WO2025120326A1

Movatterモバイル変換

Info

Publication number: WO2025120326A1
Application number: PCT/GB2024/053045
Authority: WO
Inventors: Brian Desmond GREEN; Cristina De Castro
Original assignee: Aramune Technologies Ltd
Current assignee: Aramune Technologies Ltd
Priority date: 2023-12-07
Filing date: 2024-12-06
Publication date: 2025-06-12
Anticipated expiration: 2026-06-07
Also published as: GB2636364A; GB202318732D0

Abstract

A polysaccharide, or a composition thereof, wherein the polysaccharide comprises: a backbone of beta-(1-6)-linked galactose residues and terminal residues, wherein the backbone comprises one or more first galactose residues and one or more second galactose residues, and one or more first sidechain comprising one or two arabinofuranose residues, wherein the or each first sidechain is alpha-(1-3)-linked to a first galactose residue of the backbone, and one or more second sidechain comprising a three or more alpha-(1-5)-linked arabinofuranose residues, wherein the or each second sidechain is alpha-(1-3)-linked to a second galactose residue of the backbone. The polysaccharide finds applications including as a feed supplement or a medicament, providing immunomodulatory effects and/or enhanced feed efficiency.

Description

[Formula (I)] The repeating units of the arabinan polysaccharide may comprise or consist of blocks referred to herein as blocks “A”, “E” and “F”. Thus, the repeating units of the arabinan polysaccharide may further comprise block E and/or block F. The repeating units may further comprise block E, which may be represented by Formula (II), as follows: [Formula (II)] The repeating units may further comprise block F, which may be represented by Formula (III), as follows: [Formula (III)] Each block (i.e. block A, block E and block F) comprises a backbone. Each block may be linked by an alpha-(1-5)-glycosidic bond, specifically the backbone of each block. Thus, the repeating units may comprise Formula (I) linked to Formula (II) by an alpha- (1-5)-glycosidic bond. The repeating units may comprise Formula (I) linked to Formula (III) by an alpha-(1-5)-glycosidic bond. The repeating units may comprise Formula (II) linked to Formula (III) by an alpha-(1-5)-glycosidic bond. Preferably block F (if present in the repeating unit) is the least abundant block of A, E and F. Preferably block A is the most abundant block of A, E and F. Block F (if present in the repeating unit) may be about two, about three, about four, about five, about six, about seven or about less abundant than block A. Block F (if present) may be between about one and about two times less abundant than block A. Preferably for every occurrence of F there is between about 4 and about 6 occurrences of A, and for every occurrence of F there is between about 1 and about 2 occurrences of E. Thus, the ratio of A:E:F in the repeating units may be about 2-8:1-3:1 or about 3-7:1-2:1 or about 4- 6:1-2:1. Most preferably the ratio of A:E:F is about 4-6:1-2:1. The ratio of A:F in the repeating units may be about 2-8:1 or about 3-7:1 or about 4-6:1. Most preferably the ratio of A: F is about 4-6:1. The ratio of A:E in the repeating units may be about 2-8:1 or about 2-7:1 or about 2-6:1. Most preferably the ratio of A:F is about 2-6:1. The repeating unit may comprise Formula (IV), defined herein as follows:

