Aloe extract for microbial neutralization

Abstract

The present invention relates to a composition comprising a polysaccharide derived from Aloe vera having agglutinating activity against microorganisms, and its use in the prevention or treatment of infectious diseases. The composition has an average molecular weight in the range of 30-100 kDa and comprises 80-100% w / w mannose and 0-5% w / w glucose.

Description

Technical Field

[0001] The present invention relates to an aloe extract for neutralizing microorganisms. In particular, it relates to an aloe extract that promotes the neutralization of microorganisms by promoting the aggregation of microorganisms.

Background Art

[0002] Pathogenic microorganisms have developed tools to break through the natural host defense system, colonize, establish themselves in our tissues, and invade. Most of the problems with microorganisms begin when harmful microorganisms attach to human epithelial surfaces such as the skin and mucous membranes. To effectively bind to the host tissue surface, many microorganisms have multiple adhesin proteins on their outer surface. These adhesins bind to receptor molecules on the surface of host epithelial cells. The anti-adhesion strategy aims to block the interaction between microbial adhesins and host epithelial cell receptors by blocking these receptors to prevent microbial colonization of host cells. Since microbial colonization is the first important step in the infection process, pathogens are rendered harmless by effectively blocking microbial attachment through the anti-adhesion strategy. However, a drawback of anti-adhesion therapies is that most pathogenic microorganisms have genes encoding multiple types of adhesin proteins. As a result, in order to succeed in blocking, it is necessary to block each of these adhesin proteins.

[0003] Colony formation can also be hindered by microbial aggregation, also known as clustering. Aggregation is the process by which microorganisms become entangled in a network by agglutinating compounds that form crosslinks between individual microorganisms. Aggregating factors have been described. Roche et al. 2015 Mucosal Immunol. 8:176 describes how antibodies play a major role in colonization and defense against infection by the physical process of microbial aggregation by antibodies. WO2017 / 117500 describes an oral composition containing mucin-coated silica to promote bacterial aggregation and bacterial clearance from the oral cavity. US2016 / 346436 describes a method for inducing bacterial aggregation, comprising contacting bacteria with nanocrystalline cellulose (NCC) or an NCC hydrogel composition to induce aggregation, thereby reducing the bacteria's ability to adhere to a surface or form a biofilm. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Extracts from aloe vera have been used as medicinal ingredients. Vu et al. Pharmaceutics (2020) 12: 644 discloses an aloe vera extract consisting of 57–77% mannose, 15–22% glucose, and 5–7% galactose, with a molecular weight of 150–800 kDa. This extract is incorporated into dental sponges to stimulate the formation of dental pulp tissue. EP 1 323 738 describes a negatively charged aloe vera polysaccharide fraction containing 0–40% glucose and 60–100% mannose. The polysaccharides in this fraction have a molecular weight of 100–300 kDa and block bacterial adhesion to tissues. Known antimicrobial agents from aloe vera are based on anti-adhesion and are unlikely to block all microbial adheren proteins. Agglutinating factors from aloe vera have not been disclosed to date. [Brief explanation of the drawing]

[0005] [Figure 1A] Agglutination of Staphylococcus aureus stained with neutral IEX fraction D0 using FITC. [Figure 1B] Absence of agglutination in Staphylococcus aureus stained with negatively charged IEX fraction D1 via FITC. [Modes for carrying out the invention]

[0006] Detailed description of the invention In one embodiment, the present invention relates to a composition having agglutination activity against microorganisms. This composition comprises a polysaccharide derived from aloe vera.

[0007] One advantage of the composition according to the present invention is that it has agglutinative activity against microorganisms, thereby immobilizing them into aggregates or clusters. These aggregates neutralize the microorganisms. One reason for this is that as microorganisms form clusters, their ability to resist adhesion is reduced. When microorganisms aggregate in clusters, there is less surface available for attachment to the host epithelium because the surface area of ​​the attached cell membrane is significantly reduced. Aggregates can be removed from an individual's body more easily than single microorganisms. Thus, the agglutinative effect supports the epithelium's natural defense mechanism against microbial invasion. Another advantage is that since the bacteria are not killed, antibiotic resistance is not induced. Yet another advantage is that the composition according to the present invention can be used for the aggregation of a wide range of different microorganisms. Both bacteria, fungi (including yeast), and viruses can aggregate.

[0008] In one embodiment, the microorganism is a Gram-negative bacterium, a Gram-positive bacterium, or a fungus. Suitable examples of bacteria or fungi that can agglutinate include those selected from the group consisting of Bacillus, Bacteroides, Clostridium, Enterococcus, Escherichia, Listeria, Neisseria, Pseudomonas, Salmonella, Staphylococcus, Streptococcus, Yersinia, Aspergillus, and Candida. In a preferred embodiment, the microorganism is selected from the group consisting of Candida, Enterococcus, Escherichia, Helicobacter, Klebsiella, Lactobacillus, Propionobacterium, Staphylococcus, or Streptococcus. In one embodiment, the microorganism to be agglutinated is selected from the group consisting of Candida albicans, Staphylococcus aureus, Escherichia coli, and Helicobacter pylori. In another embodiment, the microorganism is selected from the group consisting of Escherichia coli ATCC 25922, Enterococcus faecalis, Candida albicans ATCC 10231, Klebsiella pneumoniae, Lactobacillus crispatus DSMZ 20584, Lactobacillus gasseri DSMZ 20243, Lactobacillus rhamnosus, Propionibacterium acnes, Streptococcus mytis, and Streptococcus pyogenes. In yet another embodiment, the microorganism is Staphylococcus aureus ATCC 35556, Staphylococcus aureus genotype B (GTB), vancomycin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, or multi-resistant Staphylococcus aureus.

