Title of Invention

NEGATIVELY CHARGED POLYSACCHARIDE DERIVABLE FROM ALOE VERA AND A PROCESS FOR PREPARING THE SAME

Abstract

The present invention relates to a new composition of matter is provided comprising negatively charged polysaccharides which can be derived from Aloe vera and a process to prepare that composition of matter by sub fractionation of an extract of Aloe vera, passing the formed subfraction over a positively charged column and eluting the same with a salt solution. Optionally the Aloe vera is pre purified over aolumn. This composition of matter and also the extract comprising the same which is formed after pre purification or ultra filtration of an Aloe vera extract is useful as a food supplement or in dietary foods, for use in personal care and in cosmetics, especially to prevent an infection with the bacteria Helicobacter pylon, Pseudomonas aeruginosa, Streptococcus mutans or Streptococcus sanguis.

Full Text

Negatively charged polysaccharide derivable from Aloe vera The present invention relates to a composition of matter comprising polysaccharides derivable from Aloe vera and a method to prepare said composition of matter, a plant or animal exti*act comprising said composition of matter and a method to prepare said plant or animal extract and the application thereof as food supplement, in personal care and in pharmaceutical use. Aloe is a member of the lily family comprising over two hundred different aloe species. Aloe barbadensis Miller or Aloe Curacao is generally recognized as the 'true aloe" because of its wide use and most effective healing power. Aloe vera contains two major liquid sources, a yellow latex (exudate) and the clear gel (mucilage). The mucilaginous jelly from the parenchymal cells of the plant is referred to as Aloe vera gel. Aloe vera gel is about 98.5 % water by weight. More than 60 % of the total solid is made up of polysaccharides of carbohydrate origin. Since the earliest days of recorded history man has made use of whole leaves exudates and fresh gels obtained from Aloe vera because it is responsible for a range of biological activities including antibacterial, antiviral and anti-inflammatory activities. In was the traditional medicine of many cultures and used inter aha for leprosy, burns and allergic conditions. Other aloe species with healing power are for instance Aloe arborescens, Aloe vahombe, Aloeferox and Aloe saponaria. In the literature a lot of different polysaccharides mentioned to be responsible for said biological activities have been described. For instance in US patent 4,861 761 a one-step method for the preparation of a pure therapeutically active polysaccharide called Aloeferon with a molecular weight of about 70 kD has been disclosed. In US patent 5,118,673 said biological activities are ascribed to Acemannan, a polysaccharide extracted from Aloe vera gel comprising maimose molecules which are for about 91 % 0-acetylated. Besides maimose another glysosyl component namely galactose is present in a ratio of about 20 : 1. The molecular weight is on the average about 1000 kD. This non-toxic polymer is also said to be effective in the suppression of tumours. However, recently Nirmal Pugh et al. described in the Journal of Agricultura i Food Chemistry, 49,1030 - 1034 (2001) a new high-molecular-weight poiysaceharido from Aloe vera with potent immuno stimulatory activity. The molecular weight is reported to be 4000 - 5000 kD. The major glycosyl components are glucose (37.2 %), galactose (23.9 %), mannose (19.5 %), and arabinose (10.3 %). It is stated that although this polysaccharide comprises only 0.015 % of the original dry weight, its biological activity in this assay accounts fully for the activity in the crude Aloe juice. It is proposed that the much lower immuno stimulatory activity of acemannan is due to a very potent substance (most likely Aloeride polysaccharide) that is present in trace amounts as a "contaminant". Therefore, up to now it has not been established for sure which fraction of Aloe vera causes the biological activity of said plant. An object of the present invention is to isolate a novel composition of matter derivable from Aloe vera or a plant or animal extract which is suitable as food supplement or in dietary foods, in personal care or in cosmetics, or in pharmaceutical use, especially to prevent the adhesion of microorganisms in tissues. Another object of the invention is to provide processes by which such a composition of matter or extract may be isolated. It was found that a new negatively charged polysaccharide fraction isolated from Aloe vera and mainly comprismg marmose showed a