[Formula IV] wherein each star of Formula (IV) corresponds to an arabinofuranose, preferably an L- arabinofuranose, more preferably an alpha-arabinofuranose, most preferably an alpha- L-arabinofuranose. The repeating unit of the arabinan polysaccharide may be represented by any one of the combinations shown in the table below: AAAAE AAAAF AAFAAE AAEAAF AAAEA AAAFA AFAAAE AEAAAF AAEAA AAFAA FAAAAE EAAAAF AEAAA AFAAA AAAFEA AAAEFA EAAAA FAAAA AAFAEA AAEAFA AAAAEF AAAAFE AFAAEA AEAAFA AAAEFA AAAFEA FAAAEA EAAAFA AAAEFA AAAFEA AAFEAA AAEFAA AAEFAA AAFEAA AFAEAA AEAFAA AEFAAA AFEAAA FAAEAA EAAFAA EFAAAA FEAAAA AFEAAA AEFAAA AAAFAE AAAEAF FAEAAA EAFAAA Each cell of the table above represents an embodiment of a repeating unit of the arabinan polysaccharide. Thus, the repeating unit of the arabinan polysaccharide may be represented by any one of the 48 examples shown in the table above. The arabinan polysaccharide may comprise “n” repeating units, e.g. “n” repeating units of Formula (I) or Formula (IV) or any of the 48 examples shown in the table above. “n” may be 2 or more, 3 or more, 4 or more, 5 or more 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, or 40 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, or 1000 or more. “n” may be about 5 to about 1000, about 10 to about 500, or about 15 to about 250, or about 15 to about 230, or about 15 to about 220. Preferably “n” is about 15 to about 220 or about 15 to about 230. The arabinan polysaccharide can be used to modulate the immune response of a subject, preferably to stimulate the immune response. The inventors have found that with increasing molecular weight, the modulation of the immune response increases as well per weight amount of arabinan polysaccharide. The average molecular weight of the arabinan polysaccharide or the repeating unit of the arabinan polysaccharide may be at least about 3 kDa, at least about 5 kDa, at least about 10 kDa, at least about 15 kDa, at least about 20 kDa, at least about 25 kDa, at least about 30 kDa, at least about 35 kDa, at least about 40 kDa, at least about 50 kDa, at least about 60 kDa, at least about 65 kDa, at least about 70 kDa, at least about 75 kDa, at least about 80 kDa, at least about 85 kDa, at least about 90 kDa, at least about 95 kDa, at least about 100 kDa, at least about 105 kDa, at least about 110 kDa, or at least about 115 kDa. The arabinan polysaccharide may have an average molecular weight of about 3 kDa to about 200 kDa, 4 kDa to about 180 kDa, 5 kDa to about 160 kDa, 6 kDa to about 140 kDa, about 7 kDa to about 120 kDa, about 8 kDa to about 120 kDa, about 9 kDa to about 120 kDa, about 10 kDa to about 120 kDa, about 20 kDa to about 120 kDa, about 30 kDa to about 120 kDa, about 40 kDa to about 120 kDa, about 50 kDa to about 120 kDa, about 60 kDa to about 120 kDa, about 70 kDa to about 120 kDa, about 80 kDa to about 120 kDa, about 90 kDa to about 120 kDa, about 100 kDa to about 120 kDa or about 110 kDa to about 120 kDa. Preferably the arabinan polysaccharide has a molecular weight of 10 kD to 120 kDa. The arabinan polysaccharide may be administered several different routes, including, for example, oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), transdermal, intracisternal, intraperitoneal, vaginal and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route. Preferably the arabinan polysaccharide is administered orally. The arabinan polysaccharide is non-toxic, particularly in humans. The arabinan polysaccharide is non-toxic at doses up to 100 µg/ml, particularly in humans. The arabinan polysaccharide may or may not be a rhamnogalacturonan, such as rhamnogalacturonan-I of rhamnogalacturonan-II. Pharmaceutical Compositions Pharmaceutical compositions according to the invention may further comprise a pharmaceutically acceptable salt or other form thereof. Pharmaceutical compositions according to the invention may comprise one or more pharmaceutically acceptable excipients, such as carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants, and/or solubility enhancers. Pharmaceutical compositions according to the invention may comprise a pharmaceutically acceptable salt and optionally one or more pharmaceutically acceptable excipients. The pharmaceutical compositions can be formulated by techniques known in the art. The pharmaceutical compositions can be formulated as dosage forms for oral, parenteral, such as topical, transdermal, intramuscular, intravenous, subcutaneous, intradermal, intraarterial, intracardial, nasal, inhalation or aerosol administration (e.g. nasal or oral inhalation). The pharmaceutical composition may be formulated as a dosage form for oral administration. In the present context, the term “pharmaceutically acceptable salt” is intended to indicate salts which are not harmful to a patient. Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfgionic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p- aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in J. Pharm. Sci. 1977, volume 66, issue 2. Examples of metal salts include lithium, sodium, potassium, magnesium salts and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts and the like. The pharmaceutical compositions according to the invention may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19^th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. In addition, the compounds of the invention may form solvates with water or common organic solvents. Such solvates are also encompassed within the scope of the present invention. The pharmaceutical composition may be sterile. The composition may further comprise a buffer system, preservative(s), tonicity agent(s), chelating agent(s), stabilizers and surfactants, which is well known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20^th edition, 2000. The composition may also further comprise one or more therapeutic agents active against the same disease state. Methods to produce controlled release systems useful for compositions of the current invention include, but are not limited to, crystallization, condensation, co- crystallization, precipitation, co-precipitation, emulsification, dispersion, high pressure homogenisation, encapsulation, spray drying, microencapsulating, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes. General reference is made to Handbook of Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99: Protein Composition and Delivery (MacNally, E.J., ed. Marcel Dekker, New York, 2000). It will be appreciated that the preferred route of administration may depend on factors such as the nature of the condition to be treated and the general condition and age of the subject to be treated. The polysaccharide of the invention may be administered by oral, rectal, nasal, inhalation (e.g. nasal inhalation or oral inhalation), pulmonary, topical (including buccal and sublingual), transdermal, intracisternal, intraperitoneal, vaginal and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route. Preferably the polysaccharide is administered orally. For topical use, sprays, creams, ointments, jellies, gels, inhalants, dermal patches, implants, solutions of suspensions, etc., containing the compounds of the present invention are contemplated. For the purpose of this application, topical applications shall include mouth washes and gargles. Compounds of the invention may be used in wafer technology, wafer technology, such as the biodegradable Gliadel polymer wafer, which is useful for brain cancer chemotherapy. Pharmaceutical compositions for oral administration include solid dosage forms such as hard or soft capsules, tablets, troches, dragees, pills, lozenges, powders and granules and liquid dosage forms for oral administration include solutions, emulsions, aqueous or oily suspensions, syrups and elixirs, each containing a predetermined amount of the active ingredient, and which may include a suitable excipient. Compositions for oral use may be prepared according to any known method, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch or alginic acid; binding agents, for example, starch, gelatine or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. Tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in U.S. Patent Nos. 4,356, 108; 4, 166,452; and 4,265,874 to form osmotic therapeutic tablets for controlled release. Formulations for oral use may also be presented as hard gelatine capsules where the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules wherein the active ingredient is mixed with water or an oil, for example peanut oil, liquid paraffin, or olive oil. Aqueous suspensions may contain the active compounds in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide such as lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin. Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as a liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavouring, and colouring agents may also be present. The pharmaceutical compositions of the present invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example a liquid paraffin, or a mixture thereof. Suitable emulsifying agents may be naturally occurring gums, for example gum acacia or gum tragacanth, naturally occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavouring agent and a colouring agent. The pharmaceutical composition may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non- toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3- butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed using synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a solution or suspension for the administration of the prolactin receptor antagonist in the form of a nasal or pulmonal spray. As a still further option, the pharmaceutical compositions containing the compound of the invention can also be adapted to transdermal administration, e.g. by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosal, e.g. buccal, administration. Pharmaceutical compositions for parenteral administration include sterile aqueous and non-aqueous injectable solutions, dispersions, suspensions or emulsions as well as sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use. A medicament may be a composition (e.g. a pharmaceutical composition or an edible composition). A medicament may be a prescription drug, a non-prescription drug, an over the counter medicine, a dietary supplement, a dietary food, a clinical food, an edible product, a tablet, a capsule, a pill, and food products such as beverages or any other suitable food product. The medicament may be an injectable substance or an inhalable substance, such as a nasal spray. Edible Compositions The polysaccharide according to the invention may be added to an edible composition or a pharmaceutical composition in a specific salt form. The edible composition according to the present invention may take any physical form. In particular, it may be a food product, a beverage, a dietary food product, or a clinical food product. It may also be a dietary supplement, in the form of a beverage, a tablet, a capsule, a liquid (e.g. a soup or a beverage, a spread, a dressing or a dessert) or any other suitable form for a dietary supplement. The edible composition may be in a liquid or a spreadable form, it may be a spoonable solid or soft-solid product, or it may be a food supplement. Preferably the edible composition is a liquid product. The edible product may suitably take the form of e.g. a soup, a beverage, a spread, a dressing, a dessert, a bread. The term “spread” as used herein encompasses spreadable products such as margarine, light margarine, spreadable cheese-based products, processed cheese, dairy spreads, and dairy- alternative spreads. Spreads as used herein (oil-in-water or water-in-oil emulsions) may have a concentration of oil and/or fat of between about 5% and 85% by weight, preferably between 10% and 80% by weight, more preferred between 20% and 70% by weight. Preferably the oil and/or fat are from vegetable origin (such as but not limited to sunflower oil, palm oil, rapeseed oil); oils and/or fats of non-vegetable origin may be included in the composition as well (such as but not limited to dairy fats, fish oil). The polysaccharide and thus the edible composition according to the invention may be a prebiotic. A prebiotic may be defined as a composition that induces growth and/or activity of beneficial microorganisms, such as fungi and/or bacteria, colonising the intestine. Growth and/or activity of beneficial microorganism may be increased by the polysaccharide acting as a substrate for the beneficial microorganisms. It is common for edible compositions (e.g. animal feeds) to be provided as a composition including other components, for example as a mixture. The edible composition may therefore also include one or more feed materials, such as fermentation products (e.g. spent yeast), molasses, potato, grain (e.g. spent grain, and/or maize, soybean, wheat, oats, barley, and/or rice), seaweed, fodder (e.g. hay, straw, silage, compressed and pelleted feeds, oils, mixed rations, grains and legumes), peanut shell, bean pods (e.g. soy bean pods), corn bract and/or corn cobs. Preferably the feed material comprises fermentation products, potato, molasses, grain, seaweed, fodder, peanut shell, bean pods, corn bract and/or corn cobs. The edible composition may also include vitamins, minerals, chemical preservatives and/or antibiotics. The edible composition may include these components in an amount of 30 wt% or more, such as 50 wt% or more, or 70 wt% or more, such as 90 wt% or more, or 95 wt% or more, such as 98 wt% or more. The amount may be 99.9 wt% or less, such as 99.5 wt% or less, or 99 wt% or less, for example 98.5 wt% or less, or 98wt% or less, for example 95 wt% or less. The amount of these components in the edible composition may be from 30 to 99.9 wt%, such as from 50 to 99.5 wt%, or from 70 to 99 wt%. For example, the edible composition may comprise fermentation products, molasses, grain, seaweed, fodder, peanut shell, bean pods, corn bract and/or corn cobs in an amount of 30 wt% or more, such as from 50 to 99.5 wt%. The edible compositions (e.g. animal feed and/or nutraceutical compositions) may include the polysaccharide in what might seem like quite low concentrations. However, due to the amount of feed that animals can eat, the overall amount of the compound eaten can be sufficient to provide a technical effect. Therefore, the edible composition (e.g. animal feed) may include the polysaccharide in an amount of 0.000001wt% or more, such as 0.000005wt% or more, or 0.00001 wt% or more, preferably 0.00005 wt% or more, or 0.0001 wt% or more, for example 0.0002 wt% or more, or 0.0003 wt% or more, or 0.0004 wt% or more. The amount of the polysaccharide in the edible composition (e.g. animal feed) may be 10 wt% or less, such as 5 wt% or less, or 1 wt% or less, such as 0.5 wt% or less, or 0.1 wt% or less, such as 0.05 wt% or less, such as 0.01 wt% or less, preferably 0.005 wt% or less, or 0.001 wt% or less, for example 0.0008 wt% or less, or 0.0007 wt% or less, or 0.0006 wt% or less. The amount of the polysaccharide in the edible composition may be from 0.000001wt% to 10 wt%, for example from 0.00001 wt% to 1 wt%, or from 0.00005 wt% to 0.005 wt%, preferably from 0.0001 wt% to 0.001 wt%, or from 0.0003 wt% to 0.0008 wt%. The polysaccharide in the edible composition (e.g. animal feed) may have been extracted from a plant that contains the compound. The polysaccharide may be added to the edible composition as plant material that contains the polysaccharide. Therefore, the composition preferably contains plant material (e.g. root, stem, leaf and/or flower) that contains the polysaccharide. The composition may contain the plant material (e.g. root, stem, leaf and/or flower) in a dry (i.e. dehydrated) amount of 0.0001 wt% or more, such as 0.001 wt% or more, or 0.005 wt% or more, such as 0.01 wt% or more, preferably 0.02 wt% or more, or 0.05 wt% or more, or 0.08 wt% or more, such as 0.09 wt% or more, or 0.1 wt% or more, or 0.15 wt% or more. The dry amount of plant material (e.g. root, stem, leaf and/or flower) in the composition may be 20 wt% or less, such as 10 wt% or less, or 5 wt% or less, preferably 2 wt% or less, for example 1 wt% or less, or 0.8 wt% or less, such as 0.6 wt% or less, or 0.4 wt% or less, or 0.3 wt% or less. The dry amount of the plant material (e.g. root, stem, leaf and/or flower) in the composition may be from 0.0001 wt% to 20 wt%, such as from 0.005 wt% to 5 wt%, preferably from 0.02 wt% to 2 wt%, such as from 0.08 wt% to 1 wt%. The edible composition may either contain the polysaccharide in an amount of from 0.000001wt% or more (e.g. from 0.000001wt% to 10 wt%, or from 0.0001 wt% to 0.001 wt%), or contain plant material comprising the polysaccharide in a dry amount of from 0.0001 wt% or more (e.g. from 0.0001 wt% to 20 wt%, or from 0.02 wt% to 2 wt%), and contain other components that are vitamins, minerals, chemical preservatives, antibiotics, fermentation products (e.g. spent yeast), molasses, grain (e.g. spent grain, and/or maize, soybean, wheat, oats, barley, and/or rice), seaweed, fodder (e.g. hay, straw, silage, compressed and pelleted feeds, oils and mixed rations, and sprouted grains and legumes), peanut shell, bean pods (e.g. soy bean pods), corn bract and/or corn cobs, where the other components are included in an amount of 30 wt% or more (e.g. from 30 to 99.9 wt%). The skilled person will appreciate that any remainder can made up with other components, such that the total composition adds up to 100%. The plant material (e.g. root, stem, leaf and/or flower) may be whole or processed. The plant material may be partially or totally dehydrated, or may not be dehydrated. The animals being fed the animal feed may include livestock. The livestock may be ruminants or non-ruminants. The livestock may be cattle, sheep, goats, pigs, horses, donkeys, zebu, bali cattle, yak, water buffalo, gayal, reindeer, camel (e.g. Bactrian camel, Arabian camel), llama, alpaca, poultry (e.g. chicken), rabbit, and/or guinea pig. The animals may include pets (companion animals), such as dogs, cats, rabbits, ferrets, pigs, rodents (e.g. gerbils, hamsters, chinchillas, rats, mice, and guinea pigs), birds (e.g. parrots, passerines, and fowls), reptiles (e.g. turtles, lizards, snakes, and iguanas), equine (e.g. horses, ponies and donkeys) aquatic pets (e.g. fish, freshwater snails, and saltwater snails), amphibians (e.g. frogs and salamanders), and/or arthropods (e.g. tarantulas and hermit crabs). The animals may be for meat-based food products, non- meat-based food products (e.g. eggs, milk), and/or non-food products such as wool and leather. The edible composition may comprise: a polysaccharide or composition thereof of the first and/or third aspects; and a nutraceutically acceptable carrier, diluent or excipient. The carrier may, for example, be water or an aqueous fluid such as saline or a sugar solution. However, the skilled person will be well aware of carriers, diluents or excipients that are edible and/or nutraceutically acceptable. An aqueous composition may comprise at least 50% w/w water. An aqueous solution may be a solution comprising at least 50% w/w water. An aqueous suspension may be a suspension comprising at least 50% w/w water. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. The aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. It will be appreciated that the terms “treatment" and “treating” relate to the management and care of a subject for the purpose of combating a condition, such as a disease or a disorder. These terms are intended to include the full spectrum of treatments for a given condition from which the subject is suffering, including alleviating symptoms or complications, delaying the progression of the disease, disorder or condition, alleviating or relieving the symptoms and complications, and/or to cure or eliminating the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of a subject for the purpose of combating the disease, condition, or disorder and includes the administration of the ligand to prevent the onset of the symptoms or complications. Cell Culture The polysaccharide according to the invention may be used as a cell culture additive or as a cell culture agent, for use as or adding to a culture media. The culture media may further comprise a cell, biological tissue, a biological organ, a biological system or an organism. The culture media may be a cloning medium or a hybridoma feeder supplement (for the purpose of enhancing cell cloning efficiency or hybridoma growth and survival rates). According to another aspect, there is provided a method of culturing a cell, a biological tissue, a biological organ, a biological system or an organism, the method comprising: placing a cell, a biological tissue, a biological organ, a biological system or an organism in a culture media; and placing into the culture media: a polysaccharide or a composition thereof according to the invention, or a plant of the Malvales order or part thereof. All of the embodiments and features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects or embodiments in any combination, unless stated otherwise with reference to a specific combinations, for example, combinations where at least some of such features and/or steps are mutually exclusive. Examples Purification of Polysaccharides Primary Method Figure 3 of the accompanying drawings shows a procedure for purifying samples of Malva Sylvestris. A precipitation with cold aqueous ethanol 80% was performed on 1.0 g of PM straw (straw of M. Sylvestris obtained from a source in Poland) extract. Therefore, the mallow raw sample was dissolved in 20 ml of water to which 80 ml of ethanol was added (to reach 80% in volume) and left at 4°C for 16 h. After centrifugation, 757 mg of supernatant (PM/16/A, yield: 75.7%) and 241 mg of precipitate (PM/16/B, yield: 24.1%) were collected. The total recovery after precipitation was 99.8%. The extract has been found to reduce in activity when spray dried with carriers such as maltodextrin, although the activity returned upon extraction from the maltodextrin. This extraction process was found to be reproducible by comparison of NMR data. For this reason, a number of supernatants and precipitates obtained in the same way were combined (as shown in Figures 1a and 1d) to provide PM/17/A, which was combined with PM/16/A, and PM/17/B, which was combined with PM/16/B. The PM/16/B and PM/17B fractions were purified by Sephacryl HR-300 size exclusion chromatography (V=78.9 ml, flow= 13.2 ml/h and ammonium bicarbonate 50 mM as eluent), splitting the purification in 5 homologous loadings. Therefore, PM/16/B (241 mg) was dissolved in 6 ml of eluent and centrifuged to remove any insoluble material and the supernatant obtained (179 mg in 6 ml) was then loaded in three successive chromatographic runs (2 ml for each loading) (Figure 3e, loading and collecting scheme for PM/16/B sample). Each chromatographic run was monitored with a refractive index detector (sensitivity of 5×e^-4), from which was obtained the chromatographic profiles of each run, such as that in Figure 3f (S-300 chromatogram of PM/16/B first load and showing how each fraction was collected). After each column, four fractions were collected (Figure 3e): PM/18/A/1 (6.4 mg), PM/18/B/1 (18 mg), PM/18/C/1 (3.8 mg), PM/18/D/1 (25.1 mg), PM/18/A/2 (6.3 mg), PM/18/B/2 (18.2 mg), PM/18/C/2 (7.2 mg), PM/18/D/2 (21.2 mg), PM/18/A/3 (5.8 mg), PM/18/B/3 (3.8 mg), PM/18/C/3 (6.0 mg) and PM/18/D/3 (20.2 mg). PM/18/A3 was later used to acquire the 2D NMR data, while the PM/19/A1 and A2 were pooled together to run a separation by ion exchange chromatography. Similarly, PM/17/B (156 mg) was dissolved in 4 ml of eluent and centrifuged to remove insoluble components. The supernatant, 90 mg, was loaded in two 2 ml steps in the same chromatographic system used previously (Figure 3g). After each run, the chromatographic profile was recorded, which was the same for both, so the first one is given as an example to show how the four fractions were collected after each column (Figure 3h): PM/19/A/1 (4.7 mg), PM/19/B/1 (2.8 mg), PM/19/C/1 (5.2 mg), PM/19/D/1 (20.3 mg), PM/19/A/2 (3.1 mg), PM/19/B/2 (1.5 mg), PM/19/C/2 (4.4 mg) and PM/19/D/2 (13.4 mg). All fractions were stored at -20°C and the PM/18/A/3 fraction was subject to both linkage analysis and two-dimensional NMR analysis. The samples PM/19/A/1 and PM/19/A/2 were pooled (total weight: 7.8 mg), dissolved in 1 ml of water and loaded in column with Q-Sepharose fast flow resin (volume of 1.9 ml, flow = 4 ml/h, eluent: NaCl in stepwise gradient, three resin bed volume with each of these solutions: 10 mM, 100 mM, 200 mM, 400 mM, 700 mM and 1 M). Six fractions were collected: named: PM/20/A_10mM, PM/20/B_100mM, PM/20/C_200mM, PM/20/D_400mM, PM/20/E_700mM and PM/20/F_1M; each was desalted by size exclusion chromatography on Biogel P-10 (Volume=18.5 ml, Flow rate: 12 ml/h and with H₂O eluent). Each desalting gave a chromatogram like that in Figure 3i (this one was collected for example by the 200 mM sample): the first peak was always labelled with “1”, the region between the first and the second peak as “2”, and the second peak (the salts) as “3”. All peaks were inspected by¹H NMR except the “3”. Secondary Method Five commercial-scale aqueous extracts of M. sylvestris plant material were prepared under a range of different process methods. A sample of each commercial extract was subjected to cold EtOH (80%) purification in order to separate the low molecular weight carbohydrate-fractions from the high molecular weight fractions. Approximately 200 mg of each commercial sample (shown in the Table) was dissolved in 10 ml of water. 40 ml of cold ethanol was added (to reach 80% ethanol content by volume in total volume of 50ml). The mixture was left for 17h at 4°C to allow precipitation to occur. Centrifugation (7000 rpm, 4°C, 30’) was used to separate the supernatant from the precipitate. The supernatant and the precipitate were recovered and their mass weights are reported in the table below. The total yield of each precipitate was actually greater than 100% because the supernatant tended to retain moisture. The supernatant was sticky. Sample (mg) Supernatant (mg) Precipitate (mg) Commercial extract 1 (256) 179 84 Commercial extract 2 (237) 165 79 Commercial extract 3 (237) 163 84 Commercial extract 4 (226) 175 75 Commercial extract 5 (234) 153 84 The supernatants and the precipitates were each subjected to¹H-NMR analysis and compared with the starting material (raw M. sylvestris). Spectra were acquired at 298K and at 600MHz. Samples were dissolved in a small amount of D2O. The¹H-NMR profile of raw M. sylvestris was almost identical to those of the commercial extracts. The different commercial extracts can be considered equivalent to each other on the basis of their¹H-NMR profiles. The ethanolic supernatants of the commercial extracts were found to possess low molecular weight sugar molecules, such as sucrose and traces of glucose, exactly as for laboratory extracts. The¹H-NMR profiles of the ethanolic precipitates of commercial extracts resemble those of the ethanolic precipitates of laboratory extracts, i.e. high-molecular-weight arabinogalactans. Characterisation of Polysaccharides 0.4 mg of PM /11/A (early elution from S-300) and 0.5 mg of PM/11/B (sample regarding the first true peak from S-300 chromatogram) were subjected to derivatisation as acetylated methyl glycosides (AMG). All sugars were recognised by their typical fragmentation pattern and identified by comparison with AMG standards. Both fractions were composed of neutral sugars (glucose, galactose, mannose, xylose, arabinose), a deoxysugar (rhamnose) and acidic sugars (glucuronic and galacturonic acid). The two fractions were qualitatively similar to each other because they contained the same type of sugars, but the proportions between the sugars were not the same, which suggest the presence of a mixture of glucans. This is in agreement with the proton spectra profiles, which were similar for the two samples, except for the intense signal at ca. 4.2 ppm, which was absent in PM/11/A and probably not related to any glycan (note that this signal was in PM/10/B, the original mixture later divides in PM/11/A and PM/11/B by chromatography). PM/11/A and PM/11/B were derivatised as Partially Methylated Acetylated Alditols (PMAA), using 0.7 mg of A-fraction and 0.8 mg of B-fraction respectively. All sugars were recognised by their typical fragmentation, and the types of variously bound sugars present are in agreement with the compositional analysis, i.e. pentoses and hexoses, deoxy sugars, and uronic acids. This procedure was less effective on PM/11/B due to the presence of the contaminant, a signal at 4.2 ppm in the proton spectrum. 0.6 mg of PM/18/A/3 was derivatised as Partially Methylated Acetylated Alditols and the spectrum obtained was compared with previous homologous fractions spectra. All the differently linked-sugars were recognised by their typical fragmentation pattern and the sugar composition of PM/18/A/3 is very similar compared to the other homologous fractions. However, the chromatographic pattern is different in terms of the relative intensities of the various peaks for all fractions, which means that the PM sample is heterogeneous, which can be due to the presence of different glycans or to their non- regular structure or to a combination of both possibilities. 