[0009] In one embodiment, the composition according to the present invention is obtained from aloe, particularly Aloe barbadensis mirror or Aloe curaçao, also known as Aloe vera. The composition according to the present invention can also be obtained from other aloe species, including Aloe arborescens, Aloe baombe, Aloe ferox and Aloe saponaria. The composition can be obtained from aloe inner leaf fillets, which are a transparent gel of aloe leaves. In one embodiment, the composition is obtained from inner leaf fillets containing 10% w / w or more polysaccharides after freeze-drying of the gel. The composition can be decolorized.

[0010] The compositions according to the present invention may also be obtained from other plants, such as cranberry, ginseng, plantain, echinacea, garcinia, arnica, angelica, hibiscus, licorice, and moringa.

[0011] Preferably, the composition is uncharged at pH 7. This composition does not bind to an anion exchange column such as Q-Sepharose at pH 7 and passes through it.

[0012] The compositions according to the present invention contain, or may consist of, polysaccharides having an apparent molecular weight in the range of 30 to 100 kDa or greater than 1000 kDa. Preferably, the compositions according to the present invention contain, or consist of, polysaccharides having an apparent molecular weight in the range of 50 to 100 kDa. In one embodiment, the polysaccharides in the composition according to the present invention have an apparent molecular weight in the range of 30 to 100 kDa, preferably 50 to 100 kDa. In another embodiment, the polysaccharides in the composition according to the present invention have an apparent molecular weight greater than 1000 kDa. These fractions exhibit agglutination activity against microorganisms such as Staphylococcus aureus or Escherichia coli. The apparent molecular weight can be determined by methods known in the art, for example, by ultrafiltration.

[0013] As shown in the following examples, the compositions according to the present invention differ from acemannan, which contains relatively little mannose and more glucose. Acemannan is preferably prepared by ethanol precipitation. In one embodiment, acemannan is prepared by adding 4 parts 96% ethanol to 1 part aloe juice, mixing the mixture, incubating without stirring for at least 4 hours and up to 24 hours to precipitate the polysaccharides, centrifugally separating the mixture at >3500 xg for 15 minutes, and recovering a pellet containing the precipitated polysaccharides. The pellet may be dried, for example, by freeze-drying and dissolved in an aqueous solution, preferably milli-Q water.

[0014] Unless otherwise indicated, the weight figures referred to in this application are preferably based on dry weight.

[0015] The distribution of polysaccharides in the composition according to the present invention is 80-100% by weight of mannose and 0-5% by weight of glucose, preferably 90-98% by weight of mannose and 0-5% by weight of glucose, of the total polysaccharides. In one embodiment, the distribution of polysaccharides in the composition according to the present invention is 85-95% by weight of mannose and 3-4% by weight of glucose, of the total polysaccharides. In another embodiment, the distribution of polysaccharides in the composition according to the present invention is 90-98% by weight of mannose and 2-4% by weight of glucose, of the total polysaccharides.

[0016] The polysaccharide may further contain 0-5% by weight of galactose and 0-5% by weight of fucose, for example, 1.0-5.0% or 2.5-5% by weight of the total polysaccharide.

[0017] Preferably, the polysaccharides in the composition do not contain or contain very few negatively charged polysaccharides such as galacturonic acid or glucuronic acid, for example, 0-3% w / w of the total polysaccharides in the composition.

[0018] Preferably, the composition contains little to no anthraquinone, for example, less than about 0.05% w / w or less than about 0.001% w / w.

[0019] In one embodiment, the distribution of polysaccharides in the composition according to the present invention is 85-95% w / w mannose, 3-4% w / w glucose, 0-5% w / w galactose, and 0-1.5% w / w fucose, based on the weight of the total polysaccharides. In another embodiment, the distribution of polysaccharides in the composition according to the present invention is 90-98% w / w mannose, 2-4% w / w glucose, 0-5% w / w galactose, and 0-1.5% w / w fucose, based on the weight of the total polysaccharides.

[0020] Preferably, the polysaccharides in the composition contain mannose:glucose in a weight ratio in the range of 25:35.

[0021] The polysaccharide content of the composition according to the present invention is preferably in the range of 0.5 mg / ml to 10 mg / ml, 1.0 mg / ml to 10 mg / ml, 1.0 mg / ml to 5.0 mg / ml, or 1.0 mg / ml to 3.5 mg / ml, etc., and is at least 0.5 mg / ml, at least 1.0 mg / ml or at least 2.0 mg / ml.

[0022] The mannose content of the composition according to the present invention is preferably in the range of 0.5 mg / ml to 10 mg / ml, 1.0 mg / ml to 10 mg / ml, 1.0 mg / ml to 5.0 mg / ml or 1.0 mg / ml to 3.5 mg / ml, etc., and is at least 0.5 mg / ml, at least 1.0 mg / ml or at least 2.0 mg / ml.

[0023] The galactose content of the composition according to the present invention is preferably at most 0.15 mg / ml, more preferably at most 0.10 mg / ml.

[0024] The composition according to the present invention contains or consists of a polysaccharide having an apparent molecular weight in the range of 30 to 100 kDa or >1000 kDa, preferably in the range of 50 to 100 kDa, and the polysaccharide contains 80 to 100% w / w or 90 to 98% w / w mannose and 0 to 5% w / w glucose of the total polysaccharide.

