oxacycloalkyl, C(O)C _2-10 alkenyl, C(O)C _2-10 oxaalkenyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, Ph, CH ₂ Ph, (O)Ph, C(O)CH ₂ Ph, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl, C(O)C ₂ - ₁₀ oxacycloalkyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C _2-10 oxacycloalkyl, C(O)C ₁ - ₁₀ alkyl, C(O)C _1-10 oxaalkyl, C(O)C _3-10 cycloalkyl, C(O)C _2-10 oxacycloalkyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C _3-10 cycloalkyl, C(O)C _1-10 alkyl, C(O)C ₁ - ₁₀ oxaalkyl, C(O)C _3-10 cycloalkyl;

Preferably:

R ¹ is independently H, C _1-10 alkyl, C _1-10 oxaalkyl, C(O)C _1-10 alkyl, C(O)C _1-10 oxaalkyl;

Preferably, where R ¹ is C _1-10 alkyl, C _1-10 oxaalkyl, C(O)C _1-10 alkyl or C(O)C _1-10 oxaalkyl, R ¹ forms a ring structure with the 6-membered ring A or the 6-membered ring B. Preferably, the ring structure is derivatised.

Preferably:

R ¹ is independently H, C _1-10 alkyl, C(O)C _1-10 alkyl; Preferably, where R ¹ is C _1-10 alkyl or C(O)C _1-10 alkyl, R ¹ forms a ring structure with the 6- membered ring A or the 6-membered ring B. Preferably, the ring structure is derivatised.

Formulations

In one or more examples of the feed preservatives described herein, the feed preservative further comprises one or more preserving agents.

In one example, the one or more preserving agents are one or more antimicrobial compounds selected from arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine, and andrographolide. In one example, the one or more preserving agents is baicalin.

It will be understood that references to an antimicrobial compound herein (for example a berberine alkaloid, ursane-like triterpenid, phenylpropanoid, arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine, and andrographolide), encompass, where permitted, all isomeric forms, racemates, amorphous or crystalline forms, solvates, acceptable salts, solvates of said salts, and prodrugs thereof.

Arecoline

Arecoline is a major alkaloid constituent extracted from the traditional Chinese medicine Semen arecae, semen of Areca catechu L. According to Chinese Pharmacopoeia the content of Arecoline in S Semen arecae is not less than 0.2%. Semen arecae is manufactured primarily in Hainan province China, however Areca catechu L. is produced in southern Yunnan, Fujian, Guangxi, and southern Taiwan and other areas.

Arecoline is an odourless oily, colourless liquid with a boiling point of 209 °C. It produces a solution when mixed with water and ethanol. Its molecular formula is C ₈ H ₁₃ NO ₂ , molecular weight is 155.19. Its hydrobromide salt (C ₈ H ₁₄ BrNO ₂ , MW = 236.11) has a melting point of 170-175 °C and is diffluent in both water and ethanol. The molecular structures of arecoline and arecoline hydrobromide are shown in Figure 18.

Baicalin and baicalein

Baicalin is a major flavonoid constituent found in the traditional Chinese medicine, Scutellaria Root, the root of Scutellaria baicalensis Georgi. According to Chinese Pharmacopoeia the content of Baicalin found in Scutellaria Root is 9.0%. Scutellaria Root is manufactured mainly in Northeast China; Hebei, Shanxi, Henan, Shanxi, Neimeng province et al., although Scutellaria Root can be grown in most provinces of northern China.

Baicalin is a pale yellow powder, and is bitter in taste. It is diffluent inN,N-dimethylformamide, and soluble in alkaline solution, such as sodium bicarbonate, sodium carbonate, sodium hydroxide (however baicalin is unstable in alkaline environment). It is almost insoluble in water. Its molecular formula is C ₂₁ H ₁₈ O ₁₁ , molecular weight is 446.36. The molecular structure of baicalin and its aglycone, baicalein, are shown in Figure 18.

Anemonin

Amemonin is the dry root of the Ranunculaceae plant Clematis chinensis osbeck, Clematis ssp Hexapetala Pall and Clematis manshurica rupr. The dry root and rhizome is called Clematidis radix et rhizome and according to the Chinese Pharmacopoeia contains 4.6% B1 Pulsatilla glycosides. Clematidis radix et rhizoma is produced in Jiangsu, Zhejiang, Jiangxi, Anhui and other provinces, with Clematis Hexapetala production mainly in the northeast and Shandong while Manshurica Rupr lotus production is primarily in the northeast. Other sources include the dry root of Ranunculus japnicus thunb, R Sceleratus L, Anemone hupehensis lem and Pulsatilla chinesis.

Anemonin is a white powder of bitter taste. Its melting point is around 158 °C and it is slightly soluble in cold water, soluble in hot water, and soluble in hot ethanol. Its molecular formula is C ₁₀ H ₈ O ₄ with a molecular weight of 192.16. The molecular structure of anemonin is depicted in

Figure 18.

Matrine and oxymatrine

Matrine and its N-oxide derivative, oxymatrine, are alkaloids extracted from the legumes ( Fabaeceae ) and the dry root and fruit of the plant Sophora (Sophora flavescens var.

Flavescens). According to the Chinese pharmacopoeia 2010, no less than 1.2% of Matrine is extracted from Sophora.

Matrine is a white odourless powder of bitter taste. The molecular formula is C15H25N2), with a molecular weight of 249. Similarly, oxymatrine is a white powder of bitter taste. The molecular formula is C ₁₅ H ₂₄ N ₂ O ₂ , with a molecular weight of 264. The molecular structures of matrine and oxymatrine are set out in Figure 18.

Andrographolide Andrographolide is a labdane diterpenoid that is produced by the Andrographis paniculata plant. According to the Chinese Pharmacopoeia, Andrographis paniculata consists of no less than 0.8% of the active substance.

Andrographolide is a colourless, powder with a crystalline appearance and is bitter in taste. It is soluble in boiling ethanol, poorly soluble in ethanol at ambient temperature, and almost insoluble in water. It has a melting point of 224-230°C and decomposes upon melting. Its molecular formula is C ₂₀ H ₃₀ O ₅ , and has a molecular weight of 350.44. The molecular structure for andrographolide is shown in Figure 18.

In one example, the one or more preserving agents are one or more essential oils or components thereof. In one example, the one or more essential oils or components thereof is one or more monoterpenoids. In one example, the one or more monoterpenoids is selected from the group consisting of: thymol, curcudiol, curcuphenol, m-cymene, o-cymene, p-cymene, isomenthone, isomenthol, menthone, menthol, limonene, phellandrene, piperitone, terpinolene, topanol A, comosusol A, sydonol, carvacrol, terpinene and sabinene. In one example, the one or more monoterpenoids is thymol.

It will be understood that references to a preserving agent herein (e.g. thymol), encompass, where permitted, all isomeric forms, racemates, amorphous or crystalline forms, solvates, acceptable salts, solvates of said salts, and prodrugs thereof.

Thymol

Thymol is a natural monoterpene phenol derivative. It is found in oil of thyme and can be extracted from, for example, Thymus vulgaris and other various plants. It can be isolated as a white crystalline solid. It has a molecular formula of C ₁₀ H ₁₄ O and its molecular weight is 150.22. It is a derivative of p-cymene and is isomeric with carvacrol. The molecular structure for thymol and related monoterpenes that share similar characteristics and similar bioactivities and are suitable for the animal feed preservatives/feeds/uses of the disclosure are shown in Figure 19.

It will be understood that the scope of combinations of the antimicrobial compounds of this disclosure with other agents includes in principle any combination of an antimicrobial compound as described herein with any agent, composition or substance suitable for use as a preserving agent. When combined in the same formulation it will be appreciated that the antimicrobial compounds of the disclosure have requisite stability and compatibility with each other and the other components of the formulation so that they may be formulated for administration. When formulated separately they may be provided in any convenient formulation, conveniently in such a manner as are known for such compounds in the art. It should be understood that the formulations may include other agents as disclosed herein and agents conventional in the art having regard to the type of formulation in question.

For example, the feed preservative comprises a berberine alkaloid. For example, the feed preservative comprises a ursane-like triterpenoid. For example, the feed preservative comprises a phenylpropanoid. In one example, the phenylpropanoid is a honokiol-like phenylpropanoid.

In one example, the phenylpropanoid is a piceid-like phenylpropanoid.

In one example, the feed preservative comprises berberine or an acceptable salt thereof. In one example, the acceptable salt is berberine sulfate. In one example, the acceptable salt is berberine chloride. In one example, the feed preservative comprises ursolic acid or an acceptable salt thereof. In one example, the acceptable salt is the sodium salt of ursolic acid. In one example, the acceptable salt is the potassium salt of ursolic acid. In one example, the feed preservative comprises ursolic acid or an acceptable salt thereof. In one example, the acceptable salt is the sodium salt of ursolic acid. In one example, the acceptable salt is the potassium salt of ursolic acid. In one example, the feed preservative comprises piceid or an acceptable salt thereof. In one example, the feed preservative comprises honokiol or an acceptable salt thereof. In one example, the feed preservative comprises baicalin or baicalein.

Combination feed preservatives

The present disclosure contemplates feed preservatives that comprise two or more antimicrobial agents i.e. feed preservatives that comprise a combination of antimicrobial agents (“combination feedpreservatives”). It would be appreciated that such combination feed preservatives can be prepared using conventional procedures.

In this regard, the present disclosure contemplates a feed preservative comprising any two or more of the following: a berberine alkaloid, a ursane-like triterpenoid, a honokiol-like phenylpropanoid, a piceid-like phenylpropanoid, baicalin or baicalein. In this regard, the present disclosure contemplates a feed preservative comprising any three or more of the following:

• a berberine alkaloid, a ursane-like triterpenoid, a honokiol-like phenylpropanoid, a piceid-like phenylpropanoid, baicalin or baicalein.

In this regard, the present disclosure contemplates a feed preservative comprising any four or more of the following:

• a berberine alkaloid, a ursane-like triterpenoid, a honokiol-like phenylpropanoid, a piceid-like phenylpropanoid, baicalin or baicalein.

In this regard, the present disclosure contemplates a feed preservative comprising the following:

• a berberine alkaloid, a ursane-like triterpenoid, a honokiol-like phenylpropanoid, a piceid-like phenylpropanoid and baicalin or baicalein.

Dual agent feed preservatives

For example, the present disclosure relates to a combination feed preservative comprising two antimicrobial agents i.e. “dual agent” feed preservatives.

In one example, the feed preservative comprises a berberine alkaloid and a ursane-like triterpenoid. In one example, the feed preservative comprises a berberine alkaloid and a honokiol-like phenylpropanoid. In one example, the feed preservative comprises a berberine alkaloid and a piceid-like phenylpropanoid. In one example, the feed preservative comprises a berberine alkaloid and baicalin or baicalein.

In one example, the feed preservative comprises a ursane-like triterpenoid and a honokiol-like phenylpropanoid. In one example, the feed preservative comprises a ursane-like triterpenoid and a piceid-like phenylpropanoid. In one example, the feed preservative comprises a ursane-like triterpenoid and baicalin or baicalein.

In one example, the feed preservative comprises a honokiol-like phenylpropanoid and a piceid- like phenylpropanoid. In one example, the feed preservative comprises a honokiol-like phenylpropanoid and baicalin or baicalein. In one example, the feed preservative comprises a piceid-like phenylpropanoid and baicalin or baicalein. Triple agent feed preservatives

For example, the present disclosure relates to a combination feed preservative comprising three antimicrobial agents i.e. “triple agent” feed preservatives.

In one example, the feed preservative comprises a berberine alkaloid, a ursane-like triterpenoid and a honokiol-like phenylpropanoid. In one example, the feed preservative comprises a berberine alkaloid, a ursane-like triterpenoid and a piceid-like phenylpropanoid. In one example, the feed preservative comprises a berberine alkaloid, a ursane-like triterpenoid and baicalin or baicalein. In one example, the feed preservative comprises a berberine alkaloid, a piceid-like phenylpropanoid and honokiol-like phenylpropanoid. In one example, the feed preservative comprises a berberine alkaloid, a piceid-like phenylpropanoid and baiclain or baicalein. In one example, the feed preservative comprises a berberine alkaloid, a honokiol-like phenylpropanoid and baicalin or baicalein. In one example, the feed preservative comprises a ursane-like triterpenoid, a piceid-like phenylpropanoid and baicalin or baicalein. In one example, the feed preservative comprises a ursane-like triterpenoid, a honokiol-like phenylpropanoid and baicalin or baicalein. In one example, the feed preservative comprises a ursane-like triterpenoid, a piceid-like phenylpropanoid and a honokiol-like phenylpropanoid. In one example, the feed preservative comprises a piceid-like phenylpropanoid, a honokiol-like phenylpropanoid and baiclain or baicalein.

The disclosure also relates to feed preservatives comprising a synergistically effective amount of any one or more antimicrobial agents as described herein.

In one or more examples of the animal feed preservatives described herein, the feed preservative further comprises one or more acceptable excipients. The one or more acceptable excipients are preferably one or more vehicles or one or more acceptable additives. In one example, the one or more additives are selected from the group consisting of: buffers, solubilisers, gelling agents, viscosity enhancers, preservatives, oils, antioxidants, emulsifiers, foam forming agents, isotonic agents, a propellant gas, thickeners and combinations thereof. In one or more example of the feed preservatives described herein, the feed preservative further comprises an additive that masks a bitter flavour of the one or more antimicrobial agents. In one or more examples of the feed preservatives described herein, the feed preservative further comprises an additive that masks a bitter flavour of the one or more preserving agents. The animal feed preservatives of the disclosure may also contain other ingredients. Non-limiting examples of ingredients are listed hereafter. A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate which may be used as a diluting agent; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; and a liquid carrier, may be added. Various other ingredients may be present as coatings or to otherwise modify the physical form of the veterinary composition. The veterinary compositions may contain methyl and propylparabens as preservatives, a dye and flavouring agents such as cherry or orange flavour. Information on additives and excipients that are suitable for pharmaceutical applications may be found in, Remington [4] Information on additives and excipients that are suitable for for veterinary applications may be found, for example, in the Merck Veterinary Manual (online at www.merckvetmanual.com) or the CRC Handbook of Food, Drug and Cosmetic Excipients, 2005. Preferably, ingredients are government approved (e.g. FDA- approved) or GRAS substances.

Feed preservative (and feed) formulations may be prepared by any method known in the art, for example by bringing into association an active ingredient, or combination of active ingredients, with acceptable excipient(s).

Feed preservatives (and feeds) of the present disclosure may be formulated for administration by any appropriate route depending on the animal subject. For example by the oral (including buccal or sublingual) and nasal routes. Therefore, the feed preservatives of the disclosure may be formulated, for example, as tablets, capsules, powders, granules, lozenges, creams or liquid preparations, such as oral or sterile parenteral solutions or suspensions. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association an active ingredient, or combination of active ingredients, of with acceptable excipient(s). Such formulations may be prepared as enterically coated granules, tablets or capsules suitable for oral administration and delayed release formulations. The combinations of active ingredients are proposed for both liquid delivery as well as in granules for mixing through animal feeds.

Animal feed preservatives are proposed for administration in animal feed or in the drinking water of an animal. Thus, administration of a feed preservative may occur via the feed of an animal. The feed preservative may be in the form of a mash, granule, crumble, pellet or powder. In one example, administration occurs via granules for mixing through animal feeds. Administration of a composition may occur via the drinking water of an animal. The feed preservative may be in an aqueous form. The feed preservative may be in the form of a liquid or syrup. In one example, the feed preservative is dissolved in the drinking water for administration. In one example, the feed preservative may be added as suspension to the drinking water for administration.