surprisingly higher biological activity than the corresponding not charged or only weakly charged polysaccharide fractions, wliich fractions do not bind to a positively charged column. This higher biological activity has been found for subfractions with all apparent molecular weights. The present invention provides such a composition of matter comprising polysaccharides derivable from Aloe vera with the following characteristics: a) the polysaccharides comprise 70 - 90 % D-mannose with a range between 60-100 %, 30 - 10 % D-glucose with a range between 40 - 0 % and 0 - 10 % other monosaccharides, b) the polysaccharides are negatively charged and c) the polysaccharides bind to a positively charged column. With the second indicated broader range is meant that a polysaccharide with a weight percentage of indicated monosaccharides within the broad ranges belong to the scope of the invention, but that those with such a weight percentage within the small ranges are preferred. All percentages relating to a composition of matter relate to weight percentages. Preferably, in said composition of matter the polysaccharides have an average molecular weight of about 100 - 300 kD. However as also other subfractions with an average molecular weight of either 10 - 50 kD, or 50 -100 kD or higher than 300 kl) show a considerable biological activity, these subfractions also form an aspect of the present invention. Preferably, the ratio of D-mannose and D-glucose in said polysaccharides is within the range of about 5 to 20, preferably 7-10. The present invention also provides a process to prepare said composition of matter by the follownig process steps: a) sub fractionation of a plant or animal extract, for instance an Aloe or A loe wre extract in two fractions, one with an apparent molecular weight of > ± 5 kD, name J subfraction I and one with an apparent molecular weight of b) passing of subfraction I over a positively charged column as for instance a DEAE-Sepharose column, a DEAE-Sephadex column or a DEAE-cellulose column, c) eluting the part of subfraction I bound to said column with a salt solution, 1^ -r instance with a sodium chloride solution resulthag in subfraction I-Dj d) desalting and ultra filtration of I-Dj, for instance through a PMIO membraru- under nitrogen pressure, to concentrate I-Djto about 0.1 of the original volume of the Aloe vera extract e) optionally preparation of subfractions of I-Dj with desired apparent niolccuhn weights of > 300 kD, 100 - 300 kD, 50 - 100 kD and 10 - 50 kD, particularly by sequential ultra filtration over a XM-300, XM-100, XM-50 and finally a PM-10 membrane or by preparative FPLC over a Superose column. Preferably, a pre purification step is carried out before process step "a' over a Sephadex G-25 column. In an article of A. Femenia et al.. Carbohydrate Polymers 39, 109 -117 (19*'^-^). also extracts of Aloe vera have been described, however said extracts are not further fractionated and not fiirther separated with the aid of a positively charged colunm. The present invention also provides as a suitable substance a plant or anima! extract, especially an Aloe extract, more especially being an extract from Aloe vera, indicated as NAG-25 (no affinity for Sephadex G-25) extract, which comprLses the composition of matter as defined above in a concentration of 5 -10, especially 8 time:; higher and of low molecular weight compounds of about 2 times lower than the extraci:; i known in the art. Generally such a plant or animal NAG-25 extract (to be understood :i, plant NAG-25 or animal NAG-25 extract) will be a sap. According to a further aspect of the invention a process has been provided to prepare such a plant or animal NAG-25 extract by purification of the corresponding untreated plant or animal extract over a Sephadex G-25 column to remove materials with affinity for said column. By "corresponding" is meant the same species of plan! ■■ n animal. Such a NAG-25 extract comprises all high molecular compounds without air. affinity for the polysaccharide matrix of the positively charged column used but als^^ less low molecular weight compounds than expected. If necessary the resulting exirac; i; further concentrated by a factor of 5 to 50 by the removal of water resulting in the plaia or animal NAG-25 extract according to the present invention, also indicated with 2QRide. Generally, if the starting compound is a spray dried powder a concentrated solution may be obtained by starting in a low volume of water in which case no funh; r concentrating steps are necessary. Preferably, if a plant is used to prepare the composition of matter or the N At i -21-extract according to the invention this is an Aloe plant, especially an Aloe vera. However an extract containing the negatively charged polysaccharides can also be gained from other plants. Biologically active polysaccharides have been found in Vaccinnium macrocarpon (Cranberry), Panax ginseng, Plantago, Echinacea, Garcim;!, Arnica, Angelica, Hibiscus, Glycyrrhiza, Morinda etc. If an animal is used especially fishes and slugs are suitable. However besides extracts from plants and animals als.