[0025] In one embodiment, the composition according to the present invention is preferably uncharged at pH 7 and contains a polysaccharide having an apparent molecular weight in the range of 30 to 100 kDa or >1000 kDa, preferably in the range of 50 to 100 kDa, in the range of 0.5 mg / ml to 10 mg / ml, 1.0 mg / ml to 10 mg / ml or 1.0 mg / ml to 3.5 mg / ml, and the polysaccharide contains 80 to 100% w / w or 90 to 98% w / w mannose and 0 to 5% w / w glucose of the total polysaccharide weight.

[0026] In one embodiment, the composition is preferably uncharged at pH 7 and contains a polysaccharide in an amount of 0.01 to 100% w / w of the weight of the composition, such as 0.05 to 100% w / w, 40 - 80% w / w, 0.01 to 30% w / w, 5 to 80% w / w, 5 to 50% w / w, 10 to 30% w / w, 0.05 to 10% w / w, 0.05 to 1.0% w / w or 0.1 to 1.0% w / w. The apparent molecular weight of the polysaccharide is in the range of 30 to 100 kDa or >1000 kDa, preferably in the range of 50 to 100 kDa. The polysaccharide contains 80 to 100% w / w or 90 to 98% w / w of mannose and 0 to 5% w / w of glucose based on the weight of the total polysaccharide.

[0027] In another embodiment, the composition is preferably uncharged at pH 7 and contains 0.1 to 3% w / w of a polysaccharide having an apparent molecular weight >1000 kDa. The polysaccharide contains 80 to 100% w / w or 90 to 98% w / w of mannose and 0 to 5% w / w of glucose.

[0028] In yet another embodiment, the composition is preferably uncharged at pH 7 and contains 0.05 to 0.5% w / w of a polysaccharide having an apparent molecular weight in the range of 30 to 100 kDa, preferably in the range of 50 to 100 kDa. The polysaccharide contains 80 to 100% w / w or 90 to 98% w / w of mannose and 0 to 5% w / w of glucose based on the weight of the total polysaccharide.

[0029] The composition according to the present invention may further contain a carrier such as a pharmaceutically acceptable carrier.

[0030] The ability of a composition to agglutinate microorganisms can be expressed by its minimum agglutination concentration (MAC), which is the lowest concentration of polysaccharides in a sample that convincingly demonstrates microbial agglutination. In one embodiment, the MAC is determined by a 2x dilution series. A lower MAC indicates a stronger composition. The composition preferably has a MAC of less than 8 mg / ml, less than 5 mg / ml, less than 2 mg / ml, less than 0.1 mg / ml, less than 0.01 mg / ml, less than 0.005 mg / ml, less than 0.003 mg / ml, or less than 0.001 mg / ml when tested against Staphylococcus aureus ATCC 35556, for polysaccharides / sample. For example, 0.001 mg / ml to 7 mg / ml, 0.001 mg / ml to 5 mg / ml, 0.001 mg / ml to 1 mg / ml, 0.001 mg / ml to 0.1 mg / ml, 0.001 mg / ml to 0.010 mg / ml, 0.001 mg / ml to 0.005 mg / ml, 0.001 mg / ml to 0.003 mg / ml, or 0.001 mg / ml to 0.002 mg / ml. This is preferably tested with a suitable assay such as a fluorescence assay.

[0031] The quantification of polysaccharides may be carried out by any suitable means, such as measuring the dry weight using freeze-drying, or by hydrolysis of the polysaccharide followed by chromatography using, for example, HPAEC-PAD.

[0032] The Maximal Aggregating Dilution (MAD), the highest dilution of a sample that still exhibits agglutination, can be determined by using an equal volume of the microbial sample and performing a 2-fold serial dilution, for example, with PBS. In one embodiment of the present invention, the MAD value of the composition according to the present invention is preferably at least 100, at least 500, or at least 1000, for example, 100-2000, 500-2000, or 1000-2000, when tested against Staphylococcus aureus ATCC 35556.

[0033] In one embodiment, the composition according to the present invention preferably has a MAC of 0.001 mg / ml to 0.010 mg / ml, 0.001 mg / ml to 0.005 mg / ml, 0.001 mg / ml to 0.003 mg / ml or 0.001 mg / ml to 0.002 mg / ml and a MAD value of 500 to 2000 or 1000 to 2000 when tested against Staphylococcus aureus ATCC 35556.

[0034] The compositions according to the present invention are typically aqueous liquids when isolated. They can be stored in any form. In one embodiment, it is stored in solid form, or, for example, as a freeze-dried powder. In another embodiment, it is stored in liquid form, for example, as an aqueous liquid or gel.