Dose

It will be appreciated that the exact total effective amount of antimicrobial agent, and the ratio of the individual antimicrobial agents, depends on a number of factors including the animal subject (e.g. chicken versus pig), animal body weight, route of administration and whether the feed is an aqueous form or a solid form such as a crumble, granules, mash, pellets or a powder.

Thus, the amounts or dose of an antimicrobial compound in the feed preservatives may be increased or decreased accordingly. When an antimicrobial agent of the disclosure is used in combination with an additional antimicrobial agent or an additional preserving agent the ratio of the individual antimicrobial compounds may be increased or decreased accordingly. Further, the dose of each agent may differ from that when the agent is used alone.

In this regard, and with respect to a dual agent feed preservative, when an antimicrobial compound of the disclosure (a first antimicrobial agent) is used in combination with a second antimicrobial agent in a feed preservative, the dose of the first agent may differ from that when the first agent is used alone; the dose of the second antimicrobial agent agent may differ from that when the second antimicrobial agent is used alone; or the dose of the first antimicrobial agent may differ from that when the first antimicrobial agent is used alone and the dose of the second antimicrobial agent may differ from that when the second antimicrobial agent is used alone. In one example, when an antimicrobial compound of the disclosure (a first antimicrobial agent) is used in combination with a second antimicrobial agent the dose of the first antimicrobial agent is lower than that when the first antimicrobial agent is used alone. In one example, the dose of the second antimicrobial agent agent is lower than that when the second antimicrobial agent is used alone. In one example, the dose of the first antimicrobial agent is lower than that when the first antimicrobial agent is used alone and the dose of the second antimicrobial agent is lower than that when the second antimicrobial agent is used alone. In one example, there is a synergistic effect between the first antimicrobial agent and the second antimicrobial agent.

In one example of a dual agent feed preservative, the berberine alkaloid and the ursane-like triterpenoid are in a ratio ranging from about 99.9:0.1 to 0.1:99.9, wherein the ratio is a berberine alkaloid:ursane-like triterpenoid ratio by mass. In one example, the berberine alkaloid and the ursane-like triterpenoid are in a ratio ranging from about 99.9:0.1 to 66.7:33.3. In one example, the berberine alkaloid and the ursane-like triterpenoid are in a ratio ranging from about 66.7:33.3 to 50.0:50.0. In one example, the berberine alkaloid and the ursane-like triterpenoid are in a ratio ranging from about 50.0:50.0 to 0.1:99.9.

In one example, the berberine alkaloid and ursane-like triterpenoid are in a ratio selected from about 99.9:0.1; 95.7:4.3; 95.2:4.8; 95.0:5.0; 94.3:5.7; 90.0:10.0; 85.0:15.0; 80.0:20.0; 75.0:25.0; 70.0:30.0; 66.7:33.3; 65.0:35.0; 62.5:37.5; 60.0:40.0; 55.0:45.0 or 50.0:50.0; 45.0:55.0; 40.0:60.0; 35.0:65.0; 33.3:66.7; 30.0:70.0; 25.0:75.0; 20.0:80; 15:85; 10:90; 5:95; or 0.1:99.9.

In one example, the berberine alkaloid and ursane-like triterpenoid are in a ratio of about 94.3:5.7. In one example, the berberine alkaloid and ursane-like triterpenoid are in a ratio of about 62.5:37.5. In one example, the berberine alkaloid and ursane-like triterpenoid are in a ratio of about 50.0:50.0.

In one example, the ursane-like triterpenoid is ursolic acid. In one example, the ursane-like triterpenoid is an acceptable salt of ursolic acid. The acceptable salt may be selected from potassium ursolate or sodium ursolate. In one example, the acceptable salt is potassium ursolate. In one example, the acceptable salt is sodium ursolate.

In one example, the berberine alkaloid is berberine or an acceptable salt thereof. The acceptable salt may be selected from berberine sulfate or berberine chloride. In one example, the acceptable salt is berberine sulfate. In one example, the acceptable salt is berberine chloride.

In one example of a triple agent feed preservative, the feed preservative comprises a berberine alkaloid, a ursane-like triterpenoid and a phenylpropanoid. In this regard, the feed preservative may comprise:

■ a high amount of a berberine alkaloid, a low amount of a ursane-like triterpenoid, and a low amount of a phenylpropanoid;

■ a high amount of a berberine alkaloid, a medium amount of a ursane-like triterpenoid, and a low amount of a phenylpropanoid; or

■ a high amount of a berberine alkaloid, a low amount of a ursane-like triterpenoid, and a medium amount of a phenylpropanoid.

Another example of a triple compound combination feed may comprise: ■ a medium amount of a berberine alkaloid, a medium amount of a ursane-like triterpenoid, and a low amount of a phenylpropanoid;

■ a medium amount of a berberine alkaloid, a low amount of a ursane-like triterpenoid, and a medium amount of a phenylpropanoid;

■ a medium amount of a berberine alkaloid, a medium amount of a ursane-like triterpenoid, and a medium amount of baicalin, such that the berberine alkaloid, ursane-like triterpenoid and baicalin are in equal amounts;

■ medium amount of a berberine alkaloid, a high amount of a ursane-like triterpenoid, and a low amount of a phenylpropanoid; or

■ a medium amount of a berberine alkaloid, a low amount of a ursane-like triterpenoid, and a high amount of a phenylpropanoid.

Another example of a triple compound combination feed may comprise:

■ a low amount of a berberine alkaloid, a low amount of a ursane-like triterpenoid, and a high amount of a phenylpropanoid;

■ a low amount of a berberine alkaloid, a medium amount of a ursane-like triterpenoid, and a high amount of a phenylpropanoid;

■ a low amount of a berberine alkaloid, a high amount of a ursane-like triterpenoid, and a medium amount of a phenylpropanoid;

■ a low amount of a berberine alkaloid, a high amount of a ursane-like triterpenoid, and a low amount of a phenylpropanoid; or

■ a low amount of a berberine alkaloid, a medium amount of a ursane-like triterpenoid, and a medium amount of a phenylpropanoid.

Thus, example combination feeds may comprise a berberine alkaloid:ursane-like triterpenoid:phenylpropanoind in ratios ranging from: 99.0:0.5:0.5; 90:9:1, 80:19:1; 70:29:1; 60:39:1; 50:49:1; 50:40:10, 40:40:20, 90:1:9, 80:1:19; 70:1:29; 60:1:39; 50:1:49; 50:10:40; 40:20:40 to 33.3:33.3:33.3, where the berberine alkaloid ranges from a high to medium amount.

Thus, example combination feeds may have an berberine alkaloid: ursane-like triterpenoid:phenylpropanoid in ratios ranging from 33.3:33.3:33.3 to 20:40:40, 10:40:50, 10:50:40, 1:49:50, 1:50:49, 1:39:60; 1:60:39; 1:29:70, 1:70:29; 1:19:80; 1:80:19; 1:9:90; 1:90:9 to 0.5:0.5:90, where the the berberine alkaloid ranges from a medium to low amount.

Animal subject Preferably, the animal is human. The animal is preferably non-human. Preferably, the non- human animal is a food-producing animal. The food-producing animal is preferably selected from a chicken or a pig. Preferably, the animal is an aquatic animal The aquatic animal is preferably finfish. Preferably, the aquatic animal is shellfish. Shellfish are preferably selected from crustaceans or molluscs. Preferably, crustaceans are selected from the group comprising crabs, crayfish, lobsters, prawns, and shrimp. Molluscs are preferably selected from the group comprising clams, mussels, oysters, scallops and winkles. Preferably, the animal is a mammal. The mammal preferably is a human, horse, dog, cat, sheep, cattle, pig or primate. Preferably, the animal is a bird. The bird is preferably chickens, geese, turkeys or ducks. Chickens include, for example, broiler chickens (broilers), broiler breeders, chicks, fryers, roosters and layer hens (layers).

Feed safety and residue levels

Human and animal drugs and animal feed additives are highly regulated for safety reasons. In Australia, the Therapeutic Goods Administration (TGA) is responsible for regulating therapeutic goods for human use while the Australian Pesticides and Veterinary Medicines Authority (APMVA) is responsible for the assessment and registration of pesticides and veterinary medicines. In the US, the Food and Drug Administration (FDA) is responsible for the approval of human and animal drugs and feed additives which are governed by the Federal Food, Drug, and Cosmetic Act (FD&C Act).

The FD&C Act requires that compounds intended for use in food-producing animals are shown to be safe and that food produced from animals exposed to these compounds is shown to be safe for consumption by people. In particular, the use in food-producing animals of any compound found to induce cancer when ingested by people or animal is prohibited by statute (21 CFR Part 500, Subpart E - Regulation of carcinogenic compounds used in food-producing animals) unless certain conditions are met (the so-called “Diethylstilbestrol (DES) Proviso”). Under the DES proviso use of a suspected carcinogenic compound is not prohibited if it can be determined by prescribed methods of examination that “no residue” of that compound will be found in the food produced from food-producing animals under conditions of use reasonably certain to be followed in practice.

For illustration, despite the safe use of berberine alkaloids as dietary supplements for humans, berberine has come under suspicion that it is a carcinogenic agent from a study carried out in rodents by the US National Centre for Toxicological Research [13]. Thus, if the FDA decides that berberine should be regulated as a carcinogenic compound, US statue prohibits the use of berberine in food-producing animals unless the “no residue” DES proviso applies.

The term “no residue” refers to any residue remaining in the edible tissues of food-producing animals that is so low that it presents an insignificant risk of cancer to consumers. More specifically, an insignificant risk of cancer is defined as a 1 in 1 million increase in risk.

A “safe” residue level of an antimicrobial agent, e.g. berberine, as used herein, is one that poses an insignificant risk of disease, particularly cancer.

The residue level of an antimicrobial agent, e.g. a berberine alkaloid, ursane-like triterpenoid or a phenylpropanoid (such as a honokiol-like phenylpropanoid or a piceid-like phenylpropanoid) may be determined by experiment relying on mass analysis of the component. An exemplary protocol for determining the residue level of an antimicrobial agent using LC-MS/MS is as follows. Animals are administered a nominated dose of the antimicrobial agent included in their feed as a preservative or are administered regular feed without the antimicrobial agent (i.e. control groups). Administration is continued for a specified period when tissue collection and analysis occurs. Selected groups of animals are either fed upto tissue collection or fed beyond tissue collection on regular feed to examine residues after a specified washout period. Muscle tissue and/or tissue from an organ may be collected. The organ may be liver, kidney or skin.

For example, muscle tissue, liver and/or kidney tissue is collected. Skin tissue may also be collected. IRP001 chloride is extracted from the tissue. The residual mass of IRP001 chloride is determined using a LC-MS/MS assay. The assay is fully validated during each assay run with accuracy, and limits of detection (LLOD) and quantitation (LLOQ) assessed.

A “Residue study” is provided elsewhere (Example 3) and describes the determination of residual berberine in chicken tissue. In brief, samples of muscle tissue (from breast, leg and thigh) or organ tissue (liver and kidney) were excised from each bird after euthanasia. A known weight of tissue (approximately lg) was homogenised in 2 mL water. Samples were centrifuged and a known volume of the supernatant was removed for analysis of berberine by LC-MS/MS to provide the residue level of berberine in tissue (ng of berberine per g of tissue). Wash-out periods may be incorporated into this protocol.

In one or more examples of the feed preservatives described herein, there is a low residue level of the one or more antimicrobial agents in the tissue of an animal after the feed preservative is administered to the animal. In one or more examples of the feed preservatives described herein, there is a safe residue level of the one of more antimicrobial agents in the tissue of an animal after the feed preservative is administered to the animal.

In one or more examples of the feed preservatives described herein, there is a low residue level of the one or more antimicrobial agents in the tissue of an animal after the feed preservative is administered to the animal and a washout period. In one or more examples of the feed preservatives described herein, there is a safe residue level of the one or more antimicrobial agents in the tissue of an animal after the feed preservative is administered to the animal and a washout period.

In one example, there is a low residue level of the one or more antimicrobial agents in the muscle tissue of an animal after the feed preservative is administered to the animal. In one example, there is a safe residue level of the one or more antimicrobial agents in the muscle tissue of an animal after the feed preservative is administered to the animal.

In one example, there is a low residue level of the one or more antimicrobial agents in the muscle tissue of an animal after the feed preservative is administered to the animal and a washout period. In one example, there is a safe residue level of the one or more antimicrobial agents in the muscle tissue of an animal after the feed preservative is administered to the animal and a washout period.

In one example, there is a low residue level of the one or more antimicrobial agents in the liver and muscle tissue of an animal after the feed preservative is administered to the animal. In one example, there is a safe residue level of the one or more antimicrobial agents in the liver and muscle tissue of an animal after the feed preservative is administered to the animal.

In one example, there is a low residue level of the one or more antimicrobial agents in the liver and muscle tissue of an animal after the feed preservative is administered to the animal and a washout period. In one example, there is a safe residue level of the one or more antimicrobial agents in the liver and muscle tissue of an animal after the feed preservative is administered to the animal and a washout period.

In one example, there is a low residue level of the one or more antimicrobial agents in the liver of an animal after the feed preservative is administered to the animal. In one example, there is a safe residue level of the one or more antimicrobial agents in the liver of an animal after the feed preservative is administered to the animal. In one example, there is a low residue level of the one or more antimicrobial agents in the liver of an animal after the feed preservative is administered to the animal and a washout period. In one example, there is a safe residue level of the one or more antimicrobial agents in the liver of an animal after the feed preservative is administered to the animal and a washout period.

Preferably, there is a low residue level of the berberine alkaloid in the animal after the administration period. There is preferably a safe residue level of the berberine alkaloid in the animal after the administration period. Preferably, the animal is a chicken.

Preferably, there is a safe residue level of the berberine alkaloid in the muscle tissue of the chicken after the administration period. The residue level is at least below about 13 ng of the berberine alkaloid per g of muscle tissue

Preferably, the residue level is about 10 ng of the berberine alkaloid per g of muscle tissue. The residue level is preferably about 5 ng/g.

Preferably, the berberine alkaloid has been administered in the feed of the chicken at a rate of about 0.3 g/kg. The residue levels of the berberine alkaloid in the muscle tissue of the chicken are preferably as follows: about 6.1 ng/g in the muscle tissue in the breast of the chicken; about 5.5 ng/g in the muscle tissue in the lower leg of the chicken; and about 11.6 ng/g in the muscle tissue in the upper leg of the chicken.

Preferably, the berberine alkaloid has been administered in the feed of the chicken at a dose of less than about less than 0.1 g/kg.

Preferably, the berberine alkaloid has been administered in the feed of the chicken at a dose of about 0.03 g/kg. 35. The residue levels of the berberine alkaloid in the muscle tissue of the chicken are preferably as follows: below 2 ng/g in the muscle tissue in the breast of the chicken; below 2 ng/g in the muscle tissue in the lower leg of the chicken; and below 2 ng/g in the muscle tissue in the upper leg of the chicken. Preferably, there is a low residue level of the berberine alkaloid in the muscle tissue of the animal after the administration period and a washout period. There is preferably a safe residue level of the berberine alkaloid in the muscle tissue of the animal after administration period and a washout period.