* extracts of lower organisms like seaweed, sponges and mushrooms should be consid'-'i ed for this patent application as belonging to the scope of the invention. Therefore for in is patent application the wording plant and animal also comprise lower organisms. According to still a further aspect of the invention an ^/oe extract, especially an Aloe vera extract is provided which has been ultra filtrated preferably with a cross t1.v,v method over a membrane to prepare subfractions with a desired apparent molecuha-weight as indicated above, but with both charged and uncharged polysaccharides, binding and not binding to a positively charged column. Also this Aloe ultra filter extract comprises the new negatively charged polysaccharides according to the invention. Preferably, said charged and uncharged polysaccharides are further separaud in charged and uncharged polysaccharides by passing over charged filters, from whici i the charged polysaccharides are indicated with 2QRide. The new negatively charged polysaccharides, the plant or animal NACr-25 extracts and the Aloe ultra filter extract comprising a high percentage of said negat:\; ; charged polysaccharides according to the invention herein described have a hioji biological activity and may be applied as food supplement or in dietary foods, iov instance to prevent the adhesion of bacteria, particularly in the mucous layer of the human gastric epithelium. Furthermore, said negatively charged polysaccharides, d) plant or animal NAG-25 extracts and tho Aloe ultra filter extracts comprising ihe :;::[:: can be applied for personal care and cosmetic use to prevent infections of detrimentai and harmful microorganisms, for instance in dental care as in toothpaste to prevciu gingivitis and caries. Furthermore, the charged polysaccharides and said extiacts comprising the same can probably suitably be applied in liquids for instance to pruicr; eye lenses, in sprays and tonics, and in drops, creams and gels to look after the skin. hair, eyes and ears. Finally, said polysaccharides and said extracts comprising ihc :ninv: are to be applied in pharmaceutical use, especially as a medicament or adjuvaiis in a pharmaceutical composition to prevent or cure infections with infectious microorganisms like viruses, fimgi and bacteria or in prevention and healing of inflammadons, and probably in immuno tlierapy and in wound healing. An infection with four of said bacteria, the Helicobacter pylori, Pseudomonas aeruginosa, Streptococcus mutaiis and Streptococcus sanguis bacteria, can particularly be com? ■ ir.;-. i by these polysaccharides. Throughout this patent application all percentages relating to a composition ! i matter relate to weight percentages. Furthermore, by "corresponding" is meant as starting material the same species of plant or animal as the resulting extract. Furthermore, unless otherwise indicated, by "extract" is meant extract by water. The infection of the stomach hy Helicobacter pylori is one of world's mo . common bacterial infections. A minority of infected individuals develops a gastro duodenal disease associated with said bacterium. Examples thereof are the dove 1 o|) i u>. 11! of peptic ulcer disease, chronic and atrophic gastritis mucosa-associated lymphoid u:::;nc lymphomas and gastric cancer. Adhesion of Helicobacter pylori to the mucosa is liiaiicu to the apical surface of the mucosa epithelial cells and to cells lining the gastric pii.s, particularly in the bottom part of the stomach. Different adhesins have been found ti) mediate this binding, by recognition of proteins or specific glycoconjugates, i.e. niiu:in ;. present on the eukaryotic cell surface (D. liver et al., Science 279, 373 - 377 (19981) Therefore, at least part of the adhesion of Helicobacter pylori seems to be glycoconjugate dependent. However until now the effectivity of a plant NAG-25 or animal NAG-25 extract, as for instance an^/oe extract or an ultra filtration.4/ot^ exiriici containing negatively charged polysaccharides according to the invention to combat ;i!i infection with Helicobacter pylori has nowhere been described. hi an established ELISA assay it appeared that Aloe