5.8 mg of PM/18/A/3 (dissolved in 550 µl of D₂O) were used to record the full set of the 2D-NMR spectra and the fully attributed HSQC spectrum. By analysing the HSQC spectrum, it first appears that the sample contains some starch, then there are two different anomeric regions: that of the a-arabinofuranose (Araf) units (5.3 – 5.0 ppm) and of the b-galactose (Gal) and b-glucuronic acid (GlcA) units (4.55 – 4.4 ppm). The inspection of the long range correlation and the NOEs effects revealed different structural motifs. In detail: B (terminal arabinofuranose or t-Araf) and B’ (5-linked Araf or 5-Araf) are both linked to O-3 of a 3,6-Gal (H) unit. There are other two types of a- Araf units, F and F’: the first is 5-linked, while the other is terminal and both are connected to O-5 of a Araf unit. There are also other types of Araf units, including one 2,5-linked (residue C) and others that seem to be terminal, namely with no substituents. All the arabinofuranose units are a configured at the anomeric centre. Regarding the other sugars, beside 3,6-Gal, it is possible to recognize 6-Gal (L’) and t-Gal (M), along with a 4OMe-GlcA (L), namely a glucuronic acid with a methyl group linked at O-4. These other units are all in the pyranose form and b configured at the anomeric centre. All the Gal units are 1^6 linked and the terminal non-reducing end can be either a t- Gal or 4OMe-GlcA. Hence, these data suggest the presence of an arabinogalactan, with a backbone made by b-(1^6) Gal, substituted at O-3 with an arabinan a-(1^5) linked, with chains of different length. However, the spectrum is very complex which could be due to the presence of a non-regular glycan or of a mixture of different polysaccharides (an arabinan plus a galactan). To solve this issue and to gain more information, the fractions analogue to PM/18/A/3 - namely PM/19/A/1 and PM/19/A/2 - have been pooled and further separated by anion exchange chromatography. All fractions were stored at -20°C. PM/20/A/1 (0.5 mg) and PM/20/C/1 (0.3 mg) were dissolved in 550 µl of D2O and used to record full 2D-NMR spectra. These two fractions were selected because of the different pattern in the anomeric signals of the arabinofuranose units and Gal/GlcA residues. Comparison of spectra, along with the integration of appropriate densities in the HSQC spectra, revealed that in the sample PM/20/A1: - Had no (or very few) 4OMe-GlcA residues - The non-reducing end of the chain terminates with β-Gal - The arabinan chains are much longer than those in PM/20/C1 - Some 5-Araf (ca. 30%) are further substituted at O-2 with a t-Araf These results also revealed that in the sample PM/20/C1: - 4OMe-GlcA or t-Gal are located at the non-reducing terminal of the glycan - The arabinan chains are shorter and less branched (having no branched second sidechains) Some key densities of the HSQC spectra have been integrated (Table 1). These provide semi-quantitative values of the proportions between the sugars. Moreover, these values are accurate when the same types of sugars are compared, for instance the comparison can be done within the different types of Araf (or of Gal units), while the comparison between Araf and Gal is less accurate due to the instrumental setting used. Table 1: ratio between the major type of residues in PM/20/A1 and PM/20/C1 as evaluated by integration of key densities in the HSQC spectra. Sugar residue Sugar type PM/20/A1 PM/20/C1 B t--Araf 4 2.5 F’ t-a-Araf 1 Ca. 1 B’ 5-a-Araf 1 1 F 5-a-Araf 8 0.2 C 2,5-a-Araf 3 0 H 3,6-b-Gal 4 2 L 4OMe-b-GlcA -- Ca. 1 L’ 6-b-Gal 6 1.9 M t-b-Gal 4 0.8 Based on the values in Table 1 (and some approximations) models of the two fractions were constructed. The model of glycans in PM/20/A1 is shown in Figure 1 of the accompanying drawings. The model of glycans in PM/20/C1 is shown in Figure 2 of the accompanying drawings. The two models are made by assembling different Gal block, that follow certain proportion between them; then the branched Gal (unit H) has an arabinan linked at position 3. The length of this arabinan is not the same for the two samples. Note that <<n>> means an average number of units, e.g. <<n>> = 8 means that there will be an array of sidechains of different length, with “n” from 0 to 16. It was found that the extracts may include pectin residues, or traces thereof. Early extracts showed the presence of GalA and Rha, which are residues characteristic of pectins. These were not present in the purified fractions; however, the pectin components could have been lost during ion exchange purification. Biological results Macrophage (RAW 264.7) Assays Macrophages are important cells in the innate immune system that are found in almost all tissues. They search for foreign bodies that could be harmful to the host. When they find something potentially harmful, they become activated and undergo significant changes. This includes changes in their gene expression, an increase in their size, and increased endocytosis and hydrolytic protein production. Activated macrophages also produce and secrete many products that help them fight off threats and coordinate an adaptive immune response. Some of the enzymes produced by activated macrophages include superoxide dismutase, NADPH oxidase, and inducible nitric oxide synthase (iNOS), which converts arginine into NO. NO can, in turn, damage, kill or prevent the replication of microbes. Preparation of Macrophages Murine macrophages from the RAW264.7 cell line were purchased from European Collection of Cell Cultures (ECACC). The cells were grown in 75cm culture flasks, in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% heat-inactivated FBS, 100u/ml penicillin and 100μg/ml of streptomycin. The cells were incubated in the presence of 5% CO₂ at 37°C. All cell culture work was carried out in sterile conditions in a biological safety cabinet to prevent any contamination. Macrophage Proliferation Assay Cells were seeded in 96 well plates at a seeding density of 1x10⁵cells ml (1x10⁴ cells/well). Lipopolysaccharide (LPS) (Sigma-Aldrich) a known activator of macrophages at a concentration of 1μg/ml was used a positive control. Incubation time for assays was 24h. Cells were cultured in the presence of 5% CO₂ at 37°C and macrophage proliferation was measured using AlamarBlue. Measurement of Nitrite Concentration The iNOS enzyme is a highly active isoform of nitric oxide synthase which is involved in both the innate and adaptive immune systems. iNOS expression in macrophages is controlled by cytokines and microbial products, primarily through transcriptional induction. iNOS is present in macrophages from various species and commonly occurs in human monocytes and in macrophages from patients with infectious or inflammatory diseases. The sustained production of NO by the iNOS enzyme gives macrophages the ability to fight against various pathogens and tumour cells. When iNOS is activated, it produces nitric oxide (NO) by changing L-arginine into citrulline. NO is mediator of inflammation and is made by macrophages that have been activated in a specific way. Because NO is very reactive, it is usually measured indirectly by measuring one of its breakdown products, nitrite (NO2-). To measure the NO production of macrophage cells the nitrite (NO2-), concentration was determined in culture medium was measured using a proven chemical called the Griess reagent. A 60μl aliquot of medium from each well was transferred to a sterile 96-well plate together with 60μl of Griess Reagent. The concentration of NO2- was measured colourimetrically at λ 540nm using a Tecan Safire2 microplate reader, immediately. A standard solution of 0.1M sodium nitrite (NaNO2) was used to make serial dilutions in the range of 0.36 - 50μM to quantitate NO₂- concentrations by means of a standard curve. Measurement of IL-8 expression A modified murine macrophage cell line was used to measure IL-8 expression. Specifically, the cell line was an IL-8 Luciferase Reporter cell line, a stably transfected RAW 264.7 cell line expressing Renilla luciferase reporter gene under the transcriptional control of the IL-8 promoter. The transfected cells were grown in 75cm culture flasks in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % heat- inactivated FBS, 100 μg/mL each of penicillin and streptomycin, and 3 μg/mL of puromycin. The cells were cultured to confluency before being used in in-vitro assays in the presence of 5% CO₂ at 37°C. All cell culture work was carried out under sterile conditions in a biological safety cabinet to prevent any contamination. To determine IL-8 expression the above macrophage cells were exposed to compounds in a similar manner for NO measurements except that Griess reagent was not used but a luciferase reagent was added instead. A 50μl aliquot of the reagent mixture (assay buffer & substrate) is added to each well containing 100µl of cell suspension. The level of IL-8 expression was measured at a target λ 490nm using a Clario Star microplate reader. The method of detection was luminescence. IL-8 expression index was determined relative to the control sample following blank correction. IL-8 expression – fractions of Malva sylvestris extract Figure 4 of the accompanying drawings shows the IL-8 expression index of cultured RAW 246.7 cells when treated with E. Coli lipopolysaccharide (LPS), Medium, QUB input, or a range of concentrations for each of samples PM/18/A/3, PM/20/A1, PM/20/B1, PM/20/C1, and PM/20/D1. QUB input is an aqueous extract of M. sylvestris obtained from PM straw. QUB input is used as a positive control and as a benchmark for other samples. The polysaccharide samples were each found to increase IL-8 expression compared to the control, indicating that they are active immune modulators. Each sample was found to provide higher activity than the positive control (QUB input) for least one concentration. PM/20/A1 exhibited the highest activity, followed by PM/20/B1, then PM/20/C1, then PM/18/A/3 and PM/20/D1. The activity of the polysaccharides was found to be concentration dependent. Nitric oxide production – fractions of Malva sylvestris extract Figure 5 of the accompanying drawings shows the NO production of cultured RAW 246.7 cells when treated with E. Coli lipopolysaccharide (LPS), Medium (negative control), QUB input, for a range of concentrations for each of the samples PM/18/A/3, PM/20/A1, PM/20/B1, PM/20/C1, and PM/20/D1. QUB input is an aqueous extract of M. sylvestris obtained from PM straw used as a positive control and as a benchmark for other samples. Each of the samples showed high levels of nitric oxide production, indicating that they are active immune modulators. Each sample was found to provide higher activity than the positive control (QUB input) for least one concentration. PM/18/A/3 exhibited the highest activity, followed by PM/20/A1, then PM/20/C1, then PM/20/B1, then PM/20/D1. The activity of the polysaccharides was found to be concentration dependent. Nitric oxide production – comparison of extracts from different parts of M. sylvestris The sodium nitrite concentration (as a measure nitric oxide production, indicating immunomodulation) in RAW 264.7 cells was determined in the presence of extracts of commercial (EVRA, Lauria, Italy) spray-dried extracts of different parts of the M. sylvestris plant (root, stem, and flowers & leaves), which were each purified by size exclusion chromatography to provide a sample containing particle sizes from 1000- 2000µm and a sample containing particle sizes less than 500µm. Figure 6 