Preferably, there is a safe residue level of the berberine alkaloid in the muscle tissue of the chicken after the administration period and a washout period.

Preferably, the washout period is a period between 1 and 2 weeks. The washout period is preferably selected from a period between 1 day and 14 days; between 1 day and 7 days; between 1 day and 4 days; and between 1 day and 2 days. Preferably, the washout period is a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.

Preferably, after a washout period of 1 day the residue levels of the berberine alkaloid in the muscle tissue of the chicken are as follows: about 5.7 ng/g in the muscle tissue in the breast of the chicken; about 3.2 ng/g in the muscle tissue in the lower leg of the chicken; and about 6.0 ng/g in the muscle tissue in the upper leg of the chicken.

Preferably, after a washout period of 2 days the residue levels of the berberine alkaloid in the muscle tissue of the chicken are as follows: about 3.6 ng/g in the muscle tissue in the breast of the chicken; about 3.1 ng/g in the muscle tissue in the lower leg of the chicken; and about 4.5 ng/g in the muscle tissue in the upper leg of the chicken.

Preferably, after a washout period of 4, 7 and 14 days, the residue levels of the berberine alkaloid in the muscle tissue of the chicken are below 2 ng/g.

Preferably, the berberine alkaloid has been administered in the feed of the chicken at a dose of about 0.3 g/kg.

The level of residue is preferably at least below 13 ng of the berberine alkaloid per g of muscle tissue. The level of residue is preferably about 10 ng of the berberine alkaloid per g of muscle tissue. Preferably, the level of residue is about 5 ng/g. Preferably, the berberine alkaloid has been administered in the feed of the chicken at a dose of about greater than 0.1 g/kg.

Preferably, there is a low residue level of the berberine alkaloid in the liver and muscle tissue of the animal after the administration period. Preferably, there is a safe residue level of the berberine alkaloid in the liver and muscle tissue of the animal after the administration period.

Preferably, there is a safe residue level of the berberine alkaloid in the liver and muscle tissue of the chicken after the administration period. The residue levels of the berberine alkaloid in the liver and muscle tissue of the chicken are preferably below 2 ng/g. Preferably, the berberine alkaloid has been administered in the feed of the chicken at a dose of about 0.03 g/kg.

Preferably, there is a low residue level of the berberine alkaloid in the liver and muscle tissue of the animal after the administration period and a washout period. Preferably, there is a safe residue level of the berberine alkaloid in the liver and muscle tissue of the animal after the administration period and a washout period.

Preferably, there is a safe residue level of the berberine alkaloid in the liver and muscle tissue of the chicken after the administration period and a washout period. The washout period is preferably a period between 1 week and 2 weeks. Preferably, the washout period is a period selected from between 1 day and 14 days; between 1 day and 7 days; 1 day and 4 days; and between 1 day and 2 days. The washout period is preferably a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.

Preferably, after a washout period of 1 day the residue levels of the berberine alkaloid in the muscle tissue of the chicken are as follows: about 5.7 ng/g in the muscle tissue in the breast of the chicken; about 3.2 ng/g in the muscle tissue in the lower leg of the chicken; and about 6.0 ng/g in the muscle tissue in the upper leg of the chicken, and a residue level of the berberine alkaloid in the liver tissue of the chicken of about 8.0 ng/g.

Preferably, after a washout period of 7 days the residue levels of the berberine alkaloid in the muscle tissue in the breast, lower leg and upper leg of the chicken are below 2 ng/g and the residue level of the berberine alkaloid in the liver tissue of the chicken is about 6.5 ng/g. Preferably, after a washout period of 14 days the residue levels of the berberine alkaloid in the muscle tissue in the breast, lower leg and upper leg of the chicken are below 2 ng/g and the residue level of the berberine alkaloid in the liver tissue of the chicken is about 3.0 ng/g.

Preferably, the berberine alkaloid has been administered in the feed of the chicken at a dose of about 0.3 g/kg.

Preferably, there is a low residue level of the berberine alkaloid in the liver and muscle tissue of the animal after the administration period. Preferably, there is a safe residue level of the berberine alkaloid in the liver and muscle tissue of the animal after the administration period.

Preferably, there is a safe residue level of the berberine alkaloid in the liver and muscle tissue of the chicken after the administration period. The residue levels of the berberine alkaloid in the liver tissue and muscle tissue in the breast, lower leg and upper leg of the chicken are preferably below 2 ng/g. Preferably, the berberine alkaloid has been administered in the feed of the chicken at a dose of about 0.03 g/kg.

Preferably, there is a safe residue level of the berberine alkaloid in the liver tissue of the chicken after the administration period and a washout period. The washout period is preferably a period selected from between 1 week and 2 weeks. Preferably, the washout period is a period selected from between 1 day and 14 days; between 1 day and 7 days; between 1 day and 4 days; and between 1 day and 2 days. The washout period is preferably a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.

Preferably, after a washout period of 1 day the residue level of the berberine alkaloid in the liver tissue of the chicken is about 8.0 ng/g. After a washout period of 7 days the residue level of the berberine alkaloid in the liver tissue of the chicken is preferably about 6.5 ng/g. Preferably, after a washout period of 14 days the residue level of the berberine alkaloid in the liver tissue of the chicken is about 3.0 ng/g. The berberine alkaloid has preferably been administered in the feed of the chicken at a dose of about 0.3 g/kg.

Preferably, the administration period is 35 days.

The safety of a feed preservative (or animal feed) once administered to an animal can also be assessed by monitoring animals for any adverse effects such as behavioural changes e.g. morbidity or adverse reactions. Unanticipated events can also be monitored. Safety can also be assessed by histological examination of tissue (such as the tissue of organs e.g. skin, liver and kidneys or gastrointestinal tissue) or by investigation of blood chemistry. Safety studies including histological examination or investigation of blood chemistry are provided elsewhere (Examples 1 and 2 report safety in chickens and Example 4 reports safety in pigs).

In one or more examples of the feed preservatives described herein, the feed preservative is safe as assessed by histological examination of the tissue of an animal after administration of the feed. In one example, the histological examination comprises the analysis of lesions in the tissue. In one example, the tissue is selected from gastrointestinal tissue, kidney tissue, liver tissue, pancreatic tissue and a combination thereof. In one example, the gastrointestinal tissue is selected from duodenum, jejenum, ileum, colon and any combination thereof. In one example, the gastrointestinal tissue is selected from duodenum, jejenum and ileum.

In one example, the analysis comprises the semi-quantitative scoring of lesions in tissue to give lesion scores. In one example, the lesion scores are summed to give a lesion index. In one example, the lesions scores are summed to give a cumulative pathology index. In one example, the lesions scores are summed to give a cumulative pathology index, wherein the tissue is a combination of duodenum, jejenum and ileum. In one example, the lesion scores are summed to give a hepatitis index, wherein the tissue is liver tissue.

In one example, the histological examination comprises the analysis of Coccidia in tissue. In one example, the analysis of comprises the scoring of Coccidia in tissue to give Coccidia scores. In one example, the Coccidia scores are summed to give a Coccidia index.

In one example, the feed preservative is safe as assessed by examination of the blood chemistry of an animal after administration of the feed. In one example, the hematology and/or serum chemistry is evaluated.

Health and growth pertormance in animat subjects

In one example, there is an improvement in body condition. In one example, there is an improvement in body condition score. In one example, there is an improvement in behaviour.

In one example, there is an improvement in behaviour score. In one example, there is a reduction in morbidity. In one example, there is a reduction in mortality.

In one or more examples of the feed preservatives described herein, the feed preservative improves or maintains gastrointestinal health in the animal. In one or more examples of the feed preservatives described herein, the feed preservative improves or maintains gastrointestinal health as measured by histological examination. In one example, there is a reduction in lesion score. In one example, there is a reduction in lesion score. In one example, there is a reduction in Coccidia score. In one example, there is a reduction in Coccidia index. In one example, there is a reduction in cumulative pathology index. In one example, there is a reduction in hepatitis index. In one example, there is an improvement in fecal score. In one example, there is a reduction in fecal oocyst count.

In one or more examples of the feed preservatives described herein, the feed preservative improves or maintains growth performance in the animal. In one example, the feed preservative improves growth performance in the animal. In one example, the weight gain is increased. In one example, the daily weight gain is increased. In one example, feed intake is increased. In one example, the feed efficiency is increased. In one example, feed conversion ratio (FCR) is reduced.

The present disclosure contemplates a feed preservative where a combination of any two or more of the above health and growth performance effects is observed.

The present disclosure also contemplates a feed preservative wherein there is a synergistic effect between any one of the antimicrobial agents. The synergistic effect may be any one of the above health and growth performance effects. The synergistic effect may be a combination of any two or more of the above health and growth performance effects.

Stability

In one or more examples of the feed preservatives described herein, the antimicrobial agent is stable. In one example, the antimicrobial agent is stable within the feed preservative. In one example, the antimicrobial agent is stable within the feed preservative as measured by LCMS.

In one example, the antimicrobial agent is stable within the feed preservative for at least two weeks as measured by LCMS.

Animal feeds

The present disclosure also relates to an animal feed comprising a feed preservative as described herein.

The present disclosure also relates to an animal feed comprising one or more antimicrobial agents, wherein the one or more antimicrobial agents are selected from: one or more berberine alkaloids; one or more ursane-like triterpenoids; one or more phenylpropanoids; or mixtures thereof.

The present disclosure also relates to an animal feed comprising one or more antimicrobial agents, wherein the one or more antimicrobial agents are selected from: one or more berberine alkaloids; one or more ursane-like triterpenoids; one or more phenylpropanoids; or mixtures thereof, and wherein the feed is safe.

In one example of an animal feed, the one or more berberine alkaloids are selected from: berberine, 13-hydroxyberberine, 7,8-dihydro-13-methylberberine, 13-benzylberberine, berberrubine, fibrauretin (palmatine), tetrahydropalmatine, coralyne, coreximine, jatrorrhizine or an acceptable salt thereof. In one example, the one or more berberine alkaloids is berberine or an acceptable salt thereof. In one example, the acceptable salt is selected from berberine sulfate or berberine chloride. In one example, the acceptable salt is berberine sulfate. In one example, the acceptable salt is berberine chloride.

In one example of an animal feed, the one or more ursane-like triterpenoids are selected from: ursolic acid neoilexonol, regelin, β-boswellic acid, urmiensolide, alstoprenylene, asiatic acid, corosolic acid, uvaol, rotundic acid, lupanol, lupeol acetate, 3-oxolupenal, betulonic acid, betulinic acid, bevirimat, oleanolic acid, erythrodiol, β-amyrin, maslinic acid, α-boswellic acid, myricadiol, mupinensisone, miliacin, enoxolone, lucyin A, echinocystic acid, sumaresinolic acid, gypsogenic acid, imberic acid, carandinol, capillirol B, capillirone, cylindrin, tetrahymanol and tetrahymanone triterpenoids, Ilexpublesnin F, ixorene, azadirahemiacetal, polystanin E, mauritic acid, cycloartenol, ganoderol A, eburicol, suberosol, curcurbitacin A, B, C, D, E, I, J, K, L, O, P and Q, 11-deoxycucurbitacin I, 23,24-dihydrocucurbitacin B, colocynthin, an acceptable salt thereof and any combination thereof. In one example, the one or more ursane-like triterpenoids is ursolic acid or an acceptable salt thereof.

In one example of an animal feed, the one or more phenylpropanoids is one or more honokiol-like phenylpropanoids. In one example, the one or more honokiol-like phenylpropanoids is selected from: honokiol, isohonokiol, dehydrodieugenol, diferulic acid, magnolignan, magnolol, randainol, an acceptable salt thereof and any combination thereof. In one example, the one or more honokiol-like phenylpropanoids is honokiol or an acceptable salt thereof.

In one example of an animal feed, the one or more phenylpropanoids is one or more piceid-like phenylpropanoids. In one example, the one or more piceid-like phenylpropanoids is selected from: piceid, ethylstilbestrol, rhapontin, astringin, resveratrol, lysidiside A, hexestrol, dienestrol, chlorophorin, 3-hydroxy-5-methoxy-6-prenylstilbene-2-carboxylic acid, isorhapontin, piceatannol, pinosylvin, pinosylvin methyl ether, 4-prenylresveratrol, pterostilbene, oxyresveratrol, an acceptable salt thereof and any combination thereof. In one example, the one or more piceid-like phenylpropanoids is piceid or an acceptable salt thereof.

In one or more examples of the animal feeds described herein, the feed further comprises one or more preserving agents. In one example, the one or more preserving agents are one or more antimicrobial compounds selected from arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine, andrographolide, an acceptable salt thereof and any combination thereof. In one example, the one or more preserving agents is baicalin or an acceptable salt thereof.

In one or more examples of the animal feeds described herein, the feed further comprises an additive that masks a bitter flavour of the one or more antimicrobial agents.

In one or more examples of the animal feeds described herein, the feed further comprises an additive that masks a bitter flavour of the one or more preserving agents.

In one example, the animal feed further comprises an animal foodstuff suitable for consumption by an animal. In one example, the animal is human. In one example, the animal is a non-human animal. In one example, the animal is a food-producing animal. In one example, the food- producing animal is a chicken. In one example, the food-producing animal is a pig.

In one example, the feed is the form of a crumble, granule, mash, pellet or powder. In one example, the antimicrobial agent is present in the feed in an amount of 0.001 g/kg to 2 g/kg of feed. The total effective amount or dose of the antimicrobial agent in the feed may range from about 0.001 g/kg to about 2 g/kg. Example amounts of the total amount of antimicrobial compound in the feed are: 0.001 g/kg (0.0001 wt %); 0.003 g/kg (0.0003 wt %); 0.005 g/kg (0.0005 wt %); 0.01 g/kg (0.001 wt %); 0.03 g/kg (0.003 wt %); 0.05 g/kg (0.005 wt %); 0.1 g/kg (0.01 wt %); 0.3 g/kg (0.03 wt %); 1.0 g/kg (0.1 wt %) and 2 g/kg (0.2 wt %).

In one example, the feed is in an aqueous form. In one example, the aqueous form is selected from a liquid or syrup. In one example, the antimicrobial agent is present in the feed in an amount of 0.001 g/L to 0.1 g/L.

In one or more examples of the animal feeds described herein, there is a safe residue level of the one or more antimicrobial agents in the tissue of an animal after the feed preservative is administered to the animal. In one example, there is a safe residue level of the one or more antimicrobial agents in the muscle tissue of an animal after administration. In one example, there is a safe residue level of the one or more antimicrobial agents in the muscle tissue of an animal after administration and a washout period. In one example, there is a safe residue level of the one or more antimicrobial agents in the liver and muscle tissue of an animal after administration. In one example, there is a safe residue level of the one or more antimicrobial agents in the liver and muscle tissue of an animal after administration and a washout period. In one example, there is a safe residue level of the one or more antimicrobial agents in the liver of an animal after administration. In one example, there is a safe residue level of the one or more antimicrobial agents in the liver tissue of an animal after administration and a washout period.

In one or more examples of the animal feeds described herein, the feed is safe as assessed by histological examination of the tissue of an animal after administration of the feed. In one example, the histological examination comprises the analysis of lesions in the tissue. In one example, the tissue is selected from gastrointestinal tissue, kidney tissue, liver tissue, pancreatic tissue and a combination thereof. In one example, the gastrointestinal tissue is selected from duodenum, jejenum, ileum, colon and any combination thereof. In one example, the gastrointestinal tissue is selected from duodenum, jejenum and ileum.