vera extracts could iiiiii 1)11 the adhesion of preparations of Helicobacter pylori adhesins to salivary mucins indeed Therefore it was decided to investigate which components in the extract are responsi Ide for the inhibition. Active subfractions of Aloe vera gels were obtained by a combiiiLU ion of precipitation, molecular sieving and anion-exchange chromatography and were characterized with regard to molecular weight and sugar composition and appeared I-J \\^ novel. The present invention is illustrated by the following figures with the legends: Figure 1: Elution of the bound fraction of 200 ml AV-15 jfraction I (the > 5 kD Iraetitui of AV-15 NAG) from a DEAE-Sepharose column with a NaCl gradient from 0-2 M NaCl. The Y-axis represents the NaCl concentration (M) as well as the absorbance at 215 nm; the X-axis represents the elution volume. Zero ml represents the startmg ptMiii of the 0-3.0 M NaCl gradient which was applied after collection of the DEAE-unbour.d AV-DQ fraction followed by washing the column with 2 column volumes of Mil!i- water. Figure 2; Inhibition of adherence of mucin to Helicobacter pylori S-layer by Aloe r. /, / extracts obtained from various sources with muc, positive control containing mucine only AV, co-incubation of mucine with a 2 - 16 fold dilution range of Aloe vera extract AV-3 and AV-4 1:1 Aloe Gel products; AV-B, AV-D, AV-E and AV-F arc coxnmercialy available Aloe vera sources concentrated 40,10, 5 and 2,5 times respectively A49o„m absorbance values per well in duplo, for mucin in quattro. Figure 3: Inhibition of 100 - 300 kD subfraction of charged fraction I-Diof an A!o,' v. / ; extract with muc, positive control containing mucine only AV-2, an 1:1 Aloe-Gel product in different concentrations A49onm absorbaucc values per well in duplo, for mucin in quattro. Figure 4: Inhibition of adherence of FITC-labeled Helicobacter pylori to hmnan luiu mw slices by subfractions of Aloe vera extract AV-5 with a. regular view b. consecutive slice incubated with FITC-labeled Helicobacter pylori imd total fraction I c. consecutive slice incubated with FITC-labeled Helicobacter pylori and fraction I-DQ, identical with controls without ^/oe vera subfractions. The following abbreviations used throughout this patent application have \h.c meaning: DEAE = diethylaminoethyl FITC = fluorescein 5-isothiocyanate HPAEC-PAD = high pH anion-exchange chromatography with pulsed amperomeuic detection BCA = bicinchoninic acid ELIS A == en2yme-linked immunoassay A490nm ~ absorbancc at 490 nm NMR = nucleic magnetic resonance A DEAE-binding fraction can be isolated from the > 5 kD fraction I of A V-1' NAG-25 by DEAE-Sepharose, DEAE-cellulose and DEAE-Sephacel chromatogiapir, under elution with 0.5 or 1 M NaCl. This is exemplified by the NaCi gradient in Fiimr.-1 for the elution of the DEAE-bmding fraction of 200 ml AV-15 fraction I. Non-spoi^i li. adhesion to the polysaccharide matrix is unlikely since the NAG-25 fraction and not ilic Aloe gel is used as a starting material for the subfractionation. Therefore the NaCI -dependent elution confirms that the DEAE-binding is caused by a negative charge ow the molecules. Proteins or peptides were below detectable levels. Sugar analysis revealed that galacturonic acid is present in small amounts, but this sugar appeared ai' () to be present in the non-binding Do fraction (results not shown). So the molecular naim c of the negative charge is not known yet. All available Aloe vera extracts mhibited the interaction with the mucins in a dose-dependent way when they were co-incubated with a fixed concentration of niucai, see Figure 2. Variations in inhibitory activity reflect differences in compositions ot extracts in dependence of source or culture conditions of the Aloe vera plant but (io hardly change the proportional biological activity of the various subfractions. Apparently an ^/oe vera component or Aloe vera components compete with mucin lor the binding to the Helicobacter pylori adhesin preparation. The majority of the inhibitory activity of the ^/5 ver^ extracts appear to reside-in a subfiaction, I, with a molecular weight of at least 5 kD, according to its behavir,ur on Sephadex G-25 chromatography. Further studies were focussed on this charged fraction because of its high activity. Carbohydrate analysis and analytical permeaiioj-, chromatography on a Superdex HR-200 column revealed that polysaccharides were il i-. major components. The sugar composition depends on the Aloe vera extract but an> extract consists of homo- and hetero polymers of mannose and glucose. The bulk of the inhibitory activity could be retained and eluted specifically \'v iil) NaCl from anion-exchange columns. DEAE-Sepharose chromatography was applied in isolate this apparently negatively charged polysaccharide fraction, indicated witli fraction I-D i. Sequential ultra filtration was employed to obtain subfractions with apparent molecular weights of > 300 kD, 100 - 300 kD, 50 -100 kD and of 10 ^ 50 k [,). These subfractions were also prepared for the components that did not bind to DEAE Sepharose indicated with fraction I-DQ. All subfractions appear to contain for 90'?