of the accompanying drawings shows the results of the investigation. It was found that the extracts of the root, stem, flower and leaf of M. sylvestris each provided immunomodulatory activity. In the root, the 1000-2000µm sample provided similar activity to the <500 µm sample. In the stem, the <500 µm sample was significantly more active than the 1000-2000 µm sample. In the flowers and leaves (combined), the <500 µm sample was significantly more active than the 1000-2000 µm sample. In each case the activity increased with higher concentration (from 50 to 100µg/mL), indicating that the compounds are responsible for the activity. Cold 80% aqueous ethanol extracts were obtained for different parts (root, stem, flowers & leaves) of M. sylvestris. The M. sylvestris extracts were purified by size exclusion chromatography as described above. RAW 264.7 cells were exposed to each of the extracts and nitric oxide (sodium nitrite) production, indicating immunomodulation was determined for samples obtained, in each case, after 1 hour or after 2 hours of extraction. Figure 7 of the accompanying drawings shows the results of this study. It was found that the activity of extracts of M. sylvestris extracts were around the same as those from S. cordifolia. However, with M. sylvestris, the yields of arabinogalactans were significantly higher (15-25% by weight), and there is an increased variety of plant parts that can be selected to optimise yield and activity. Samples purified using size exclusion chromatography to <500 µm were found to have higher activity than the larger particle size samples. Nitric oxide production – comparison of sources of M. sylvestris M. sylvestris was obtained from two sources, one in Italy and another in Poland. The activity of <500µm extract samples (obtained after 1 or 2 hours of extraction) were compared as discussed above. As shown in Figure 8 of the accompanying drawings, it was found that both sources had comparable activities. Nitric oxide production – comparison with other arabinogalactans The NO production of arabinogalactans PM/20/A1 and PM/20/C1 were compared against an arabinogalactan obtained from larch wood (purchased from Sigma-Aldrich, 80% purity by HPLC). The arabinogalactan obtained from larch wood exhibited no nitric oxide production. The arabinogalactans PM/20/A1 and PM/20/C1 each showed good nitric oxide production, rising to around 40µM nitric oxide production (sodium nitrite) at a concentration of about 30µg/mL. This shows that not all arabinogalactans stimulate NO production. Nitric oxide production – comparison of extracts with purified samples Figure 9 of the accompanying drawings shows a graph of nitric oxide production (sodium nitrite) concentration (as a measure nitric oxide production, indicating activation of cells and thus immunomodulation) in RAW 264.7 cells against log concentration of a lab extract, commercial extract, commercial extract filtered >10kDa MWCO, PM/20/A1 and PM/20/C1. The following results were obtained: Sample EC50 (µg/mL) Lab extract 45.3 Commercial scale aqueous extract of 73.30 M. sylvestris Commercial scale aqueous extract of 35.6 M. sylvestris filtered >10kDa MWCO PM/20/A1 2.1 PM/20/C1 2.4 The maximum response for each of PM/20/A1 and PM/20/C1 was around 11 µg/mL. PM/20/A1 was slightly more active than PM/20/C1. The purified samples were around 35 times more active than the extracts. Nitric oxide production – combination with Sida cordifolia arabinan An arabinan was extracted from S. cordifolia as described in WO2022/090735A1 (e.g. Example 1 thereof). The nitric oxide production (sodium nitrite) was determined for arabinan alone (0.3 µg/mL) or arabinogalactan alone (0.3 µg/mL). These were compared with the nitric oxide production response when arabinan (0.3 µg/mL) combined with arabinogalactan (0.3 µg/mL). Figure 10 shows the results of this study. It was found that, when arabinan and arabinogalactan where combined the nitric oxide (sodium nitrite) produced was 49% greater than the sum of their individual responses. This may indicate a synergistic relationship between arabinan and arabinogalactan that increases immune modulation. Mechanistic Investigations The mechanism of action of the extract was assessed by investigating its effects on metabolite levels within macrophage cells using a modified method of that described in Andersen et al. (bioRxiv 2021.12.15.470649). Preparation of cells: RAW 264.7 cells were grown under typical culture condition (37°C; 5% CO2) with DMEM medium supplemented with 10% general FBS and 1% Pen/Strep, plus 1% L- Glutamine. Cells were counted the cell density adjusted with culture medium to 1x106 cells/mL in preparation for seeding. Into each well of a 6 well plate 2mL of cell suspension (1x10⁶ cells/mL) was added (i.e. 2x10⁶ cells per well). The plate was then incubated for 24h (37°C; 5% CO₂). Preparation of polysaccharides: 10mg of polysaccharide extract was accurately weighed and added 1mL ultra-pure water to obtain a 10mg/mL stock solution. Stock solutions were filter sterilised using a 0.22 μm filter. Sterilized stock solution was diluted to concentrations 100 μg/mL, 10 μg/mL and 1 μg/mL. The medium from each well of the 6 well plate was aspirated and 2mL of each polysaccharide solution was added to the wells. Plates were incubated for a further 24h 24h (37°C; 5% CO₂). Cell harvesting: After 24h the test solutions were removed and cells were washed twice with ice-cold PBS then 2mL of PBS was added to each well. Cells were detached from the plate surface using a cell scraper to re-suspend the cells in the PBS, then a small sample (50ul) was removed to determine cell densities (Invitrogen^TM, Countess^TM automated cell counter, Washington, USA). Based on the cell density results, a total of 3x10⁶ cells in suspension were collected into Eppendorf tube. Tubes were centrifuged at 100g (1000 rpm) for 5 min and the supernatant was removed leaving 3x10⁶ cells in each tube. Metabolite extraction: Each tube containing 3x10⁶ cells was snap frozen using liquid nitrogen. 100 μL of 75% ethanol was added to each tube and vortexed for 2 min, then sonicated on ice for 5min (VWRTM, Ultrasonic cleaner, Malaysia). Then 250 μL of MTBE was added to each tube and shaken at 500rpm for 30 mins at room temperature (Eppendorf Thermo Mixer C, Hamburg, Germany), then 62 μL of ultrapure H2O was added to each tube. This was vortexed for 2min and incubated at room temperature for a further 10min. Tubes were centrifuged (16000g; 4°C) then speed vacuumed at 45°C under V-AQ mode for approximately 2h until each tube was completely dry (Eppendorf Concentrator plus, Hamburg, Germany). Samples were reconstituted in 60 μL of 85% ethanol; 15%PBS soluble. Samples then underwent metabolite profiling using a BIOCRATES Quant500 kit in accordance with the manufacturer’s instructions. Three concentrations of the extract were compared against three concentrations of two comparable polysaccharides - arabinan (from Sida cordifolia), and fucoidin (from seaweed). ‘High’ = (100μg/mL); ‘Medium’ = (10μg/mL); ‘Low’ (1μg/mL). E. Coli LPS was tested at 0.03μg/mL as it is extremely potent. Principal Components plots showed that the overall metabolic responses of macrophage cells to the three plant polysaccharides. The three polysaccharides were found to behave more similarly to each other than they did to E.coli LPS. At high concentrations there was a modest divergence in responses between fucoidin and the extracts of M. sylvestris and Sida cordifolia. There was less of a divergence at low/medium concentrations. At medium/higher concentrations the responses did differ more from the controls. Anti- inflammatory macrophages display enhanced OXPHOS metabolism, fatty acid oxidation, glutaminolysis, tryptophan catabolism with release of kynurenine, and synthesis of polyamines. The extract increased the concentration of the polyamines putrescine, spermidine and spermine in macrophages compared to Sida, LPS and Fucoidin. The extract increased the synthesis of glutamine in macrophages compared to Sida, LPS and Fucoidin. The extract also increased acylcarnitine levels, indicating a probable increase in beta- fatty acid oxidation. Separate studies were conducted using compounds known to interact with several cell signalling proteins. The cells seeded in 96-well plates were pre-treated for 12h with culture medium or culture medium containing inhibitory compounds before being challenged with polysaccharide extracts. It was found that the NO response of M. sylvestris polysaccharide extract may be mediated by TLR-4 (toll-like receptor 4). Existing compounds are known to interact with TLR-4. For instance, naloxone and mianserine bind a pocket or active site of within the TLR4/MD2 complex and halt signalling. MD2 and CD14 are accessory co-factors that dimerize with TLR4, and signalling pathways may differ depending on their involvement. TAK242 interferes with TLR4’s intracellular binding of its adaptor proteins. The IL-8 responses of arabinan and M. sylvestris polysaccharide extract appear to be mediated by multiple pattern recognition receptors. The mechanism of action of arabinan or M. sylvestris polysaccharide extract was found to involve the TLR-4/MD2 binding pocket to some extent. When using antibodies to block MD2 and/or CD14, there was no significant effect on NO responses for arabinan or M. sylvestris extract. However, IL-8 expression in the presence of arabinan was enhanced by blocking either of these co-factors. It was found that neither arabinan nor M. sylvestris acted via MYD88. Using PDTC, TPCK and BAY 117082, which each inhibit NF-κB activation, responses of both arabinan and M. sylvestris extract were found to involve nuclear factor kappa- light-chain-enhancer of activated B cells (NF-κB). Mitogen activated protein (MAP) kinase inhibitor studies with U0126, SB 203580 and SP600125 were used to determine that NO production caused by arabinan and M. sylvestris extract involves the action of JNK (c-Jun N-terminal kinase; aka MAPK-8). Neither involves the action of MAP kinase or p38α/β (MAPK-14 / MAPK-11). M. sylvestris-enhanced IL-8 secretion was found to be reduced by a variety of agents including Pep-Inhi-MYD88 Control, TLR2 inhibitor, TLR4 inhibitor, Dectin-1 inhibitor, Mannan