In one or more examples of the animal feeds described herein, the analysis comprises the semi- quantitative scoring of lesions in tissue to give lesion scores. In one example, the lesion scores are summed to give a lesion index. In one example, the lesions scores are summed to give a cumulative pathology index, where the tissue is a combination of duodenum, jejenum and ileum. In one example, the lesion scores are summed to give a hepatitis index, wherein the tissue is liver tissue.

In one example, the histological examination comprises the analysis of Coccidia in tissue. In one example, the analysis comprises the scoring of Coccidia in tissue to give Coccidia scores. In one example, the Coccidia scores are summed to give a Coccidia index.

In one or more examples of the animal feeds described herein, the feed is safe as assessed by examination of the blood chemistry of an animal after administration of the feed. In one example, hematology and/or serum chemistry is evaluated.

In one or more examples of the animal feeds described herein, the feed preservative improves or maintains gastrointestinal health in the animal. In one example, the feed improves or maintains gastrointestinal health as measured by histological examination.

In one or more examples of the animal feeds described herein, the feed improves or maintains growth performance in the animal. In one example, the feed improves growth performance in the animal. In one example, the weight gain is increased. In one example, the daily weight gain is increased. In one example, feed conversion ratio (FCR) is reduced.

The present disclosure contemplates a feed where a combination of any two or more of the above health and growth performance effects is observed.

The present disclosure also contemplates a feed wherein there is a synergistic effect between any one of the antimicrobial agents. The synergistic effect may be any one of the above health and growth performance effects. The synergistic effect may be a combination of any two or more of the above health and growth performance effects.

In one or more examples of the animal feeds described herein, the antimicrobial agent is stable.

In one example, the antimicrobial agent is stable within the feed. In one example, the antimicrobial agent is stable within the feed as measured by LCMS. In one example, the antimicrobial agent is stable within the feed for at least two weeks as measured by LCMS.

It would be understood that preparation of feed preservatives and feeds can be accomplished using conventional procedures. Guidance on feed formulation is provided by, for example, the Food and Agriculture Organization of the United Nations at (www.fao.org). It would be recognised that formulation of feeds is dependent on the animal subject. For example, animal feed for a monogastric mammal, such as a pig, typically comprises concentrates as well as additives e.g. supplements whereas animal feed for a ruminant mammal, such as cattle, generally comprises forage (including roughage and silage) and may further comprise concentrates as well as supplements.

Thus, the present disclosure contemplates animal feed preservatives or animal feeds formulated so that they are suitable for use in any one of the animal subjects as defined herein. For example, the present disclosure contemplates a feed preservative formulated for use in chickens (a chicken formulated feed preservative). For example, the present disclosure contemplates a feed formulated for use in chickens (a chicken formulated feed). For example, the present disclosure contemplates a feed preservative formulated for use in pigs (a pig formulated feed preservative). For example, the present disclosure contemplates a feed formulated for use in pigs (a pig formulated feed).

It will be appreciated that the exact effective amount of an antibiotic agent in the feed depends on various factors including the animal subject (e.g. human or chicken versus pig), route of administration, body weight and the form of the feed (i.e. whether the feed is an aqueous form or a solid form such as a crumble, granules, mash, pellets or a powder). Accordingly, the amounts of the antimicrobial agent in the prepared feed may be increased or decreased to suit the above factors. In combined feeds comprising more than one active antimicrobial agent it will be appreciated that the total effective amount of active agents and the ratio of the individual agents may be varied to suit the above factors.

Animal feeds may include various ingredients e.g. vitamins, minerals (e.g. calcium, phosphorus, trace elements such as zinc, selenium and chromium, sodium), enzymes (e.g. phytases to improve nutrient digestibility), essential oils, direct fed microbial (to maintain gastrointestinal microbiota balance and health), organic acids, amino acids (e.g, methionine, lysine and threonine) which can act as supplements and can be provided in a premix.

Other ingredients include auxiliary components and excipients as described above for the feed preservatives of the disclosure including: binders, anti-oxidants, preservatives, coloring agents, pigments and dyes, flavouring agents, such as sweeteners, which may be used to mask the bitterness of feed ingredients to improve feed palatability, vehicles, diluting agents, emulsifying and suspending agents, attractants, and medications including growth enhancers, immunostimulants, hormones and antimicrobials. In addition, excipients are chosen for their suitability in preparing feed forms such as mash, granules, crumbles, pellets, powders and lickblocks. For example, cornstarch or polyvinylpyrollidone (PVP) are suitable for forming a granular feed product. Preferably, ingredients are government approved (e.g. FDA-approved) or GRAS substances.

Guidance on animal feed preservatives and animal feeds, ingredients and excipients is also provided by the Food and Agriculture Organization of the United Nations at (www.fao.org) and other resources, for example, the Merck Veterinary Manual (online at ww w.merckvetmanual.com) and the CRC Handbook of Food, Drug and Cosmetic Excipients, 2005.

Uses

The present disclosure also relates to use of one or more antimicrobial agents as an animal feed preservative, wherein the one or more antimicrobial agents are selected from: one or more berberine alkaloids; one or more ursane-like triterpenoids; one or more phenylpropanoids; or mixtures thereof.

The present disclosure also relates to use of one or more antimicrobial agents in the preparation of an animal feed preservative, wherein the one or more antimicrobial agents are selected from: one or more berberine alkaloids; one or more ursane-like triterpenoids; one or more phenylpropanoids; or mixtures thereof.

The present disclosure also relates to use of one or more antimicrobial agents in the preparation of an animal feed, wherein the one or more antimicrobial agents are selected from: one or more berberine alkaloids; one or more ursane-like triterpenoids; one or more phenylpropanoids; or mixtures thereof.

The present disclosure also relates to use of an animal feed preservative as described herein in the preparation of an animal feed.

Development of antimicrobial compounds for their use in feed preservatives is described below with reference to challenge studies which can assess the ability of the compounds to act as preservatives ([14] to [19]).

Challenge Study: Growth of yeast and mould in pelletised poultry feed with and without antimicrobial treatment

Introduction

Challenge Studies are used to determine Food Safety implications and Shelf Life Stability of foods, especially those stored under ambient and refrigerated conditions [14 to 19]. These studies are conducted to aid in new R&D products, new formulations of existing product lines and to test existing products against new organisms of concern. Example organisms are listed in Table

9.

Procedure

All yeast and mould cultures will be grown up and harvested according to standard protocols such as they those employed by Diebel Laboratories, Inc (DLI), Gainesville, FL. Harvested mould cultures will be filtered through sterile cheese cloth to form a spore suspension. An “Inoculation Cocktail” will be established by combining the individual harvested spore suspensions into a single mixture. After culture is filtered, a drop of lactophenol aniline blue will be placed on a slide. A drop of culture will be dispensed into the lactophenol aniline blue and mixed. A cover slip will be placed over the mixture, and examined under the microscope for minimal hyphae presence. If a significant amount of hyphae are observed, the culture will be filtered again. Once spore culture is verified, it will be diluted to achieve the target inoculum level listed below.

Control and treated product will be aseptically weighted out into large sterile whirl-pak bags in 50g units and labeled with each pull day. Separate 50g samples of product, in sterile whirl-pak bags, will be inoculated with 0.25 mL of the mould cocktail (0.5% of total volume). Into each whirl-pak bag, the mould cocktail inoculum will be added drop-wise to several different locations within the bag to aid in homogenising the inoculum. Bags will then be massaged to mix the inoculum throughout the product. Bags will also be rolled closed. The target inoculum level will be approximately 10 ² - 10 ³ CFU/g of product. Day 0 sample bags will immediately be plated. On each successive pull day, 450 mL of Butterfields Buffered Phosphate Diluent (BUT) will be added and homogenised for 1 - 2 minutes. The samples will then be plated immediately. These plates will be spread-plated onto Potato Dextrose Agar with chlortetracycline additive for enumeration of mould and YM agar for enumeration of yeast. Plates will be incubated at 25 °C for 5 days, after which they will be enumerated.

The subsequent inoculated samples will be stored under at ambient temperature in a high humidity chamber (a closed chamber with an open pan of water to ensure high ambient humidity), and pulled on Weeks 1, 2, 3, 4, 6, 8, 10 and 12 following the procedure outlined above. All pulls will consist of triplicate samples. Samples will also be observed for visible mold growth.

Negative controls, consisting of uninoculated product, will be sampled and plated for Yeast/Mold at the beginning and end of the study to determine background flora presence. As with the inoculated samples, Yeast/Mould control samples will be incubated at 25°C for 5 days before enumeration Table 9 List or example organisms

EXAMPLES

EXAMPLE 1 - poultry safety study

An aim of the study was to determine the feasibility of phytogenic compounds as used as preservatives in feed for poultry by conducting a general target poultry safety study. Thus, the study evaluated the safety of three compounds (IRP001, IRP002 and IRP003) in broilers reared to market weight through examination of histology.

Experimental description and protocols

Experimental Design

The experiment consisted of 64 pens of 20 male broiler chickens. Treatments were replicated in eight (8) blocks and the eight (8) treatments were randomised within each block. Treatments (1 pen per treatment) are shown in Table 10. A randomization procedure for pen assignment for treatments and blocks was carried out by Southern Poultry Research, Inc.

Table 10 Treatments

Legend

IRP001 = berberine chloride

IRP002 = ursolic acid in protonated form

IRP003 = piceid Apart from the above in- feed medications no concomitant drug therapy was used during the study.

Bird weights by pen were recorded at Days 0, 21, 35 and 42. Feed consumption was recorded at Days 21, 35 and 42. Samples for histology were collected at Day 42.

Floor Pen Description and Management The test house was divided into pens of equal size, arranged along a central aisle. Each pen is

20.25 (4.5’ X 4.5’) sq. ft. and has 2 feet high side walls with bottom 1/2 foot being of solid wood to prevent bird migration. The pens were prepared for use in the study according to SPR SOP. All flooring of each pen had approximately 4 inches clean pine shavings. The pen was the experimental unit. All pens were numbered consecutively and identified on pen cards. The temperature of the building was monitored. Environmental conditions during the trial

(temperature) were appropriate (optimum) to the age of the animals. Illumination was provided by fluorescent bulbs placed above the pens. The lighting scheme was 24 hours of light per day from Day 0 to Day 42.

Standard floor pen management practices were used throughout the experiment. Animals and housing facilities were inspected twice daily, observing and recording the general health status, constant feed and water supply as well as temperature, removing all dead birds, and recognising unexpected events. Birds found dead during the study were noted on the Daily Mortality Record, and were not replaced. Pen number, the date of mortality, sex, weight, and diagnosis were recorded. All birds and feed were buried in SPR's pit following standard operating protocols and practice. Records of disposition were included in the source data. Compounds IRP001, IRP002 and IRP003 were sourced from JiaHe (Shaanxi, China).

Birds

One-day-old male Cobb 500 chicks were obtained from Cobb-Vantress hatchery, Cleveland, GA and 1280 chicks allocated to the study. All chicks were spray vaccinated with a commercial coccidia vaccine at the recommended level prior to placement. Twenty male broiler chicks were placed in each pen. Accountabilities of all test animals and any extra birds were recorded on an animal disposition form. The birds were sexed at the hatchery. The breeder flock history and vaccination record at the hatchery were recorded. The broilers were not vaccinated at the farm. Feed

All feeds were provided by Southern Poultry Research, Inc. (SPR) amd manufactured at the SPR feed mill. Diet specifics of the feed used in the study are described below ( Table 11 and Table 12) .

Quantities of all basal feed and items used to prepare treatment batches were documented. Each batch of feed was mixed and bagged separately. Each bag was identified with the study number, date of mix, type of feed, and correct treatment number. Complete records of feed mixing and test article inventories were maintained.

Feeding Schedule

Starter feed was fed from Day of Treatment (DOT) 0 to 21. On DOT 21, non-consumed Starter feed was weighed by pen and discarded. Grower feed was issued and fed until DOT 35. On

DOT 35, non-consumed Grower feed was weighed by pen and discarded. Finisher feed was fed until DOT 42. On DOT 42, non-consumed Finisher feed was weighed by pen and discarded.

Feed Samples

Treatment feed samples (~ 150g each) were collected and blended: one each from the beginning, middle, and end of each batch of treatment diet. Samples were retained by SPR until directed to ship or discarded 2 months post submission of report.

Diets Diet specifics are shown in Table 11 and Table 12.

Table 11 Nutrition

The main ingredients used were corn, soybean meal and animal by product.

Table 12 Ingredients 1Vitamin mix provided the following (per kg of diet): thiamin·mononitrate,2.4mg;nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg; vitamin B12 (cobalamin),12.0 μg; pyridoxine·HCL, 4.7 mg; D-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfite complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 27.5 ug; trans-retinyl acetate, 1,892 ug; all-rac α tocopheryl acetate, 11 mg; ethoxyquin, 125 mg. 2Trace mineral mix provided the following (per kg of diet): manganese (MnSO ₄ ·H ₂ O), 60 mg; iron (FeSO ₄ ·7H ₂ O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO ₄ ·5H ₂ O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe0 ₃ ), 0.3 mg. The basal feed did not contain any probiotic/ prebiotic feed additives, NSPases, coccidiostats or antibiotic growth promoter. All diets contained phytase.

The diets were provided ad libitum in one tube-type feeders per pen. From day 0 until day 7, feed was also supplied on trays, directly placed on the litter. Water was provided ad libitum from one Ziggity nipple line per pen (six available nipples/ pen).

Assessment of general effects

Twice daily observations were recorded during the study for general flock condition. Observations included were the availability of feed and water, temperature control, and any unusual conditions. The birds were watched closely for any abnormal reactions. Feed intake, bodyweight (BW) and feed conversion ratio (FCR) were recorded and compared between groups to determine treatment effects. Bodyweight was recorded on Days 0, 21, 35 and 42. The mean initial weight of the chicks for all groups was not significantly different. FCR was calculated by the following formula [21]:

Intestinal Pathology and Histology

On the day of study completion (D42), five birds from each pen were humanly euthanised and upper, mid and lower gut sections plus liver lobe were collected and stored in neutral buffered formalin. Theses samples were shipped to Veterinary Diagnostic Pathology, LLC for analysis.

Duodenum, some with pancreas, jejunum, and ileum from chickens at 42 days of age, were submitted fixed in formalin for histologic examination. Sections of tissue (2 mm) were trimmed from the submitted tissue, placed in cassettes, and processed for paraffin-embedded 5 μm sections stained with hematoxylin and eosin (H&E). All intestinal sections were kept intact in circular form to ensure uniformity of assessment. Tissues were examined microscopically for lesions and for parasites. A lesion panel was developed for each tissue, and lesions were semi- quantitatively scored for severity per 0, normal; 1, minimal severity; 2, mild severity; 3, moderate; 4, marked and 5, severe. Coccidia if present were identified to species (if possible) and scored according to previous work [21; 22].

For each bird, a coccidia index was calculated by summing the coccidia scores from each section of intestine. A cumulative pathology index was calculated by summing all lesion scores for all sections of intestine. The total enteritis index was calculated by subtracting the coccidia index from the cumulative lesion index, leaving a number representing inflammation and repair.