[0035] In another embodiment, the present invention relates to a method for agglutinating microorganisms to prevent infection or colonization, particularly colonization of mucous membranes, and to promoting the neutralization of microorganisms in the body of a subject. The subject may be a human, particularly a person taking medication, an elderly person, or a woman. The subject may be an animal, particularly a mammal such as livestock, a sporting animal, or a pet, and includes cats, cattle, dogs, horses, fish, goats, poultry, rabbits, sheep, pigs, and lambs. The method involves bringing microorganisms into contact with a composition according to the present invention. In one embodiment, a composition according to the present invention is used in a method for preventing or treating mastitis in cattle, for example, by formulating it into a gel and applying it intramammary gland or mammary gland. Mastitis is an inflammation of the mammary gland and is typically caused by bacteria such as Escherichia coli, Staphylococcus or Streptococcus, particularly Staphylococcus aureus, Streptococcus uberis, Streptococcus disgalactie (SDY), or Streptococcus galactie (SAG). Mastitis can be asymptomatic and subclinical, or clinical with symptoms such as swelling or redness of the udder, or milk coagulation or milk flaking. Symptoms of mastitis can be mild, moderate, or severe. In preferred embodiments, this method is used to prevent or inhibit mild or moderate mastitis in dairy cows such as cattle, goats, or sheep, particularly mild or moderate mastitis in cattle. Conventional methods for treating mastitis involve administering antibiotics for 3-4 days, followed by a 3-4 day washout period to remove the antibiotics. Antibiotics are usually delivered by a mammary syringe for injecting the antibiotics into one or more teats of the udder. The effectiveness of the antibiotics is limited. Approximately 40% of cattle show a recurrence of mastitis after one week. Using the compositions according to the present invention allows for better treatment. In one embodiment, a gel containing 1 to 3 g / l of polysaccharides, preferably about 2 to 3 g / l of polysaccharides, is used to treat mastitis, preferably mastitis involving Staphylococcus aureus, in particular mastitis involving Staphylococcus aureus. A suitable gel is an acrylic acid polymer (Carbopol) based gel.

[0036] In another embodiment, the compositions according to the present invention are used, for example, in a method for preventing or treating bovine uterine inflammation by washing the uterus of an animal with the gel of the present invention, which contains 0.5 to 2 g / l of polysaccharides, preferably about 1 to 2 g / l of polysaccharides. The gel may further contain a diluent, such as saline solution, to allow for a sufficient amount to wash the uterus, and may also contain a gelling agent, such as 2 to 4% w / w xanthan gum, to prevent immediate leakage from the uterus.

[0037] The compositions according to the present invention have agglutination activity against microorganisms, meaning that microorganisms agglutinate upon contact with the compositions according to the present invention. This can be tested and visualized by labeling the microorganisms with suitable dyes, such as fluorescent dyes like fluorescein isothiocyanate (FITC), Hoechst 33342, carboxyfluorescein succinimidyl ester (CFSE), or the green fluorescent nucleic acid stain SYTO-24, all of which are commercially available. Depending on the agglutination activity, dilution ratio, and microorganism, agglutination may begin immediately after contact between the microorganisms and the compositions according to the present invention, within 1 second, 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or 3 hours.

[0038] Aggregation activity may manifest as the appearance of clusters of microorganisms rather than individual cells. Aggregation is usually stable enough to delay the inspection of aggregation, and may be observed for at least 3 hours, at least 4 hours, at least 5 hours, or at least 16 hours, at least 18 hours, or at least 24 hours after contact of the microorganisms with the composition according to the present invention. In one embodiment, aggregates may be observed for 5 to 16 hours, 5 to 24 hours, or 5 to 48 hours after contact of the microorganisms with the composition according to the present invention.

[0039] In one embodiment, agglutination in a fluorescence assay is preferably performed with a composition having a polysaccharide content of 0.001 mg / ml to 7 mg / ml, 0.001 mg / ml to 5 mg / ml, 0.001 mg / ml to 1 mg / ml, 0.001 mg / ml to 0.1 mg / ml, 0.001 mg / ml to 0.010 mg / ml, 0.001 mg / ml to 0.005 mg / ml, 0.001 mg / ml to 0.003 mg / ml, or 0.001 mg / ml to 0.002 mg / ml when tested against Staphylococcus aureus ATCC 35556.

[0040] In another embodiment, the present invention relates to the use of compositions according to the present invention. The compositions can be used in or as dietary supplements or in therapeutic diets. The compositions according to the present invention can also be used in personal care or cosmetics, such as dental care, ear care, eye care, hair care, nose care, skin care or vaginal care. In one embodiment, the composition is used in dental care in toothpaste for the prevention or treatment of gingivitis or tooth decay. In another embodiment, the composition is used in eye care to protect the lens of the eye from microbial colonization.

[0041] The compositions according to the present invention may also be applied in pharmaceutical uses, particularly as agents or adjuvants in pharmaceutical compositions, or in methods for preventing or treating infections caused by infectious microorganisms such as bacteria, fungi (including yeast), or viruses. Infection refers to the invasion or proliferation of microorganisms that are not normally present in the body, or that are present in abnormal amounts. Such infectious microorganisms are sometimes called pathogens.

[0042] Infections can be subclinical or clinical, with obvious symptoms. The effects of treatment or prevention can be permanent or temporary, lasting for days, weeks, months, or years. The effects can be complete or partial, such as immobilizing some or all of the microorganisms. This may lead to the prevention or reduction of the invasion or growth of some or all of the microorganisms. In one embodiment, 5% to 100%, 5% to 60%, or 60% to 100% of the microorganisms are immobilized.

[0043] The composition can be used in release coatings to prevent colonization or biofilm formation on biological or inert surfaces. For this purpose, surfaces can be coated with the composition according to the present invention for immediate or controlled release. The composition induces microbial aggregation on the coating, thereby preventing colonization or biofilm formation on the surface. When the coating is released, the microbial agglutinants on the coating are also released in an aggregated form and can be easily removed from the body. Surfaces that may be treated in this manner include medical devices, particularly the surfaces of indwelling medical devices such as catheters, implantable electronic devices (CIEDs), heart valves, fixators, joint replacements, stents, tracheostomies, and wound drains. The devices may include or be composed of metals, ceramics, or plastics or non-biodegradable synthetic polymers such as polytetrafluoroethylene (PTFE, Teflon®), or biodegradable polymers such as collagen, hyaluronic acid, polylactic acid, or polyurethane. The medical devices are preferably indwelling devices or implants intended to remain in the body for days, weeks, months, or years.