inhibitor, CD-14 antibody, MD-2 antibody, PDTC, TPCK, BAY 117082, U0126, SP600125, Naloxone and Mianserin. Hydrolysis studies The samples PM/20/A1 and PM/20/C1 were prepared for treatment with α-L- arabinofuranosidase to remove the terminal arabinose units from the arabinogalactan side chains. One third of the total weight of each polysaccharide sample was retained for comparative purposes, whilst two thirds was subjected to enzymatic hydrolysis. Each sample was dissolved in a stoichiometric volume of acetate buffer (pH=4.7) and a stoichiometric aliquot of enzyme was added twice. The reaction was conducted at 40°C for 48 h, after which it was stopped by denaturing the enzyme at 100°C for 15 min. At the end, a precipitate was formed and removed from the supernatant by centrifugation at (6000 rpm, 4°C, 15 min). The supernatant and the precipitate were then subjected to 1H-NMR investigation (to confirm the removal of arabinose) after which they were loaded onto a P-10 column in order to purify the hydrolysed polysaccharide from low molecular weight molecules such as arabinose and the buffer salts. The purified polysaccharides were then tested (along with the original PM/20/A1 and PM/20/C1 polysaccharides) for their ability to stimulate NO production and IL-8 gene expression. Each polysaccharide was tested at a concentration of 33µg/mL. In essence, this removed the sidechains of the polysaccharides, leaving only the backbone. Figure 11A of the accompanying drawings shows the results for the nitric oxide production. This shows that, for each of the polysaccharides, hydrolysis of the arabinofuranose groups caused an almost complete reduction in the stimulation of NO production. This shows that the sidechains are essential for NO production. The backbone alone does not lead to increased NO production, and therefore immunomodulation. Figure 11B of the accompanying drawings shows the results for the IL-8 gene expression. For PM/20/A1, as with the NO production, the hydrolysis of the arabinofuranose groups caused a reduction of the IL-8 gene expression to below the control level. For PM/20/C1, there was no significant change in the expression of the IL-8 gene by the hydrolysed polysaccharide. A key difference between the hydrolysed version of PM/20/A1 and PM/20/C1 is that PM/20/C1 has an unusual terminal 4-methoxy GlcA unit. This indicates that one or more of the sidechains and the 4-methoxy GlcA unit are required in order to stimulate IL-8 gene expression. In Vivo studies – Piglets A 3-week controlled dose-dependent trial in 192 piglets has been performed. Piglets were given access to either drinking water alone or drinking water with dissolved M. sylvestris extract (“AF200”, equivalent to 1.35, 2.70 or 5.40 g/kg/day). After 21 days, the piglets were challenged with E.coli LPS to simulate a disease/infection. Multi- timepoint tissue and blood sampling was used to determine the safety and immunological function of the compositions. Samples were specifically taken at days 0, 7 (pre- and post-challenge), 14 and 21 (pre- and post-challenge). The body weight (BW) of each piglet was determined and averaged (mean), and correlated with the average feed intake. This was used to determine the feed conversion ratio (FCR) for each group. The results are shown in the table below: Initial BW (kg) Final BW (kg) Feed intake (kg) FCR, feed/gain Control 7.411 ± 1.192 11.642 ± 2.308 0.439 ± 0.173 2.358 ± 0.892 M. sylvestris extract 7.403 ± 1.134 11.288 ± 1.669 0.288 ± 0.0287 1.648 ± 0.0804 (1.35g/kg/day) 3 weeks dosing of piglets in their drinking water shows a positive trend in Feed Conversion Ratio (FCR) and Feed Conversion Efficiency (FCE) at doses of 1.35g and 5.4g/kg. FCR in piglets dosed with 1.35g/Kg AF200 improved by 28%, and similar results were observed for piglets dosed at 5.40g/kg. The lower the FCR the more efficiently the feed is converted into body weight, and the lower the cost of livestock production. FCE in piglets dosed with 1.35g/kg AF200 improved by 27%, and similar results were again observed for piglets dosed at 5.40g/kg. Therefore, piglets provided with the polysaccharide of the invention more efficiently convert feed into weight, and therefore attract a higher value. Postmortem analysis of piglets following the study measured the hight of the jejunal villus and the depth of jejunal crypts. Figure 12 of the accompanying drawings shows the results for the jejunal villus height. It can be seen that there is a significant increase (16%) in the height of jejunal villus for piglets treated with the polysaccharides of the invention at a dose of 1.35g/kg, compared to the control. Figure 13 of the accompanying drawings shows the results for the jejunal crypt depth. It can be seen that there is a significant decrease (16%) in the depth of jejunal crypts for piglets treated with the polysaccharides of the invention at a dose of 1.35g/kg, compared to the control. Thus, 3 weeks oral dosing of piglets with M. sylvestris extract significantly increased jejunal villus height and shortened crypt depth. Similar statistically significant morphological improvements were also observed in piglet ileum. It is widely accepted that increased villus height leads to improved nutrient absorption and animal performance. Shallower crypt depth indicates the prolonged survival of villi with less need for their renewal. This shows how the present invention can enhance the growth of subjects, such as piglets. Lysozyme is an enzyme involved in innate immunity. The lysozyme activity for the control (water) group and the 2.70 and 5.40 g/kg/day group at 7 days were determined. It was found that there was a significant increase in the lysozyme activity for both of the groups treated with the extract: an 83% increase for the group treated with a dose of 2.70g/kg/day, and an 86% increase for the group treated with a dose of 5.40g/kg/day. The average white blood cell counts for the control (water) group and the 1.35 g/kg/day group at 21 days were determined. It was found that there was a significant increase in the overall white blood cell count (39% increase). This was found to be driven by significant increases in lymphocyte count (an enzyme involved in innate immunity, 45% increase) and monocyte count (30% increase) in the piglets fed the extract. Piglets were dosed M. sylvestris extract orally over a period of 8 days. The expression of 42 randomly selected immune system genes in the spleen was measured by PCR before and after the treatment. The expression of more than half of the genes was altered in a statistically significant amount. Figure 14 of the accompanying drawings shows a graph of the results for dosing at 2.7g/kg (left) and a graph of the results for dosing at 5.4g/kg (right). The genes shown inside the dashed boxes represent genes where expression was significantly altered compared with piglets dosed with 0g/kg. Gene names of significantly altered genes are shown. The table below lists the porcine immunological genes in the spleen that were significantly (P<0.05) altered by 8 days of oral treatment of piglets with Malva sylvestris extract. CCL2 IFNGR1 IL1B2 NFKB1 TLR2 TLR7 TNFRSF1A CD40 IL10 IRAK4 NFKBIA TLR3 TLR8 TNFRSF1B CSF2 IL12B MAPK8 TICAM1 TLR4 TLR9 TRAF6 CXCL10 IL1B1 MYD88 TLR1 TLR6 TNF This underscores the system-wide immunomodulatory activity of polysaccharides of the invention, and also confirms that actions occur through a diversity of immune system receptor/pathways such including various toll-like receptors (TLRs), interleukins, cytokines, and signal transducers. Piglets fed with doses of 2.70g/kg/day and 5.40g/kg/day each showed significant increases in the lysozyme activity after the LPS challenge administered at 21 days compared to before the challenge (37% increase and 81% increase respectively). Piglets in the control group did not display a significant increase in lysozyme activity following the challenge. This indicates that the piglets the groups that were fed the extract possessed improved immunomodulation compared to the control group. The piglets tolerated the extract well and mortality was unaffected by the extract. In Vivo studies – Broiler chickens A 3-week controlled dose-dependent trial in 330 broiler chickens has been performed. Broiler chickens were dosed by oral gavage once per day. The subjects were gavaged with either water or the extract (1.35, 2.70 or 5.40 g/kg/day). After 7 and 21 days, the birds were challenged with E.coli LPS to simulate a disease/infection. Multi-timepoint tissue and blood sampling was used to determine the safety and immunological function of the compositions. Samples were specifically taken at days 0, 7 (pre- and post- challenge), 14 and 21 (pre- and post-challenge). The average white blood cell counts for each group after 21 days was determined. It was found that there was a significant (25.3%) increase in heterophils in the broiler chickens fed the extract at a dose of 5.40g/kg/day for three weeks, compared to the control group. Heterophils are specialised white blood cells that are primary components of innate immunity. The most numerous granulocytic leukocytes in the peripheral blood of poultry. Heterophils rapidly migrate from the peripheral blood to the site of infection/injury. The monocyte count and overall white blood cell count for this group also increased (25.7% increase and 11.1% increase respectively) after 21 days. The birds tolerated the extract well and mortality was unaffected. In Vivo studies - Tilapia A 3-week controlled trial in tilapia has been performed. The fish were fed with standard feed coated with extract once per day at a dose of 2.7g/kg/day. The weight of the fish was measured after one week, and compared to a control where no extract was administered. Figure 15 of the accompanying drawings shows these results. The weight of the fish after one week was significantly increased by the extract. The plasma lysozyme activity of the fish was monitored over the course of the study. Figure 16 of the accompanying drawings shows these results. The plasma lysozyme activity was increased after 1 week and after 3 weeks of administration of the extract, compared to the control group. The fish tolerated the extract well and mortality was unaffected.