On the day of study completion (day 42), five (5) birds from each pen were humanely euthanised and upper, mid and lower gut sections plus liver lobe were collected and stored in neutral buffered formalin. These samples were shipped to Veterinary Diagnostic Pathology, LLC for microscopic lesion analysis. Lesions were scored for severity as 0, lesion absent or within normal; 1, minimal severity; 2, mild severity; 3, moderate severity; 4, marked severity; 5, severe. Lesion scores were recorded in a spreadsheet. A hepatitis index was calculated by summing all lesion scores from each liver.

Data Entry and Analysis Source data were entered with indelible ink. Entries were legible, signed or initialed, and dated by the person making the observation entry. Each sheet of source data was signed by the person(s) attributed to the data. Any mistakes or changes to the source data were initialled and dated and a correction code or statement added as to why the changes were made.

Locations of Source Data The original source data sheets and the final report were sent to Sponsor. An exact copy of the file and the final report were retained.

Disposal of Birds and Feed

All birds and feed were buried following SOPs. Records of disposition were included in the source data. Results

Feed Intake, FCR and Average Weight Gain

Table 13 summarises the general effects of the three phytogenic compounds in poultry. All birds appeared normal and no adverse effects or unanticipated events occurred. This is reflected in the results showing no ill effects of the compounds on feed intake, FCR or average weight gain. In fact, a slight improvement in FCR was found when a phytogenic compound was added to the feed compared to the control group.

Table 13 General effects of phy to genic compounds on poultry

Intestinal Pathology and Liver Histology

The effect of the three phytogenic compounds on intestinal pathology is summarised below (Berberine, Table 14; Ursolic acid, Table 15; Piceid, Table 16).

Table 14 Liver pathology of berberine treated birds

Table 15 Liver pathology of ursolic acid treated birds

Table 16 Liver pathology of piceid treated birds

Livers in control and treatment chickens had mild lesions without differences observed in the liver lesion index among groups. These changes included mild lymphocytic hepatitis in the portal regions, and extramedullary hematopoiesis, within normal limits for a production environment. The gastrointestinal histologic lesions identified were within normal limits for broiler chick in a production environment, as were the liver histological lesions. In summary, the results indicate that berberine, usolic acid and piceid cause no discernible adverse effect in poultry when used as a preservative in poultry feed.

Summary This study shows that Berberine, Ursolic Acid and Piceid, caused no discernible adverse effect in poultry when used as a preservative in poultry feed. Interestingly, the study suggests there is an added benefit in using these compounds as the results show a potential for better production performance and general health of poultry with the tested phytogenic compounds showing slight improvement in performance and intestinal pathology. EXAMPLE 2 - poultry safety study

This study evaluated the safety of five phytogenic compounds in broilers reared to market weight through examination of histology.

Summary and Conclusion

Histology results are shown in Table 17. Table 17 Histology 8 IRP003 0.005 8 10.4 7.4 3.0

9 IRP003 0.05 9 4.0 4.0 0.0

10 IRP003 0.5 10 4.6 4.6 0.0 11 IRP004 0.006 11 5.6 5.4 0.2 12 IRP004 0.06 12 5.2 3.4 1.8

13 IRP004 0.6 13 3.6 3.4 0.0

14 IRP005 0.014 14 6.0 6.0 0.0

15 IRP005 0.14 15 6.2 6.2 0.0

16 IRP005 1 16 6.0 6.0 0.0

From above, Cumulative Pathology and Enteritis scores were equal are lower than Treatment 1 Nil group. In conclusion, all gastrointestinal tract (GIT) histologic lesions identified were within normal limits for broiler chickens in a production environment. All liver histologic lesions identified were within normal limits for broiler chickens in a production environment. In adverse or unanticipated events were observed.

Experimental description and protocols

The study used the same methodology as Example 1 unless other stated.

Experimental Design

The experiment consisted of 16 treatments (1 pen per treatment, Table 18). Table 18 Treatments

Legend IRP001 = berberine chloride IRP002 = ursolic acid in protonated form IRP003 = piceid IRP004 = honokiol IRP005 = baicalin

Floor Pen Description and Management

The experimental house was divided into pens of equal size, arranged along a central aisle. The birds were kept in 16 pens with each having an area of 4 x 10 = 40 ft ² , with clean wood shavings as bedding with a thickness of approximately 4 inches. Each pen had 5 feet high side walls with bottom 1 1/2 feet being of solid wood to prevent bird migration.

The temperature of the building was monitored. Environmental conditions during the trial (temperature) were appropriate (optimum) to the age of the animals. Illumination was provided by fluorescent bulbs placed above the pens. The diets were provided ad libitum in one tube-type feeder per pen. From DO until D7, feed was also supplied on a tray placed on the litter of each pen. Water was provided ad libitum from one Plasson drinker per pen.

Standard floor pen management practices were used throughout the experiment. Animals and housing facilities were inspected twice daily, observing and recording the general health status, constant feed and water supply as well as temperature, removing all dead birds, and recognising unexpected events. Birds found dead during the study were noted on the Daily Mortality Record, and were not replaced. Pen number, the date of mortality, sex, weight, and diagnosis were recorded.

Birds

Day of hatch male Cobb 500 chicks were obtained and 160 chicks were allocated to the study. Ten male broiler chicks were placed in each pen. Accountabilities of all test animals and any extra birds were recorded on animal disposition form. The birds were sexed at the hatchery. The breeder flock history and vaccination record at the hatchery were recorded. Bird weights by pen were recorded on DO and 42.

EXAMPLE 3 - residue study in chickens The aim of this study was to determine tissue residues of the naturally occurring plant compound IRP001 chloride (berberine chloride) when administered orally via feed to commercial broiler chickens.

Summary

Broiler chickens received either 0.3g/kg or 0.03g/kg IRP001 chloride mixed into their feed, or received regular feed without additive (i.e. control groups). Treatment began immediately after the birds were housed in pens (in groups of 10) and treatment continued for 35 days. Birds were either euthanised on day 35 for tissue collection or were fed beyond day 35 on regular feed for up to 7 days to examine residues after a washout period. Two other groups received IRP001 chloride feed additive for 28 days at either 0.3g/kg or 0.03g/kg mixed into their feed (i.e., 0.3 g IRP001 chloride in 1 kg of feed or 0.03 g IRP001 chloride in 1 kg of feed) and were subsequently fed on regular food for a washout period of 14 days prior to euthanasia and tissue collection.

IRP001 chloride was extracted from lg samples of three muscle tissues taken from each bird (in each case from breast, upper leg and lower leg). The residual mass of IRP001 chloride was determined using LC-MS/MS. The method allowed IRP001 to be detected with a lower limit of 2 ng IRP001/g tissue. The assay was fully validated during each assay run and proved to be quantitative to be better than ± 20 % accuracy at 5 ng/g tissue. Levels lower than 2 ng IRP001/g were found to be within the baseline noise of the assay and were below the lower limit of detection (LLOD), i.e. IRP001 was not detectable.

In one embodiment, the method was optimised so that IRP001 chloride could be detected with certainty at 2ng/g tissue. The assay was fully validated during each assay run and proved to be quantitative to better than +20% accuracy at 4ng/g or 5ng/g tissue. Levels of lng/g tissue or below were found to be within the baseline noise of the assay and were below the lower limit of quantitation (LLOQ).

Residues of berberine were detectable and quantifiable after feeding for 35 days at the high IRP001 chloride concentration. The mean residue levels (n = 3) at the high feed additive concentration after 35 days feeding without washout were 6.1ng, 5.5ng and 11.6ng per gram of tissue in breast, lower leg and upper leg tissue respectively. A washout effect was evident at the high feed additive concentration in all three muscle tissues, reaching levels of approximately lng/g, below the LLOQ after 4 days washout. At the low concentration of feed additive the mean residue levels were less than lng/g, below the LLOQ, in all cases, with or without washout.

All residue levels determined in the study were below the nominated safe residue level of 13ng/g, even when measured after 35 days feeding at 0.3g IRP001 chloride/kg feed without a washout period.

The residue levels in the liver after the high feed additive concentration were above 13 ng/g without washout but below 13 ng/g after one day of washout. Given the average consumption of chicken liver is limited, the levels of IRP001 in liver do not represent cause for concern.

The data taken as a whole indicate that the risk of cancer resulting from consumption of chicken meat from IRP001 chloride-fed chickens is less than one in a million at feed additive levels equal to or less than 0.3g berberine/kg feed.

Berberine levels in chicken muscle (i.e. chicken meat) were below the LLOD after dosing at 0.03 IRP001/kg feed, or after 4 days of washout after dosing at 0.3 g IRP001/kg feed.

Introduction

As described elsewhere, in the US, the Food and Drug Administration (FDA) is responsible for the approval of human and animal drugs and feed additives which are governed by the Federal Food, Drug, and Cosmetic Act (FD&C Act).

The FD&C Act requires that compounds intended for use in food-producing animals are shown to be safe and that food produced from animals exposed to these compounds is shown to be safe for consumption by people. In particular, the use in food-producing animals of any compound found to induce cancer when ingested by people or animal is prohibited by statute (21 CFR Part 500, Subpart E - Regulation of carcinogenic compounds used in food-producing animals) unless certain conditions are met (the so-called “Diethylstilbestrol (DES) Proviso”). Under the DES proviso use of a suspected carcinogenic compound is not prohibited if it can be determined by prescribed methods of examination that “no residue” of that compound will be found in the food produced from food-producing animals under conditions of use reasonably certain to be followed in practice.

Thus, if the FDA decides that berberine should be regulated as a carcinogenic compound, US statue prohibits the use of berberine in food-producing animals unless the “no residue” DES proviso applies. The term “no residue” refers to any residue remaining in the edible tissues of food-producing animals that is so low that it presents an insignificant risk of cancer to consumers. More specifically, an insignificant risk of cancer is defined as a 1 in 1 million increase in risk.

Despite the recorded safety of berberine, a toxicology study was commissioned by the US Government (National Centre for Toxicological Research) and this study identified potential carcinogenicity in a high-dose chronic rodent study [13].

As a result to obtain GRAS status it has been necessary to estimate the maximum residue of berberine in chicken meat that would be acceptable, given the typical lifetime consumption of chicken meat. To ensure lower than a one in a million risk of cancer resulting from chicken consumption, it has been estimated that the maximum acceptable residue is 13ng berberine per gram of chicken meat (i.e. breast or leg muscle tissue).

To investigate whether the disclosed feed additive is safe and suitable for GRAS status at specified doses a suitable residue trial was conducted. Invetus Pty Ltd was contracted to conduct a trial, collect tissue and Monash University was contracted to assay tissue samples for berberine. Residue Study Design

The protocol for this study using broiler chickens is annexed to the Example as Appendix B. Two concentrations of IRP0001 chloride were investigated: 0.3g/kg feed and 0.03g/kg feed, representing high and low concentrations of feed additive.

One hundred and eighty birds were split into 18 pens, each containing 10 birds. To represent the typical farming process for broiler chickens, test birds received feed with additive for 35 days at either the high or low concentration. After 35 days one group at each additive concentration was euthanised for tissue collection (6 largest birds in each pen).

To investigate whether elimination (metabolism and excretion) of IRP001 chloride was evident when feed containing IRP001 chloride was replaced with regular feed, other groups received IRP001 chloride for 35 days and then were given regular feed for either 1, 2, 4 or 7 days prior to euthansia and tissue collection. Two additional groups received IRP001 chloride for 28 days and then regular feed for 14 days (i.e a 14 day washout). Parallel control groups were treated in exactly the same manner except that the control birds received regular feed throughout the study. In all cases, samples were taken from three regions of muscle tissue (breast, upper and lower thigh). Samples were collected, frozen and shipped for analysis. Table 19 summarises the study design showing the concentration of IRP001 used and the feeding regimen for each of the 18 groups of birds in the residue study.

Table 19 Summary of the feeding regime for each group of broilers NB Controls received regular feed without additive * asterisks indicate estimates < LLOQ Performance of the LC-MS/MS Assay

Details of the assay methods used for tissue extraction and LC-MS/MS are summarised in Appendix A. The assay of berberine was calibrated initially from simple solutions and subsequently methods for assay after tissue extraction were validated.

Berberine peaks from tissue samples could be detected at concentrations as low as 2ng/g tissue, but intereference due to tissue matrix effects and analyte carryover at lng/g tissue made quantitation of IRP001 difficult at this or lower concentrations. At 5ng/g (or 4ng/g on some occassions) the assay could be validated as accurate at ±_20% true analyte concentration. In the results section IRP001 levels greater than 5ng/g are quoted as absolute values, IRP001 levels between 2 and 5 ng/g are considered to be below the LLOQ and outputs indicating values lower than 2 ng/g are considered to be within baseline noise, below the LLOD, and as such are not detectable. In one embodiment, berberine peaks from tissue samples could be detected at concentrations as low as lng/g tissue, but intereference due to tissue matrix effects and analyte carryover at lng/g tissue made quantitation of IRP001 difficult at this or lower concentrations. At 5ng/g (or 4ng/g on some occassions) the assay could be validated as accurate at ± 20% true analyte concentration. Realistically a concentration of less than 2ng/g can be considered to be below the lower limit of quantitation (LLOQ). The lower limit of peak detection was 1-2ng/g.

Results

Tissue samples from 3 birds from each feed additive group were received by the Monash analytical team and analysed by LC-MS/MS. A single sample from each control group was assayed.

Table 20 shows mean concentration of berberine and standard deviation determined for each muscle tissue excised from 3 birds in each group. One representative from each control group was assayed and these values were found to be effectively zero, expressed in the results table as below the LLOD “<LLOD”, i.e. not detectable.

Broadly speaking the breast tissue samples, upper and lower leg muscle samples were comparable and despite the low concentrations determined the data shows distinct and logical trends. At the low feed additive concentration of 0.03g/kg feed, mean residues of berberine were not detectable in all cases, with or without washout (i.e. below the LLOD and LLOQ).

At the higher IRP001 concentration of 0.3g/kg feed, the mean berberine residues after 35 days were in the quantifiable range; 6.1 ± 1.6 for breast, 5.5 ± 3.0 ng/g for lower leg and 11.6 ± 6.6 ng/g for upper leg tissue. In both tissues a progressive washout was evident. Berberine residues fell after 1 and 2 days and after 4 days the berberine levels were below the LLOD.

Table 20 Residues of IRP001 chloride in muscle tissues

NB < LLOD = below the lower limit of detection (i.e. not detectable )

* asterisks indicate estimates < LLOQ (below the validated lower limit of quantitation)

Table 21 shows mean concentration of berberine and standard deviation determined for liver tissue excised from 3 birds in each group. One representative from each control group was assayed and these values were found to be effectively zero, expressed in the results table as below the LLOD “<LLOD”, i.e. not detectable.

Table 21 Residues of IRP001 chloride in liver tissues NB

< LLOD = below the lower limit of detection (i.e. not detectable )

* asterisks indicate estimates < LLOQ (below the validated lower limit of quantitation)

Conclusions All residue levels in muscle tissue (chicken meat) determined in the study were below the nominated safe residue level of 13ng/g, even when measured after 35 days feeding at 0.3g berberine/kg feed without a washout period. Residue levels at the lower IRP001 concentration of 0.03g/kg feed were determined to be less than 2ng per gram of tissue in all cases and can be considered to be not detectable. Residue levels in liver were above the limits of quantitation after birds were fed with 0.3g IRP001/kg feed, were reduced by washout period over 7 days, and reduced to below the limit of quantitation after a 14-day washout. Residue levels in liver after birds were fed with 0.03 g IRP001/kg feed were below the limit of detection before and after washout.