[0044] The composition can be applied topically, for example, as a cream, drop, gel, liquid, lotion, paste, shampoo, spray, or tonic. The composition can be administered orally. The composition can be dried, for example, by spray drying or freeze-drying, and formulated into suitable forms, preferably with suitable excipients, such as capsules, lozenges, powders, or tablets. The excipients must generally be safe and non-toxic and particularly suitable for use in humans or animals.

[0045] The compositions according to the present invention can be used in combination with other compounds such as nanocrystalline cellulose or silica flocculants; gelling agents or thickeners such as chitosan, caprylyl glycol, 1,2-propylene glycol, or xanthan gum; antimicrobial agents such as antibiotics; vitamins such as vitamin A, vitamin B, vitamin D, or vitamin E; and minerals such as calcium, zinc, iron, or potassium. Those skilled in the art will understand that one compound may have several different functions. For example, a vitamin may also be an antimicrobial agent. In one embodiment, the composition is used in combination with (other) aloe vera extract, antibiotics, chitosan, nutrients, or vitamins. In another embodiment, the composition is combined with a viscous aqueous carrier for the uterus for vaginal douching. The other compounds may be present in or separately from the composition, and the other compounds and the composition may be the same formulation or separate formulations. Individual formulations may be administered simultaneously or sequentially.

[0046] Those skilled in the art will understand that the concentration of the composition in a formulation may vary and depend on factors such as the formulation, application site, use, age, sex, and weight of the subject. In one embodiment, the formulation contains a composition according to the present invention in a range of 2.0 ml / l to 800 ml / l, such as 50 ml / l to 800 ml / l, 100 ml / l to 800 ml / l, 200 ml / l to 800 ml / l, or 400 ml / l to 800 ml / l.

[0047] In another embodiment, the composition according to the present invention is formulated to yield a polysaccharide concentration of 0.05 mg / ml to 10 mg / ml, 0.5 mg / ml to 10 mg / ml, or 0.8 mg / ml to 5 mg / ml in the formulation.

[0048] Those skilled in the art will understand that the dosage of formulations containing the composition according to the present invention may also vary and depend on factors such as formulation, application site, use, age, sex, and weight of the subject. In one embodiment, formulations containing the composition according to the present invention are administered at a polysaccharide dose of 0.01 to 10 mg / kg of body weight, such as 0.1 to 10 mg / kg of body weight, 0.5 to 5.0 mg / kg of body weight, or 1 to 2 mg / kg of body weight.

[0049] The formulation may contain pharmaceutically acceptable excipients such as anti-adhesion agents, antioxidants, binders, fillers, carriers, colorants, disintegrants, diluents, fillers, flavoring agents, lubricants, preservatives, solvents, surfactants, sweeteners, vehicles, or wetting agents. Excipients should not adversely affect the stability of the composition in the formulation. The term "pharmaceutically acceptable" generally means suitable for the manufacture of a composition that is considered safe and non-toxic.

[0050] Examples material and method Asemannan Production One part B21 aloe juice was mixed with four parts 96% ethanol and incubated for approximately four hours without stirring to precipitate the polysaccharides. Subsequently, the sample was centrifuged at >3500 xg for 15 minutes, and the supernatant was discarded. The pellet containing the precipitated polysaccharides was dried by freeze-drying and dissolved in milliQ water or another aqueous solution.

[0051] agglomeration Microbial cells were fluorescently labeled with fluorescein isothiocyanate (FITC, Sigma-Aldrich, St. Louis, MO, USA) or SYTO® 24 green fluorescent nucleic acid stain (Thermo Fisher, Rockford, IL, USA). FITC labeling was performed by incubation with FITC (from 2 mg / ml FITC stock in DMSO) at a final concentration of 0.2 mg / ml in 0.2 M NaHCO3 pH 9.5 buffer, shaking at 37°C for 1 hour, followed by thorough washing with phosphate-buffered saline (PBS). SYTO-24 labeling was performed in PBS for at least 30 minutes using a final concentration of 1 μM dye, followed by washing with PBS. Fluorescently labeled microorganisms were incubated in PBS on flat-bottom polystyrene 96-well plates or glass microscope slides, in the absence or presence of aloe samples or controls. Labeled microbial samples were diluted to OD 0.1 or 0.2 before use, as needed. Agglutination was examined immediately or at a convenient time using a fluorescence microscope. If agglutination was not examined immediately, the mixture was stored at 4°C until examination. Agglutination was evident from the clustering of fluorescently labeled microorganisms.

[0052] Maximum Agglomeration Dilution (MAD) The maximum agglutination dilution (MAD) is the highest dilution at which a sample still exhibits agglutination, and was determined by serially diluting an equal volume of microbial sample 2-fold with PBS or a similar solution.