Claims

CLAIMS 1. A polysaccharide, or a composition thereof, wherein the polysaccharide comprises: - a backbone of beta-(1-6)-linked galactose residues and terminal residues, wherein the backbone comprises one or more first galactose residues and one or more second galactose residues, and - one or more first sidechain comprising one or two arabinofuranose residues, wherein the or each first sidechain is alpha-(1-3)-linked to a first galactose residue of the backbone, and - one or more second sidechain comprising a three or more alpha-(1-5)-linked arabinofuranose residues, wherein the or each second sidechain is alpha-(1-3)- linked to a second galactose residue of the backbone.

2. The polysaccharide of claim 1, wherein at least one terminal residue of the backbone is a glucuronic acid residue.

3. The polysaccharide of claim 1 or claim 2, wherein the polysaccharide comprises 100 or more saccharide residues.

4. The polysaccharide or composition thereof of any preceding claim, wherein in the backbone, the proportion of galactose residues 1,3-linked to other galactose residues to galactose residues 1,6-linked to other galactose residues is 1% or less.

5. The polysaccharide or composition thereof of any preceding claim, wherein the first sidechain is represented by the following formula: →1)-[α-D-Araf-(1→5)]l-α-D-Araf wherein l is 0 or 1.

6. The polysaccharide or composition thereof any preceding claim, wherein the second sidechain is represented by the following formula: →1)-α-D-Araf-(1→5)-[α-D-Araf-(1→5)]_m-α-D-Araf {(2 ↑ 1)[α-D-Araf-(1→5)]_o-α-D-Araf}_n wherein: m is from 1 to 20; n is 10 or fewer; and o is preferably 1 or 0.

7. The polysaccharide or composition thereof any preceding claim, wherein the molar proportion of first sidechains relative to the amount of second sidechains is from 150% to 600%.

8. The polysaccharide or composition thereof any preceding claim, wherein the backbone contains one or more third galactose residues, wherein the third galactose residues are unbranched.

9. The polysaccharide or composition thereof claim 8, wherein the molar proportion of third sidechains relative to the amount of second sidechains is from 150% to 1200%.

10. The polysaccharide or composition thereof any preceding claim, wherein one or both of the termini the backbone are β-galactose and/or 6-linked galactose residues, or one terminus of the backbone is a 6-linked galactose residue and the other terminus of the backbone is a glucuronic acid residue

11. The polysaccharide or composition thereof of claim 10, wherein the glucuronic acid residue is a 4-methoxy-β-D-glucuronic acid residue.

12. The polysaccharide or composition thereof of claim 11, wherein the 4-methoxy- β-glucuronic acid residue is at the non-reducing end of backbone.

13. The polysaccharide or composition thereof any preceding claim, wherein the molecular weight of the polysaccharide is from 40 to 100kDa.

14. The composition of any preceding claim, wherein the composition comprises the polysaccharide in an amount of 1% or more by weight.

15. The composition of any preceding claim, wherein the composition is an edible composition.

16. The composition of claim 15, wherein the composition is a feed supplement.

17. The composition of claim 16, wherein the composition is a mineral lick.

18. Use of a polysaccharide or a composition thereof as defined by any one of claims 1 to 17 to increase: - the yield of an animal or herd of animals, to improve the appearance of the animal or herd thereof, and/or - the quality and/or quantity of produce of the animal or herd thereof.

19. The composition of any one of claims 1 to 14, wherein the composition is a pharmaceutical composition.

20. A polysaccharide or composition thereof for use as a medicament, wherein the polysaccharide or composition thereof is as defined by any one of claims 1 to 14 and 19.

21. A polysaccharide or composition thereof for use in the treatment, amelioration or prevention of a disease or condition of a subject, wherein the polysaccharide or composition thereof is as defined by any one of claims 1 to 14 and 19.

22. A compound or composition thereof for use in a method of treatment, wherein the method comprises administering the polysaccharide or composition thereof as defined by any one of claims 1 to 14 and 19.

23. A polysaccharide, or a composition thereof, for use in a method of treatment, amelioration or prevention, wherein the polysaccharide comprises a backbone of alpha- (1-5)-linked arabinofuranose residues, a first side chain of a single alpha- arabinofuranose residue (1-2)-linked to an arabinofuranose residue of the backbone, and a second side chain of a single alpha-arabinofuranose residue (1-3)-linked to the same arabinofuranose residue of the backbone, and wherein the method comprises administering a polysaccharide, or a composition thereof, according to any one of claims 1 to 17.