Appendix A Analytical Methods

Berberine was assayed by LC-MS/MS using tetrahydropalmitine as an internal standard. Preparation of tissue samples

1. Approximately lg of tissues were cut out and weighed into M-tubes. The tissues were stored in a freezer at -20°C until they were ready to be homogenised. 2. For each gram of tissue, 2 volumes of MilliQ water was added to the tubes.

3. The M-tubes were attached onto the GentleMACS homogeniser and the program method RNA_01_01 (60 seconds) was run 3 times to ensure that the tissue was completely homogenised.

4. The tissue homogenates were distributed into Eppendorf tubes in 200 μL aliquots. 5. To each 200 μL aliquot of tissue homogenate, 10 μL internal standard solution was added, followed by 600 μL of 100% methanol. Samples were vortexed at maximum setting for 3 x 10 seconds and then centrifuged at 10,000 rpm for 3 minutes.

6. 100 μL of supernatant was transferred into LC vials for analysis. Method validation

1. The method was validated for selectivity, linearity, LLOQ, accuracy, precision, recovery, stability and matrix effect.

2. Selectivity was assessed by preparing samples spiked with individual analyte at concentrations up to 500 ng/g with 5 replicates each. The peak signal was compared with the calibration standards (spiked with analytes) to ensure that there was no interference.

3. To evaluate LLOQ, the 5 ng/g and 10 ng/g standards were prepared at 6 replicates. The LLOQ was determined at the lowest concentration of the calibration curve which both precision and accuracy were ≤ 20%. 4. For an indication of accuracy and precision, 4 concentration levels of 20, 50, 100 and 500 ng/g were prepared (5 replicates each). Accuracy was denoted as bias (%) from the nominal concentration and precision was denoted as the relative standard deviation (RSD) of the replicates.

5. To evaluate recovery, matrix recovery samples were prepared by extracting blank tissue and then spiking with the analyte solutions to give various concentration levels up to 500 ng/g (5 replicates each). The recovery was defined by the ratio of the mean peak area of extracted samples to the mean peak area of matrix recovery samples.

6. To evaluate bench-top stability, 4 concentration levels of 20, 50, 100 and 500 ng/g were prepared at 5 replicates each, where they were kept at room temperature for 30 minutes prior to extraction. The mean peak area was compared to that of freshly-prepared standards.

7. To evaluate matrix effect (ME), 4 concentration levels of 20, 50, 100 and 500 ng/g in neat solution were prepared at 5 replicates each. ME was defined as the ratio of the mean peak area of recovery samples to that of the neat standard samples. Table 22 LCMS Assay conditions

Appendix B

COMPLIANCE This tissue residue depletion study was conducted according to the agreed protocol utilising SOPs and good scientific practice.

STUDY DESIGN a. Experimental Unit: Both the experimental and observational unit was the individual animal. The statistical unit was the treatment group. b. Animal Model: Feed intake, daily water consumption, weight change, mortality and marker residue in tissues were used as outcome parameters. c. Inclusion Criteria: Animals were selected for the study if they met the criteria outlined in below. d. Exclusion and Removal Criteria: Animals that, on receipt, are debilitated, suffering from disease, injury, or otherwise unsuitable for inclusion in the study, in the opinion of the

Investigator, were excluded. Subsequent to selection, animals that may be deemed unsuitable for continuation in the study will only be removed with the documented concurrence of the Sponsor or Investigator. The reason for any removal will be fully documented and justified in the raw data and Study Report. Any animal that is removed from the study will receive appropriate veterinary care. e. Allocation: Broiler Chicks: On receival the one hundred and eighty (1801 broiler chicks that met the inclusion criteria were sequentially allocated as they were removed from the transport container to eighteen (18) individual treatment groups, each of ten (10) birds. The method of allocation and randomisation was described in the raw data and Study Report. f. Blinding: Not applicable. INVESTIGATIONAL VETERINARY PRODUCT (IYP)

All formulation details including batch number, expiry date, receipt and usage were recorded. a. Investigational Veterinary Product: IRP001 Cl as 100% IRP001 Cl. b. Source: The IVP was supplied by the Sponsor. c. Storage: The IVP was stored at ambient temperature in a temperature designated area. The storage location and conditions of the IVP were recorded. d. Safety: A SDS or its equivalent (if available) was provided by the Sponsor. e. Assays: A Certificate of Analysis (if available) was provided for the IVP. f. Drug Disposal: The disposal of all remaining IVP was recorded.

TREATMENT a. Dose Calculation: Doses were based on fixed concentrations of IRP001 Cl in feed (0.03 or 0.1 g/kg IRP001 Cl). b. Dose Preparation: Powdered IRP001 Cl was incorporated with raw commercial feed ingredients then thoroughly mixed in, for example a “concrete mixer” type apparatus, to provide the final concentrations in feed as outlined. c. Method of Dose Administration: Study animals were dosed according to the treatment regime detailed in Table 23 below. Medicated feed was provided to chickens in the relevant treatments ad libitum as their sole source of feed.

Table 23 Treatment regime - feed conversion ratio * Euthanasia

** Note: Medicated feed is withdrawn from Groups 6 and 12 on Day 28 to allow a 14 day washout period for these groups. SCHEDULE OF EVENTS

Table 24 Schedule of events

TEST SYSTEM

Animal details were recorded in the raw data. That is: Species, broiler chickens; Number, 180; Source, commercial (one batch of 90); Age, one day old.

ANIMAL MANAGEMENT a. Animal Welfare: Study animals were managed similarly and with due regard for their welfare. Study animals were observed according to Animal Ethics Committee (AEC) requirements and a “Record of Animal Care” was completed. b. Health Management: Any routine prophylactic treatments were given as soon as possible, if necessary, and recorded (product name, batch number, expiry date, dose, route and date(s) of administration).

The study animals were observed twice daily according to the standard operating protocol (SOP) in place commencing on Day 0. Any health problem that requires further examination was recorded.

All health problems and adverse events must be reported to the Investigator within one working day. Any adverse event characterised by the Investigator as product related, results in death, is life-threatening, involves a large number of animals, or is a human adverse event, must be recorded and reported to the Sponsor and AEC within one working day.

Normal veterinary care and procedures may be performed and are described in the raw data. Concurrent medications may be administered for standard management practice and humane reasons, with prior approval from the Investigator, and Sponsor (if relevant). No treatments similar to the IVP are administered. All concurrent medications are recorded giving identity of materials used (product name, batch number and expiry date), animal ID(s), the reason for use, route of administration, dose and the date(s) administered, and are included in the raw data (Trial Log) and the Study Report.

If an injury or illness results in euthanasia or death of a study animal, this should be recorded and a post-mortem conducted, if possible, by a veterinarian. A “Post Mortem Report”, including the probable cause of death, is included in the raw data.

All health problems, adverse events and animal mortality, including their relationship to treatment, were included in the Study Report. c. Housing: Chickens were kept in purpose built chicken floor pens by treatment group in two separate and discrete controlled environment rooms at an approved animal facility. One room houses all unmedicated Groups 13 to 18 inclusive birds with the second room housing all medicated birds - Groups 1 to 12 inclusive. Each pen has a floor space of approx. 1.5m ² .

Chickens were raised on litter according to normal commercial practice.

There were 18 floor pens, 10 chickens per pen up to Day 49. The maximum chicken weight of each pen at study conclusion is well below the recommended maximum of 40 kg/m ² for meat chickens in the Australian Code of Practice.

Note - birds in Groups 13 to 18 inclusive (untreated control animals) were maintained in a similar, but physically separate isolation room to medicated Groups 1 to 12 birds thus ensuring no cross contamination during the study. d. Experimental diets: A formulated commercial starter then grower ration was fed throughout the study. A copy of a feed bag label, or equivalent, showing feed composition, was included in the raw data. e. Feed and Water Intake: Weigh and record daily feed added and calculate daily feed intake by treatment group. Measure and record daily water volume and calculate daily water intake by treatment group. f. Animal Disposal: Study animals were humanely euthanised according to AEC approval and recorded at the intervals as outlined in the Schedule of Events ( Table 24). STUDY PROCEDURES a. Trial Log: All scheduled and unscheduled events during the study were recorded.

ASSESSMENT OF EFFECTS a. Body Weights: Chickens were weighed on Days 0 (Group weight) and 7, 14, 21, 28 and 35 days - individual animal weights were recorded. Weigh scales were checked pre- and post-weighing with calibrated test weights and recorded. Body weights at study termination were compared between groups to determine treatment effects (if any). b. Examinations: Individual clinical examinations were performed on euthanasia at the time of gross pathology and tissue collection. Clinical examinations were recorded. Digital still images may be recorded as appropriate. c. Observations: Birds were inspected twice daily for general well-being, typically prior to 8am of a morning, and after 4pm of an afternoon. Thus a typical interval between observations would be 9 hours during the day, and 15 hours overnight. Birds showing abnormal clinical signs were recorded, observed closely and euthanised if deemed to be suffering significantly (e.g. marked depression with low likelihood of recovery) by the Investigator. d. Necropsy Examinations: All birds were euthanised and necropsied between Days 35 and 49 as per schedule - Table 44. e. Gross Pathology: All chickens from all Group 1 through 18 were necropsied and examined for gross visual pathological changes which were described and scored as appropriate by individual bird. f. Tissue Residue Analysis: Duplicate representative samples of liver, kidney, breast muscle (1), leg muscle (2) [upper and lower thigh] and entire skin with fat intact was collected and stored frozen (<10 degrees Celsius) from the six (6) heaviest birds in each group (Groups 1 to 18 inclusive) as per schedule, Table 24, for subsequent marker residue analysis. Groups 13 to 18 birds shall be sacrificed at Day 35 as untreated control birds with tissues collected for tissue assay requirements.

Samples were labelled with adhesive labels listing the study number, animal ID, time point, date, sample type and replicate. For residue analysis, samples were thawed and a known weight of tissue (approximately 1g) homogenised in 2 mL water. Samples were centrifuged and a known volume of the supernatant removed for analysis by LC-MS/MS.

Table 25 Analytical matrix To be analysed if required for assay validation and verification. g. Sample Storage, Transfer & Disposal: Sample storage, transfer and disposal were recorded. Replicate 1 tissue samples were shipped frozen on wet ice to the Analytical Laboratory at times outlined in the Schedule of Events. Samples were transferred according to the standard operating protocol (SOP) with an accompanying temperature data logger and frozen water vial. Replicate 2 tissue samples were retained frozen for a period of 6 months after the last sample collection time-point. Beyond that point they may be discarded at the study site's discretion unless specifically requested not to by the Sponsor's Representative.

STATISTICAL ANALYSIS

Methods were documented in the Study Report. DATA RECORDS

Protocol specifications are to supersede facility SOPs. Study forms may be added or amended as required during the study without the need for a Protocol Amendment or Deviation. a. Protocol Approval: The Protocol is to be approved and signed by all relevant personnel (see page 1) prior to study start. b. Amendments/Deviations: An amendment is a change or modification of the Protocol made prior to execution of the changed or modified task. Amendments must state the reason for the change and have documented authorisation from the Sponsor. The amendment must be signed by the Investigator, and the Sponsor.

Deviations from this Protocol or applicable SOPs are to be documented, signed and dated by the Investigator at the time the deviation(s) are identified. An assessment on the impact on the overall outcome or integrity of the study is to be made. Deviations must be communicated to the Sponsor as soon as practically possible.

All Protocol amendments and deviations are to be recorded accordingly and numbered sequentially based on the date of occurrence or date of identification. c. Notes to File: Notes to File are to be recorded accordingly to clarify events or circumstances that may not otherwise be apparent from the raw data. Notes to File must be communicated to the Sponsor as soon as practically possible. d. Change of Study Personnel: Change of the study Investigator, or other responsible study personnel, is to be recorded accordingly. e. Raw Data: All original raw data pages were paginated, identified with the study number and signed and dated by the person making the observation and by the person recording the information. f. Communication Log: The Investigator maintained copies of all correspondence relating to the study. Any telephone conversations that resulted in a change in the documentation, design, conduct, or reporting of the study, were recorded. g. Permits: The study detailed in this Protocol is to be covered by government agency permit (for example an APVMA small trial permit). STUDY REPORT

A Study Report was prepared by the Investigator, or designee. Data listings of each variable measured was included. The study Investigator’s Compliance Statement was included in the Study Report. The original signed Study report with raw data and Statistical Report appended was submitted to the Sponsor and archived.

EXAMPLE 4 - pig safety study

SUMMARY

This dose titration safety study was conducted to determine the safety of five proprietary plant extracts (IRP001, IRP002, IRP003, IRP004, IRP005) when fed to swine for twenty-eight days. Ninety-six (96) pigs were blocked by weight and gender and allotted to sixteen treatment groups to evaluate safety. Treatment allocation is summarised in Table 26.

Table 26 Study Overview

Ninety-six, recently weaned pigs arrived at the test facility. Upon arrival, the pigs were double ear tagged, weighed, and genders recorded. The pigs were blocked by gender and body weight and allocated to pens. Treatment groups were randomly assigned to sixteen pens. Each pen contained six pigs (3 males and 3 females). Respective test articles were measured out and provided in the feed from Day 0 to 28. All feed containing test article delivered to the pens were weighed throughout the duration of the study. Weekly weighbacks of unconsumed feed were recorded. All pigs had individual clinical observations of health recorded daily. On Days 0, 14, and 28, all pigs were bled (SST and EDTA). The serum samples were used to conduct serum chemistries. The EDTA blood samples were used to conduct a complete blood cell count. All pigs were weighed on a weekly basis. On Day 28, all pigs were euthanised, a gross necropsy conducted, and tissues collected for histopathology.

SUMMARY OF RESULTS

The objective of this study was to demonstrate the safety of IRP001, IRP002, IRP003, IRP004, and IRP005 when fed to swine for twenty-eight days. Based on the data collected and analysed (statistically and/or subjectively) it can be concluded that feeding of these compounds for twenty-eight days is safe. Periods of diarrhea may be evident while consuming the compounds but do not appear to affect the pigs’ ability to gain weight over a twenty-eight day period. Although, for this trial, there were statistical differences found with the hematological and blood chemistry parameters, the actual parameters were within normal ranges or similar to the control values. The clinical significance of the differences is minimal. Lastly, gross and histopathological evaluation of the major target organs does not provide any evidence that there is any toxicity to these organ systems.

STUDY OBJECTIVES

The objective of this study was to demonstrate the safety of IRP001, IRP002, IRP003, IRP004, IRP005 when fed to swine for twenty-eight days.

STUDY APPROVAL

Prior to study initiation, the Veterinary Resources, Inc. IACUC approved the protocol for this study. Table 27 Study Schedule

STUDY DESIGN

Treatment Groups

Ninety-six (96) pigs were blocked by gender and weight and randomly assigned to treatment groups (using Excel random number generator). In addition, the sixteen pens were assigned a random number, sorted in to ascending order and assigned a treatment group. Personnel delivering treatments to the pens were unblinded to treatment groups and were not permitted to be involved in any other study activities. Masking of the Study

The test feeds were prepared at the test facility by the Feed Administrator (Michael Wilgenbusch). The Feed Administrator was the only unmasked personnel during the conduct of the study.

ANIMAL MANAGEMENT Test Animals and Identification

One hundred, mixed gendered (equal number of male castrates and females), approximately three weeks old, commercial cross pigs were purchased from AMVC WeSearch, Audubon, IA. Upon arrival, each pig was tagged with a unique identification number on each ear.