[0053] Minimum aggregation concentration (MAC) The minimum agglutination concentration (MAC) is the lowest concentration of polysaccharides that still reliably induces microbial agglutination. It is determined by 2-fold serial dilution and expressed in mg or micrograms of polysaccharide / ml. A lower MAC indicates stronger microbial agglutination activity. Unless otherwise specified, sample quantification is based on dry weight measurement. [Examples]

[0054] Aloe vera aggregation activity A 10x concentrated aloe vera leaf juice and a 10x decolorized liquid made from purified inner leaf fillets containing more than 10% polysaccharides were purchased from a commercial supplier (ACTIValoe® Aloe Vera Gel 10X-D, AloeCorp, Harlingen, TX, USA). This material was dialyzed against desalinated water using a Spectra / Por 4 RC dialysis membrane tube 12-14 kDa MWCO dialysis membrane (Spectrum Medical Industries Inc., Los Angeles, USA) to remove all components less than 10 kDa. The agglutination activity of the juice fraction against Staphylococcus aureus was determined as described in "Materials and Methods" above. Agglutination activity was present in the >12 kDa fraction. The minimum agglutination concentration (MAC) of the >12 kDa fraction was measured and compared to D-mannose. The results are shown in Table 1. D-mannose did not show flocculation activity at the highest concentration tested (16 mg / ml), but flocculation was still observed even when the >12 kDa fraction of aloe vera inner leaf juice containing less than 1.28 mg / ml of polysaccharides was diluted fourfold, suggesting that further dilution while maintaining flocculation activity may have been possible.

[0055] [Table 1] [Examples]

[0056] Microbial aggregation The agglutination ability of the >12kDa fraction from Example 1 was tested with various microorganisms. The results of the maximum agglutination dilution (MAD) are shown in Table 2. This indicates that a wide range of microorganisms, from Gram-positive and Gram-negative bacteria to yeasts and some pathogens, can be agglutinated by the >12kDa fraction.

[0057] [Table 2] [Examples]

[0058] Determination of IEX characteristics of aloe extract To further characterize the fraction responsible for the agglutination activity of aloe vera against microorganisms, 10x concentrated aloe vera inner leaf juice (ACTIValoe® Aloe Vera Gel 10X-D, AloeCorp, Harlingen, TX, USA) was dialyzed against desalinated water using Spectra / Por 4RC dialysis membrane tubes (12-14 kDa MWCO dialysis membrane, Spectrum Medical Industries Inc., Los Angeles, CA) to remove all components <10 kDa. Subsequently, the dialyzed material was reconstituted with 20 mM NaPO4 pH 7.0 buffer and manually loaded onto a Q Sepharose FastFlow column (GE Healthcare, Uppsala, Sweden) and separated using a batch procedure. Two ion exchange (IEX) fractions were collected. First, uncharged flow-through in 20 mM NaPO4 buffer pH 7 was collected (D0 fraction). Next, the negatively charged compounds were eluted with 20 mM NaPO4 / 0.5 M NaCl buffer, pH 7 (Fraction D1). Polysaccharide elution was measured using refractive index detection. The fractions containing polysaccharides were pooled, dialyzed against a 12–14 kDa MWCO dialysis membrane, precipitated with 80% ethanol (incubated overnight at -20°C and centrifuged at 3000 xg for 20 minutes), and then lyophilized. Polysaccharide yields were determined based on dry weight. Fraction D0 accounted for 97% of the polysaccharides contained in the inner leaf juice of aloe vera, while fraction D1 accounted for 3% of the polysaccharides.

[0059] D0 and D1 were incubated with FITC-labeled Staphylococcus aureus cells at OD 0.2 in clear, flat-bottomed polystyrene 96-well plates (Greiner Bio-One, Monroe, NC) at a concentration of 2 mg / ml in phosphate-buffered saline (PBS) at room temperature without shaking. Staphylococcus aureus began to agglutinate within 10 minutes of adding D0 (Figure 1A). No agglutination occurred during incubation with the D1 fraction (Figure 1B). This indicates that the D0 fraction is involved in the agglutination activity of aloe vera inner leaf juice. [Examples]

[0060] Agglutination activity against Staphylococcus aureus and Helicobacter pylori To further characterize the fraction responsible for the agglutination activity of aloe vera against microorganisms, 10x concentrated aloe vera inner leaf juice (ACTIValoe® Aloe Vera Gel 10X-D, AloeCorp, Harlingen, TX, USA) was fractionated into fractions of different apparent molecular weights by continuous ultrafiltration while decreasing the molecular weight cutoff filter (MWCO) size. The starting material was initially decontaminated by centrifugation at 5,000 g for 15 minutes to remove particles. Fractions with apparent molecular weights in the ranges of >1000kDa, 300-1000kDa, 100-300kDa, 30-100kDa, 10-30kDa, and 3-10kDa were prepared by continuous centrifugal ultrafiltration using VivaSpin 20 ultrafiltration units (Sartorius, Bohemia, NY, USA) with cutoffs of 1000kDa, 300kDa, 100kDa, 30kDa, 10kDa, or 3kDa, respectively. After each run, the flow-through was used as input for the next filter. The retaining liquid of all UF filters was washed with 1000 times the volume of Milli-Q water to wash away low molecular weight compounds.

[0061] The agglutination effect of aloe fractions on Staphylococcus aureus and Helicobacter pylori was investigated. Inner leaf juice, dialysis juice, and fractions D0 and D1 of aloe vera were also tested. 50 μl of a diluted aloe vera sample in desalinated water and 50 μl of SYTO-24 labeled bacteria in 2X PBS were added to the wells of a transparent flat-bottom polystyrene 96-well microtiter plate (Greiner BioOne, Monroe, NC) in which the samples were dissolved. The plate was incubated at 4°C without shaking. Agglutination was checked by fluorescence microscopy after 16 hours, and the maximum agglutination dilution (MAD) and minimum agglutination concentration (MAC) were measured.