Test Facility and Housing

All pigs were housed at the VRI McCoskey Facility from arrival to study completion. The facility is a conventional nursery building. Heating was provided via propane heaters hung from the ceiling. Ventilation was provided via wall mounted fans, attic vents, and pit fans. The pens were approximately 4.7ft. x 10 ft. with solid sides and plastic slatted flooring. Each pen contained a five-hole plastic nursery feeder and a wall mounted double nipple waterer.

Feed

All feed was prepared on-site by the Feed Administrator. A base feed of corn/soybean meal was provided by Key Coop, Gilbert, IA and was appropriate for the age of pigs being fed. Each test article was mixed at its appropriate concentration with the base feed using a cement mixer. Feed was weighed prior to delivery to each pen. Unconsumed feed was weighed on Study Days 7, 14, 21, and 28. Feed was provided ad libitum via a five hole plastic nursery feeder. The feed was stored at ambient temperature in fibre drums lined with plastic.

Water

Water was provided ad libitum from receipt until the end of the study via nipple waterers. The water was sourced from a rural on-site well. STUDY CONDUCT

Feed Administration/Weighback

On Study Day 0, all pen feeders were emptied of any feed that was being fed during the acclimation period. On Study Day 0, feeding of feed containing the respective test article and concentration was begun. Each pen's/group's feed was weighed on a calibrated scale prior to delivery to each pen. Any additional feed provided between the weighbacks were also weighed prior to delivery. Unconsumed feed was weighed on Study Days 7, 14, 21, and 28. Feed was provided ad libitum via a five-hole plastic nursery feeder. The feed was stored at ambient temperature. Clinical Observations

All pigs were observed once daily from arrival through the end of the study. From Study Day 0 through Study Day 28, all animals were scored for clinical observations. The observations included Fecal Score, Gauntness Score, and Depression Score.

Body Weight

All pigs were weighed on Study Days -4, 0, 7, 14, 21, and 28. Weighing was done on a previously calibrated scale and checked on each day of weighing using check weights.

Blood Collection and Analysis

On Days 0, 14 and 28, blood samples were collected from each pig. Approximately, 12.0 mFs of blood were collected in to a serum separator tube and 5.0 mFs of blood collected in a tube containing EDTA (lavender top). Both tubes were submitted to the Iowa State University Clinical Pathology Faboratory for testing.

The following clinical pathology parameters were evaluated for each blood sampling period. Clinical pathology parameters on the blood samples included hematology and serum chemistry with the following parameters evaluated:

• Hematology (CBC w/ automated differential) o WBC (RDW) o RBC (Platelet Count) o Hemoglobin o MCV o MCHC o Automated Differential (Neutrophils, Lymphocytes, Monocytes, Eosinophils, Large Unidentified Cells)

• Serum chemistry (Large Animal Routine Panel - ISU Clinical Pathology) o BUN (Blood urea nitrogen) o CK (Creatine kinase) o Creatinine o AST o Glucose o Alk Phos o Total Protein o GGT o Albumin o Total Bilirubin

Necropsy

A necropsy was conducted on all pigs on Day 28. A formalin fixed section of liver, kidney, small intestine, and colon were collected from each pig. Tissue samples were submitted to ISUVDL for further histopathological examination by a pathologist.

Disposition of Animals

All pigs were disposed of via rendering by a commercial carcass disposal company (DarPro.,

Des Moines, IA).

STATISTICAL PROCESS

Data were analysed using Proc Mixed (SAS 9.4; Cary, NC). Pig was the experimental unit, with exception of feed intake where pen was the experimental unit. Dietary treatment was the fixed effect. Analysis of variance (ANOVA) was generated 5 separate times, to compare independently each dose of the IRP test article to the control. A P- value <0.05 was considered significant. Normal distribution of data and outliers were determined via Proc Univariate (SAS 9.4). RESULTS

Body Weight, Growth Performance (Average Daily Gain), and Feed Intake (ADFI)

There were no significant differences in any group body weights during any timepoints or over the entire duration of the study when compared to the control group body weights ( Tables 32 to Table 36 of Appendix).

There were statistically significant differences for the analysis of average daily gain. More specifically, differences were noted for IRP001 (for study days 7 to 14 and from study days 14 to 21), for IRP003 (for study days 14 to 21), and for IRP005 (for study days 14 to 21). There were no differences noted in the IRP002 and IRP004 groups. It should be noted that there were no differences in any of the groups when analysed over the entire duration of the study (study days 0 to 28). Numerically, the ADFI was similar to the control group (0.69 kg) with a range from 0.60 kg to 0.75 kg for all the groups. Clinical Observations

The clinical observation parameters were not statistically analysed. No abnormal observations were observed in any of the groups for gauntness and behavior observations. Abnormal fecal observations were observed in groups consuming IRP001, IRP002, and IRP005. Group 1 (IRP001 - 0.07 g/kg), Group 2 (IRP001 - 0.7g/kg), Group 5 (IRP002 - 0.5 g/kg), and Group 14 (IRP005 - 0.2 g/kg) had 1-2 pigs that were scored a 2 (diarrhea) for 1-3 days. Group 3 (IRP001 - 0.7 g/kg) had four to five pigs with a score of 2 from Study Days 9 to 12 and continued with at least one pig with a score of 2 from Study Days 13 to 23. Clinical Pathology Blood Parameters

The hematological parameters and serum chemistries with statistical differences are summarised in the tables below ( Tables 37 to Table 61 of the Appendix). Hematological parameters

Hematological parameters for IRP001, IRP002, IRP004 and IRP005 compared to a control (0.00 g/kg) with statistical differences are shown below. Table 28A Summary of hematological parameters for IRP001 fed at 0.07, 0.70, and 1.50 g/kg compared to control ( 0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05).

Table 28B Summary of hematological parameters for IRP002 fed at 0.05, 0.50, and 1.50 g/kg compared to control ( 0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ ( P < 0.05). Table 28C Summary of hematological parameters for IRP004 fed at 0.0084, 0.0800, and 0.8000 g/kg compared to control ( 0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05).

Table 28D Summary of hematological parameters for IRP005 fed at 0.02, 0.20, and 1.00 g/kg compared to control ( 0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05).

Serum chemistries

A summary of serum chemistries with statistical differences for IRP001, IRP002, IRP004 and IRP005 are given below. There were no statistical differences found for IRP003. Table 29 A Summary of serum chemistries for IRP001 fed at 0.07, 0.70, and 1.50 g/kg compared to the control (0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05). Table 29B. Summary of serum chemistries with statistical differences for IRP002 fed at 0.05, 0.50, and 1.50 g/kg compared to the control (0.00 g/kg) a' ^b ' ^c Within a row, east square means lacking a common superscript differ (P < 0.05). Table 29C Summary of serum chemistries with statistical differences for IRP004 fed at 0.0084, 0.0800, and 0.8000 g/kg compared to the control (0.00 g/kg ) a' ^b ' ^c Within a row, east square means lacking a common superscript differ (P < 0.05).

Table 29D Summary of serum chemistries with statistical differences for IRP005 fed at 0.02, 0.20, and 1.00 g/kg compared to the control (0.00 g/kg) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05).

Necropsy and Histopathological Lesions

Gross lesions were observed in three pigs at the Day 28 necropsy. Those findings are listed below:

• Pig No. 30 (Group (Gp) 4 - IRP002, 0.5 mg/kg) - pericarditis and peritonitis (chronic)

• Pig No. 46 (Gp 13 - IRP005, 0.02 mg/kg) - pericarditis and peritonitis (chronic)

• Pig No. 53 (Gp 16 - No treatment) - peritonitis (chronic) Histopathologic examination of the submitted tissues (liver, kidney, intestine, colon) did not reveal lesions compatible with toxicity. Examination of the tissues occasionally revealed mild, nonspecific and incidental findings. Tissues were unremarkable otherwise.

COLLECTION AND RETENTION OF RAW DATA All original raw data and study records generated by Veterinary Resources Inc. were forwarded to the Sponsor. An exact copy of all study records was maintained by Veterinary Resources Inc., 1523 South Bell Ave., Suite 106, Ames, IA 50010. Applicable test facility standard operating procedures (SOPs) are on file at the testing facility.

CONCLUSIONS The objective of this study was to demonstrate the safety of IRP001, IRP002, IRP003, IRP004, and IRP005 when fed to swine for twenty-eight days. Based on the data collected and analysed (statistically and/or subjectively) it can be concluded that feeding of these compounds for twenty-eight days is safe. Periods of diarrhea may be evident while consuming the compounds but do not appear to affect the pigs’ ability to gain weight over a twenty-eight day period. Although, for this trial, there were statistical differences found with the hematological and blood chemistry parameters, the actual parameters were within normal ranges or similar to the control values. The clinical significance of the differences is minimal. Lastly, gross and histopathological evaluation of the major target organs does not provide any evidence that there is any toxicity to these organ systems.

EXAMPLE 4 APPENDIX

Table 30 Normal Hematology Ranges (ISU Clinical Pathology Laboratory )

Table 31 Normal Blood Chemistry Ranges (ISU Clinical Pathology Laboratory )

Table 32 Difference of least square means for body weight, growth performance and feed intake for IRP001 fed at 0.07, 0.70, and 1.50 g/kg compared to the control (0.00 g/kg) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05). Table 33 Difference of least square means for body weight, growth performance and feed intake for IRP002 fed at 0.05, 0.50, and 1.50 g/kg compared to the control (0.00 g/kg) Table 34 Difference of least square means for body weight, growth performance and feed intake for IRP003 fed at 0.007, 0.070, and 0.700 g/kg compared to the control (0.00 g/kg) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05). Table 35 Difference of least square means for body weight, growth performance and feed intake for IRP004 fed at 0.0084, 0.0800, and 0.8000 g/kg compared to the control (0.00 g/kg) Table 36 Difference of least square means for body weight, growth performance and feed intake for IRP005 fed at 0.02, 0.20, and 1.00 g/kg compared to the control (0.00 g/kg) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05). Table 37 Difference of least square means for hematology for IRP001 fed at 0.07, 0.70, and 1.50 g/kg compared to the control ( 0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ ( P < 0.05). Table 38 Difference of least square means for hematology for IRP001 fed at 0.07, 0.70, and 1.50 g/kg compared to the control ( 0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05). Table 39 Difference of least square means for hematology for IRP002 fed at 0.05, 0.50, and 1.50 g/kg compared to the control ( 0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05). Table 40 Difference of least square means for hematology for IRP002 fed at 0.05, 0.50, and 1.50 g/kg compared to the control ( 0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05). Table 41 Difference of least square means for hematology for IRP003 fed at 0.007, 0.070, and 0.700 g/kg compared to the control (0.00 g/kg ) Table 42 Difference of least square means for hematology for IRP003fed at 0.007, 0.070, and 0.700 g/kg compared to the control (0.00 g/kg) Table 43 Difference of least square means for hematology for IRP004 fed at 0.0084, 0.0800, and 0.8000 g/kg compared to the control ( 0.00 g/kg ) Table 44 Difference of least square means for hematology for IRP004 fed at 0.0084, 0.0800, and 0.8000 g/kg compared to the control ( 0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05). Table 45 Difference of least square means for hematology for IRP005 fed at 0.02, 0.20, and 1.00 g/kg compared to the control ( 0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ ( P < 0.05). Table 46 Difference of least square means for hematology for IRP005 fed at 0.02, 0.20, and 1.00 g/kg compared to the control ( 0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ ( P < 0.05). Table 47 Difference of least square means for blood chemistry for IRP001 fed at 0.07, 0.70, and 1.50 g/kg compared to the control (0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05). Table 48 Difference of least square means for blood chemistry for IRP001 fed at 0.07, 0.70, and 1.50 g/kg compared to the control (0.00 g/kg ) Table 49 Difference of least square means for blood chemistry for IRP001 fed at 0.07, 0.70, and 1.50 g/kg compared to the control (0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05).

Table 50 Difference of least square means for blood chemistry for IRP002 fed at 0.05, 0.50, and 1.50 g/kg compared to the control (0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05). Table 51 Difference of least square means for blood chemistry for IRP002 fed at 0.05, 0.50, and 1.50 g/kg compared to the control (0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05). Table 52 Difference of least square means for blood chemistry for IRP002 fed at 0.05, 0.50, and 1.50 g/kg compared to the control (0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05).

Table 53 Difference of least square means for blood chemistry for IRP003 fed at 0.007, 0.070, and 0.700 g/kg compared to the control (0.00 g/kg ) Table 54 Difference of least square means for blood chemistry for IRP003 fed at 0.007, 0.070, and 0.700 g/kg compared to the control (0.00 g/kg ) Table 55 Difference of least square means for blood chemistry for IRP003 fed at 0.007, 0.070, and 0.700 g/kg compared to the control (0.00 g/kg )

Table 56 Difference of least square means for blood chemistry for IRP004 fed at 0.0084, 0.0800, and 0.8000 g/kg compared to the control ( 0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05). Table 57 Difference of least square means for blood chemistry for IRP004 fed at 0.0084, 0.0800, and 0.8000 g/kg compared to the control ( 0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05). Table 58 Difference of least square means for blood chemistry for IRP004 fed at 0.0084, 0.0800, and 0.8000 g/kg compared to the control ( 0.00 g/kg )

Table 59 Difference of least square means for blood chemistry for IRP005 fed at 0.02, 0.20, and 1.00 g/kg compared to the control (0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05). Table 60 Difference of least square means for blood chemistry for IRP005 fed at 0.02, 0.20, and 1.00 g/kg compared to the control (0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05) Table 61 Difference of least square means for blood chemistry for IRP005 fed at 0.02, 0.20, and 1.00 g/kg compared to the control (0.00 g/kg ) a' ^b ' ^c Within a row, least square means lacking a common superscript differ (P < 0.05).

Histopathology

Submitted tissue was evaluated for any pathological change by a pathologist blinded to the study design.

Concluding comments

Histopathologic examination of submitted tissue did not reveal lesions compatible with toxicity. Examination of tissues occasionally revealed mild, nonspecific and incidental findings. Tissues were unremarkable otherwise.

Histopathological Summary Report (Liver Evaluation)

One animal (ID 53) within treatment group 1 presented with chronic fibrosing lymphoplasmacytic hepatitis and peritonitis with intralesional bacteria; one animal (ID 30) in treatment group 5 presented with chronic fibrosing peritonitis; one animal (ID 74) in treatment group 6 presented with mild fibrosing pleuritis, one animal (ID 32) in treatment group 9 presented with mild lymphocytic periportal hepatitis; two animals (ID’s 38 and 68) in treatment group 10 presented with mild lymphocytic periportal hepatitis; one animal (ID 56) in treatment group 11 presented with mild lymphocytic periportal hepatitis; two animals (ID’s 4 and 11) in treatment group 12 presented with mild lymphocytic periportal hepatitis; one animal (ID 89) in treatment group 12 presented with moderate lymphocytic periportal hepatitis; one animal (ID 46) in treatment group 14 presented with chronic peritonitis.

Histopathological Summary Report (Kidney Evaluation)

One animal (ID 65) in treatment group treatment group 6 presented with mild multifocal lymphocytic nephritis; one animal (ID 97) in treatment group 8 presented with mild lymphocytic interstitial nephritis.

Histopathological Summary Report (Small Intestine Evaluation)

No animals demonstrated any histopathology in the small intestine.