[0062] The results are shown in Table 3. The results show that the MAD value of the D0 fraction is significantly higher (approximately 50 times) than that of the D1 fraction. This means that the D0 fraction can be diluted many times while retaining its aggregation activity. This is partly due to the fact that the D0 fraction contains far more polysaccharides than the D1 fraction (97% vs. 3% of the total polysaccharides in the juice, respectively), and partly due to the fact that the polysaccharides in the D0 fraction are more active on a w / w basis than those in the D1 fraction (as indicated by the lower MAC value of the D0 fraction). Furthermore, the aggregates formed by the D0 fraction are much larger than those formed by the D1 fraction, indicating that the D0 fraction immobilizes more microorganisms at lower concentrations than the D1 fraction. This more efficient aggregation and larger aggregate formation by the D0 fraction can lead to microbial neutralization, and the aggregates can affect the motility of microorganisms when trapped in aggregates, as they result in more restrictive movement compared to single microbial cells. This prevents microorganisms from penetrating the mucosal barrier. Aggregates are easier to remove from a system than single cells.

[0063] [Table 3]

[0064] In the ultrafiltration fractions, agglutination activity was mainly observed in the 30–100 kDa and >1000 kDa fractions. The MAC of the inner leaf juice was approximately 2–3 times that of the 30–100 kDa fraction, indicating that the purified 30–100 kDa fraction was several times potent on a weight basis than the original inner leaf juice. The 30–100 kDa fraction was also potent compared to the >1000 kDa fraction. Against both Staphylococcus aureus and Helicobacter pylori, the 30–100 kDa fraction was the most potent aloe vera fraction on a weight basis. [Examples]

[0065] Agglutination activity against Escherichia coli and Candida albicans The 30–100 kDa and >1000 kDa fractions from Example 4 were also tested against E. coli and Candida albicans, starting with 50 μl of the active sample and 50 μl of E. coli or Candida albicans at OD 0.1. The results were checked after 16 hours. The agglutination activity (MAD value) of the active UF fraction is shown in Table 4. This indicates that the 30–100 kDa and >1000 kDa fractions are also involved in agglutination activity against E. coli and Candida albicans.

[0066] [Table 4] [Examples]

[0067] Polysaccharide composition of fractions exhibiting aggregation activity Samples exhibiting flocculation ability were freeze-dried, and the active components—neutral monosaccharides, galacturonic acid, and glucuronic acid—were measured by methanolysis at 80°C. Subsequently, oligosaccharides and polysaccharides were decomposed into monosaccharides by trifluoroacetic acid (TFA) hydrolysis at 120°C for 75 minutes. Monosaccharides in the hydrolysates were determined by high-pH anion exchange chromatography (Eurofins Food Testing Netherlands BV, Heerenveen) using pulsed amperometric detection (HPAEC-PAD). The detection limit for sugars was approximately 0.05 mg / mL for individual monosaccharides in these sample solutions.

[0068] Analysis revealed that the neutral fraction D0 consisted mainly of polysaccharides, including mannose (approximately 95% w / w), and a low concentration of glucose (approximately 3.5% w / w). The negatively charged fraction D1 contained mostly galactose (approximately 32%) and a significant amount of galacturonic acid (approximately 24% w / w). Mannose and arabinose each constituted approximately 15% w / w, based on the total sugars in the D1 sample.

[0069] The polysaccharides in the most potent fractions of Examples 4 and 5, the 30-100 kDa fraction and the >1000 kDa fraction, consisted mainly of mannose (over 85% w / w), some glucose, galactose, and some fucose. The most active fraction, the 30-100 kDa fraction, contained at least 90% w / w mannose, about 3% w / w glucose, and about 1% w / w fucose. The mannose:glucose weight ratio of this fraction was 32. Thus, the polysaccharides in the D0 fraction, the 30-100 kDa fraction and the >1000 kDa fraction are involved in the aggregation activity of aloe vera inner leaf juice and consist mainly of mannose (over 80% w / w), some glucose (3-4% w / w), and small amounts of other carbohydrates.

[0070] [Table 5] [Examples]

[0071] Preparation of D0 and D1 fractions of Aloe Batch 21 Using a new batch (batch 21) of 10x concentrated aloe vera inner leaf juice (ACTIValoe® Aloe Vera Gel 10X-D, AloeCorp, Harlingen, TX, USA), the D0 fraction (B21 D0 fraction) and the D1 fraction (B21 D1 fraction) were prepared as described above in Example 3. [Examples]

[0072] B21 D0 fraction Next, the B21 D0 fraction was fractionated into fractions with different apparent molecular weights by continuous ultrafiltration while decreasing the molecular weight cutoff (MWCO) filter size, as described in Example 4. In this study, the 30-100 kDa fraction was further subdivided into 50-100 kDa and 30-50 kDa fractions for further investigation. [Examples]

[0073] Aggregation of B21 D0, D1, and D0-UF fractions The agglutination effect of the B21 aloe fraction against Staphylococcus aureus was investigated. Inner leaf juice, dialysis juice, and fractions D0 and D1 of aloe vera were also tested. 50 μl of aloe vera sample was diluted with desalinated water, and 50 μl of SYTO-24 labeled bacteria in 2X PBS was added to a 96-well clear flat-bottom polystyrene microtiter plate (Greiner BioOne, Monroe, NC). The plate was incubated at 4°C without shaking. After 16 hours, agglutination was checked by fluorescence microscopy, and the maximum agglutination dilution (MAD) and minimum agglutination concentration (MAC) were measured.