Histopathological Summary Report (Colon Evaluation)

One animal (ID 33) in treatment group 6 presented with mild cryptitis; one animal (ID 31) in treatment group 13 presented with mild cryptitis. EXAMPLE 5 - LCMS stability study

Objective

The objective of the study described in Example 5 was to determine the stability of plant extracts piceid, ursolic acid, and berberine in broiler diets as measured by LCMS. Treatments

Seven different treatments were tested in grower broiler diets. Feeds were manufactured at the O.H. Kruse Feed Technology Innovation Center, Kansas State University in accordance with GMPs (Good Manufacturing Processes).

Table 62 Experimental Feed Compositions

The basal diet was used to manufacture 110 lb. batches (50 kg) of the experimental treatment diets containing the additives. Dry ingredients were added manually and mixed for 240 seconds at room temperature. The mixed feed was then manually discharged into 55-gallon barrels. Batching data was recorded on a master formula sheet. Pellets were produced from mash diets via steam conditioning (5” diameter x 36” length) and subsequently using a pellet mill (Model CL5 California Pellet Mill Co., Crawfordsville, IN) equipped with a 5/32”x 7/8” die for broiler diets. Mash feed was then placed in the hopper of the pellet mill and the feeder was set at a constant rate to achieve approximately 2 lbs. per minute. The target conditioning temperature of 85°C was achieved by adjusting (increasing) steam addition and conditioning time was approximately 30 seconds. Pellets were then collected in cooling trays as they exited the pellet die. Pellets were cooled with ambient air for approximately 10 minutes in counter-flow cooler. A 20 lb. flush was run between each of the treatments.

Sampling

Mash feed (manufactured the day before; 0.5 lbs) was sampled by cutting the feed stream as the material was discharged to the barrels (5 sub samples) to create a composite sample. Cooled pellet samples (0.5 lbs) were collected for analysis. Feed Ingredients

The pelleted feed was not fed to poultry. A Safety Data Sheet for each product was provided by the.

Table 63 Broiler Grower Diet

Table 64 Samples Tested

Methodology

Standards with certificates of analysis were provided to Aspen. Individual analytical standards of berberine and ursolic acid were prepared in methanol. Piceid acid was prepared in 80:20 methanol: water. A combined standard was prepared from the three individual stock standards in methanol. This combined standard was used to make serial dilutions in methanol.

Samples were analysed in duplicate. Sample “A” was prepared using the pelletised samples and Sample “B” was prepared using the mash samples. lg of sample was extracted with 10 mL of methanol. “A” samples were crushed using a mortar and pestle prior to weighing. Samples were shaken for 30 min using a wrist-action shaker, allowed to settle for 10 min, with the methanol then decanted off into a separate vial.

Samples 52359-28, 29, 32, 33 and 34 were diluted 10:1 in methanol. Sample 52359-30 was diluted 100:1 in methanol and samples 52359 Blank and 52359-31 were not diluted at all. All samples were filtered through a 0.2 pm PTFE syringe filter prior to analysis.

All standards and samples were injected into a LCMS using the conditions in Table 65.

Table 65 LCMS Conditions

Samples were extracted at T0 (one day after receipt and kept on ice until extracted). The samples were then stored at room temperature. Sets were extracted one week later and two weeks later. Spikes were made to three of the samples (of both mash and pellet) and recovered quantitatively ( Tables 66-68). Sequential extractions showed the first extraction to recover 80+% of the compounds in the pellets and 90+% in the mash ( Table 69). Samples were quantified in two batches using calibration curves (CC) generated for a given batch (Figure 20 and Figure 21). Table 66 Spike Recoveries (%) of Berberine

Table 67 Spike Recoveries (%) of Ursolic Acid

Table 68 Spike Recoveries (%) of Piceid Acid

NB: All Spike Calculations for Berberine, Ursolic acid and Piceid were carried out as: recovery = (cone. In solution)/ (expected cone.) *100

Table 69 Summary of Sequential Study

Results

The results of the testing are summarised by additive in Tables 70-72. The method is estimated to be capable of detecting 0.005 g/kg of the additives. Based on the results, the compounds are stable in the feed when stored at room temperature over two weeks.

Table 70 Results for Piceid Acid

Table 71 Results for Berberine

Table 72 Results for Ursolic Acid

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

EXAMPLE 6 - berberine production

This example describes the manufacture of animal food grade quality berberine chloride by liquid extraction of Phellodendron chinense. The manfacture process is shown in Figure 22. Analysis of the manufactured berberine chloride including purity, quality and stability are also described against specifications. Summary of manufacturing process

As shown in Figure 22, a berberine chloride product can be produced by extraction from dry bark of Phellodendron chinense. The berberine chloride product is obtained as an odourless, yellow fine powder, which may be crystalline. The product has a characteristic bitter taste. In order to control possible contamination from the raw material, heavy metals (including lead, arsenic, cadmium and mercury) in the finished product are limited to comply with the EU standards for animal feed in both in method and criteria (EU, 2002; EU 2007). Residues of 127 pesticides are also assessed to ensure that residues, if present, are de minimus (US Pharmacopeia, 2010). The refinement and primary packaging steps are carried out in a clean area. With the control of water content in the product, the compliance with the microbial limit is ensured.

From the nature of extraction method and with the control to the manufacturing process and product, it is feasible to manufacture a product of animal food grade quality to meet the specifications described in Table 73.

The specifications cover a number of criteria: appearance; identification, as determined by High Performance Thin layer Chromatography (HPTLC); tests including purity e.g. sieve analysis, loss on drying, ash content, heavy metals content, solvent and pesticide residues, assay by High Performance Liquid Chromatography (HPLC) and microbiological criteria.

HPLTC Identification

Materials and methodology

Standard

Berberine Chloride (Chinese National Institutes for Food and Drug Control)

Equipment

Digital Reprostar-3 (CAMAG, Switzerland)

Automatic Development Chromatogram ADC2 (CAMAG, Switzerland)

Double slot expansion cylinder 100x100 mm Microporous membrane: 0.45 pm Preparation of solutions Preparation of standard solution

Berberine chloride standard (10 mg) is dissolved in 10 mL methanol.

Preparation of test sample solution

Ten milligrams of the test sample to be examined is accurately weighed into a 10 mL volumetric flask. Methanol is added to make up to volume and then the solution is passed through a Millipore filter (0.45 pm).

Chromatographic conditions

Stationary phase: Silica gel 60

Mobile Phase: ethyl acetate, methanol, formic acid, water (10 : 2 : 1.2 : 0.6, v/v/v/v)

Development: development for 8 cm, with saturation 15 min.

Sample volume: 10 μL

Detection: (1): Dry the plate in a current of air. Examine under 254 nm and 366 nm.

(2): Dry the plate in a current of air. Spray with 10% sulfuric acid reagent, dry in cool air, heat at 105°C for 3 min, examine under 254 nm, 366 nm and visible light.

Sample testing

Accurately sampling the standard and test sample solutions on the same plate, develop the plate under the chromatographic conditions.

HPLC Assay

Materials and methodology Standard

Berberine Chloride (Chinese National Institutes for Food and Drug Control)

Reagents

Methanol (analytic grade) Acetonitrile (chromatographic grade)

Water (distilled water)

Phosphoric acid (analytic grade)

Dodecyl sulfonic acid sodium salt (analytic grade)

Equipment

HPLC with UV detector

Analytical balance (accurate to 0.01 mg)

Flasks for preparation of mobile phase and 10-mL, 25-mL, 50-mL volumetric flasks for preparation of standard and test sample solutions

Preparation of mobile phase solution:

Acetonitrile (250 mL, chromatographic grade) and 250 mL of a 0.1% phosphoric acid aqueous solution accurately transferred into a flask, shaken, and 0.5 g dodecyl sulfonic acid sodium salt (Analytic grade) then added, sonicated for lOmin, cooled to room temperature.

Preparation of standard solution:

Accurately weigh about 10 mg of reference standard in a 50-mL volumetric flask. Add about 40 mL of mobile phase solution and sonicate for 30 min. Cool to room temperature and dilute with mobile phase solution to the volume, mix well, and filter through a 0.45 micrometer membrane. Then transfer 5.0 mL of the filtrate to a 10-mL volumetric flask, dilute with mobile phase solution to the volume, and mix well.

Preparation of sample solution:

Accurately weigh about 10 mg of sample to be examined to a 50-ml volumetric flask. Add 40 mL of mobile phase solution and sonicate for 30 min. Cool to room temperature and dilute with mobile phase solution to the volume, mix well, and filter through a 0.45 micrometer membrane.

Transfer 5.0 mL of the filtrate to a 10-mL volumetric flask, dilute with mobile phase solution to the volume, and mix well. Chromatographic conditions

Column : Intersil ODS-3 C18 (150 mmx4.6 mm, 5 mhi)

Wavelength: 265 nm Flow rate: 1.0 mL/min Temperature: room temperature

Mobile phase: acetonitrile: 0.1% phosphoric acid in water solution (50 : 50, V/V) (O.lg sodium dodecyl sulphate added to each 100 mL solution)

Injection Volume: 20 mΐ _^

System suitability: the number of theoretical plates, N, should be not less than 4000 calculated as the standard berberine chloride peak.

Sample testing

Standard solution accurately injected for three times when the chromatographic system is stable under the stated conditions. The RSD should be not more than 2.0%. Equal volumes of standard solution and sample solution are separately injected into the instrument, chromatograms recorded, and the peak responses of berberine chloride measured. Under the stated conditions, the retention time of berberine chloride is found to be approximately 6 minutes.

Calculation

Percentage of berberine chloride is calculated with the following formula:

Content (%) = [(A ₁ x W ₀ x V1 x K) / (A ₀ x V ₀ x W ₁ x (1-d))] x 100 In which:

A ₁ - Peak area of berberine chloride in the sample solution A ₀ - Peak area of berberine chloride in the standard solution W ₁ - Weight of the sample (mg)

W ₀ - Weight of the reference standard (mg) V ₁ - Total volume of the sample solution (mL)

V ₀ - Total volume of the standard solution (mL)

K - Purity of standard reference d - Loss on drying of the sample (%) Consistency of quality in batches of manufactured berberine chloride

Three batches (BN Chl20160513, Chl20160922 and Chl20170304) were manufactured, stored and tested by Shaanxi Jiahe Phyrochem Co. Ltd. (Jiahe), No. 45 Hongguang Road, Heping Industrial Park, Xi’an, Shaanxi, China.

As summarised below, the results from analysis of three nonconsecutive batches of extracted berberine chloride (BN Chl20160513, Chl20160922 and Chl20170304) demonstrate that the characters, identification, impurities and microbial limits of the samples conform to the required specifications.

The contents of berberine meet the criterion (NLT 97.0%). The impurities from heavy metals, pesticides residue and solvent residue are all below the recommended limits for feed additives (heavy metal limits are those established in the EU for animal feed (EU, 2002); pesticide residues and microbial limits conform to US Pharmacopoeia). The tests for loss on drying and total ash are acceptable against the specifications. In summary, the consistency of the product quality is well supported by these test results.

Table 73 Specifications Stability of manufactured berberine chloride

Summary

Stability study data from three nonconsecutive batches of extracted berberine chloride are presented. The study was on production size batches (Batch Number: Chl20090511, Chl20090609 and Chl20090711) manufactured by Jiahe. Thirty-six month long term studies have been completed.

Berberine chloride is stable under all storage conditions tested. All tested parameters at all time points tested in the studies under the long term conditions (25±2°C /60±10%RH) are within the specifications. Therefore, the stability study data supports a 36 month shelf life when stored at or below 25°C.

Stability data

Batches Chl20090511, Chl20090609 and Chl20090711 were manufactured and packed in double plastic bags and stored in paper drum for long term stability study. The test items include appearance, loss on drying and assay using the methods described in product specifications. The batch details and storage conditions are summarised below in Table 74.

Table 74 Batch Details and Storage Conditions

Stability study data from the nonconsecutive batches is shown in Table 75 to Table 77.

Table 75 Stability data for batch Chl20090511

Table 76 Stability data for batch CM20090609

Table 77 Stability data for batch Chl20090711 EXAMPLE 7 - piceid production

This example describes the manufacture of animal food grade quality piceid by liquid extraction of Polygonum cuspidatum. The manfacture process is shown in Figures 23 and 23A. Analysis of the manufactured piceid including purity, quality and stability is also described against required specifications.

Summary of manufacturing process

As shown in Figure 23 and Figure23A, a piceid product can be produced by liquid extraction from the dry root of Polygonum cuspidatum. The piceid product obtained is an off-white to light yellow fine powder with characteristic odor and taste. In order to control possible contamination from the raw material, heavy metals (including lead, arsenic, cadmium and mercury) in the finished product are limited to comply with the EU standards for animal feed in both in method and criteria (EU, 2002; EU 2007). Residues of 127 pesticides are also assessed to ensure that residues, if present, are de minimus (US Pharmacopeia, 2010). The refinement and primary packaging steps are carried out in a clean area. With the control of water content in the product, the compliance with the microbial limit is ensured.

From the nature of the extraction method and with the control to the manufacturing process and product, it is feasible to manufacture a product of animal food grade quality to meet the specifications described in Table 78.

The specifications cover a number of criteria: appearance; identification, as determined by High Performance Thin layer Chromatography (HPTLC); tests including purity e.g. sieve analysis, loss on drying, ash content, heavy metals content, solvent and pesticide residues, assay by High Performance Liquid Chromatography (HPLC) and microbiological criteria.

Consistency of quality in bacthes of manufactured piceid

Three batches (Batch Number: PI161220, PI170210 and PI170428) were manufactured, stored and tested by Shaanxi Guanjie Technology Co. Ltd (Guanjie) Xijing No. 3, Xijing Industrial Park, DianZi western street, Xi’an, Shaanxi, China.

As summarised below, the analyses results from the three nonconsecutive batches of extracted piceid (Batch Number: PI161220, PI170210 and PI170428) demonstrate that the characteristics, identification, impurities and microbial limits of the samples conform to the required specifications. The contents of piceid meet the criterion (NLT 98.0%). The impurities from heavy metals, pesticides residue and solvent residue are all below the recommended limits for feed additives (heavy metal limits are those established in the EU for animal feed (EU, 2002); pesticide residues and microbial limits conform to US Pharmacopoeia). The tests for loss on drying and total ash are acceptable against the specifications. In summary, the consistency of the product quality is well supported by these test results.

Table 78 Specifications Stability study of piceid

Stability study data from three nonconsecutive batches of extracted piceid are presented. The study was on production size batches (Batch Number: PI160315, PI160323 and PI160331) manufactured by Guanjie. Six month accelerated and thirty-six month long-term studies have been completed.

Piceid is stable under all storage conditions tested. All tested parameters at all time points tested in the studies under the long term conditions (25±2°C /60±10%RH) are within the specifications. Therefore, the stability study data supports at least a 24-month shelf life when stored at or below 25°C.

Stability data

Batches PI160315, PI160323 and PI160331 were manufactured and packed in double plastic bags and stored in paper drum for long term stability study. The test items include appearance, loss on drying and assay using the methods described in product specifications. The batch details and storage conditions are summarised below in Table 79.

Table 79 Batch and Storage Details

Stability study data from the nonconsecutive batches is shown in Table 80 to Table 85.

Table 80 Accelerated stability data for batch PI160315

Table 81 Accelerated stability data for batch PI160323

Table 82 Accelerated stability data for batch PI160331 Table 83 Stability data for batch PI160315

Table 84 Stability data for batch PI160323

Table 85 Stability data for batch PI160331 REFERENCES

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