[0074] The results, shown in Table 6, indicate that the MAD value of the D0 fraction is significantly higher than that of the D1 fraction (approximately 250-fold). This means that the D0 fraction can be diluted many times while retaining its aggregation activity. This is partly due to the fact that the D0 fraction contains far more polysaccharides than the D1 fraction (98.5% vs. 1.5% of the total polysaccharides in the juice, respectively), and partly due to the fact that the polysaccharides in the D0 fraction are more active on a w / w basis than those in the D1 fraction (as indicated by the lower MAC value of the D0 fraction). Furthermore, the aggregates formed by the D0 fraction are much larger than those formed by the D1 fraction, indicating that the D0 fraction immobilizes more microorganisms at lower concentrations than the D1 fraction. This more efficient aggregation and larger aggregate formation by the D0 fraction can lead to microbial neutralization, and the aggregates can influence the motility of microorganisms when trapped in aggregates, as they result in more restrictive movement compared to single microbial cells. This prevents microorganisms from penetrating the mucosal barrier. Aggregates are easier to remove from a system than single cells.

[0075] [Table 6]

[0076] In the ultrafiltration fraction, flocculation activity was mainly observed in the 50-100 kDa fraction. The highly viscous >1000 kDa fraction showed lower activity than the 50-100 kDa fraction and was of less interest as a flocculant. The MAC of the inner leaf juice was approximately three times that of the 50-100 kDa fraction, indicating that the purified 50-100 kDa fraction is several times stronger than the original inner leaf juice when expressed on a weight basis. [Examples]

[0077] Polysaccharide composition of fractions exhibiting aggregation activity Samples exhibiting flocculation ability were freeze-dried, and the polysaccharide composition was determined as described in Example 6 above (Eurofins Food Testing Netherlands BV, Heerenveen). Data analysis showed that batch 21 consisted of approximately 28% w / w mannose and 70% w / w glucose (Table 7). The B21 D0 fraction and the B21 D0 50-100kDa fraction consisted of 95-96% w / w mannose and 2-3% w / w glucose. The acemannan fraction consisted of approximately 85% w / w mannose and approximately 8% w / w glucose. Other monosaccharides were less than 1%. All percentages were based on total polysaccharides. The batch 21 flocculated fraction clearly contained more mannose and less glucose in the polysaccharide fraction than the acemannan fraction.

[0078] [Table 7] [Examples]

[0079] Treatment of mastitis Twenty-five cows were treated with a pH-neutral gel containing the composition according to the present invention. The gel, containing 2 g / l of polysaccharides, 2% w / w of Carbopol (Carbopol® 1382, Lubrizol, Cleveland, US), and 15% w / w of 1,2-propylene glycol, was injected into the cows' teats using a plastic udder injector. Mastitis was identified by visual inspection of the animals by livestock handlers. Signs of mastitis include redness or swelling of the udder or teats, coupled with general malaise in the cows. If mastitis was observed, 5-10 ml of gel was applied to the teat immediately after milking. This was repeated for three days. The results were comparable to or better than standard antibiotic treatment. After treatment with the composition of the present invention, the recurrence of mastitis appeared to be reduced compared to antibiotic treatment. [Examples]

[0080] Treatment of uterine myometritis Cattle exhibiting signs of endometritis, namely general fatigue or sluggishness, uterine discharge, or elevated body temperature, were treated by lavaging the uterus with a large amount of gel containing the composition according to the present invention. Uterine lavage was performed twice daily for 5 days using a gel containing 1.5 g / l of polysaccharides, 3% w / w xanthan gum (Xanthanal 11K, PKelco, France), and 0.3% w / w caprylyl glycol (1,2-octanediol) (Dermosoft Octiol, Dr. Straetmans Chemische Produkte GmbH, Hamburg, Germany). A significant reduction in postpartum infections was observed compared to cases without lavage. The results were comparable to those of standard antibiotic lavage.

Claims

1. A composition containing polysaccharides derived from aloe vera, It has agglutination activity against microorganisms, It contains 80-100% by weight of mannose and 0-5% by weight of glucose, A composition that is uncharged at pH 7 and has an apparent molecular weight in the range of 30 to 100 kDa.

2. The composition according to claim 1, having an apparent molecular weight in the range of 50 to 100 kDa.

3. The composition according to claim 1 or 2, comprising 90 to 98% by weight of mannose and 0 to 5% by weight of glucose of total polysaccharides.

4. The composition according to any one of claims 1 to 3, wherein the weight ratio of mannose to glucose is in the range of 25:1 to 35:

1.

5. The composition according to any one of claims 1 to 4, further comprising 0-5% w / w galactose or 0-5% w / w fucose.

6. A composition according to any one of claims 1 to 5, comprising 0.5-10 mg / ml of polysaccharides.

7. Furthermore, the composition according to any one of claims 1 to 5 further comprises other aloe vera extracts, antibiotics, chitosan, nutrients, or vitamins.

8. Use of the composition according to any one of claims 1 to 7 as a nutritional supplement, or in a nutritional supplement, or in a product for dietary therapy, in a product for personal care, or in a cosmetic product.

9. A composition according to any one of claims 1 to 7 for use as an adjuvant.

10. A composition according to any one of claims 1 to 7, for use in the treatment or prevention of an infection of the body of a human or animal.

11. The composition for use according to claim 10, wherein the infectious disease is an infectious disease in livestock, pets, sports animals, or cattle.

12. The composition according to any one of claims 1 to 7 or the composition for use according to any one of claims 9 to 11, wherein the composition is formulated as a cream, drop, gel, liquid, lotion, paste, shampoo, spray, tonic, capsule, lozenge, powder, or tablet.

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