/a, ( r particularly ft)r 95 % or more homo- or hetero polymers of mannose and glucose, oi which the polymannoses form the major components as sximmarized in table 1 here below. The remainder comprises galactose and various non-identified sugars (not sho: i: in the table). As is shown in table 1, the inhibitory activities of the DEAE-binding fraction ; are considerably liigher than of the non-binding subfractions of the Aloe vera extinci The JOO - 300 kD subfraction of I-Dj expresses the highest inhibitory activity (82 ';./, although in the assay per well, viz. 12.5 |.il, only a low amount of Aloe vera polysaccharide is present, nl. 0.325 fig of mannose and 0.045 (ig of glucose. This represents about 9.3 nM of polysaccharide in the 200 \xl end volume assuming a meiui molecular weiglit of 200 kD. The inhibition is dose-dependent and 50 % of the inhibition is reached at a 10-fold lower concentration of about 0.03 ^ig of mannose v-r well or about 0.9 nM of polysaccharide, see Figure 3. The very low amount of composition of matter recovered in the > 300 kD DEAE-binding fraction is also vci > active per pmol ofpolymannose when the high molecular weights have been taken iiiid account. The otlier two DEAE binding fractions show a much lower specific activity l-ui still higher than in the corresponding subfractions of the DEAE non-binding I-D(j. Remarkably, no inhibitory activity was detectable for the polysaccharides present in fraction I-DQ with molecular weights > 300 kD. table 1 Inhibitory activity of subfractions I-Doand I-Di on the adherence ofK pyiori Subfractions of Aloe vera extract AV-2 (see Materials) are prepared by sequential ultrafiltration starting from 25 ml of said extract AV-2. The volumL- of each subfraction is adjusted to 12.5 ml. The data are based on values in duplD obtained for equal amounts (12.5 jil) of each fraction and are expressed relaii \'c to the absorbance measured in the control wells containing mucin only. Tlic inhibitory activity of 12.5 |il of the original AV-2 extract with 813 jig glucose irl and 325 \xg maimose/ml, viz. 286 nM of polysaccharide, was 76 %. Inhibition of binding of Helicobacter pylori to gastric mucosa was demon.^tr;\!-J by incubating sequential slices of human antrum mucosa with FITC-labelled Helicobacter pylori in the absence and presence of Aloe vera subfractions. Like in il it-study of Boren et al. Science, 262,1892 -1895 (1993), selective bmding of FITC-labeled Helicobacter pylori cells was only observed on the mucosal linings of the antrum, see figures 4a and 4c. Co-incubation of the FITC-labelled Helicobacter pylov with the total weight fraction I, viz. I-DQ and I-Dj fractions of an Aloe vera extract strongly inhibited the adherence of the bacteria to the mucosa (see figure 4b) witli i: \\ sharp contrast with the absence of inhibition when subfraction I-DQ alone was co-incubated. This is another strong indication that the inhibitory activity resides in ihj negatively charged fraction. A comparable inhibition pattern has been found for Syto-13 labeled Helicohaajr pylori wherein Syto-13 is a green fluorescent stain applying two different concentralii is; of Aloe vera subfractions according to the invention on MUC5-Iabelled multi-well plates. In an article from Van den Brink et al, Gut 46, 601 - 607 (2000), "//. vyiori c^ * localizes with MUC-5AC in the human stomach" it has been described that H. pylori iii the stomach binds to a specific mucin present on the antrum part of the stomach. Therefore, the effect of Aloe vera subfractions on similar salivary mucin in an in vitr>. ■ assay system can be used as a model for the mucin-specific attachment of H. pylori xo the epithelium of the stomach. The results are shown in the tables 2 and 3. table 3 Inhibitory activity of subfraction I-Di of an AV extract on the adherence of H. py!i ^ri In the same way the effect of the adherence of an Aloe vera subfraction according to the invention on two Syto-13 labeled strains of P. aeruginosa was lesual im MUC5-labelled multi-well plates. The amount of bacteria bound to the plates \va.s dependent on the amount of coated MUC-5. The results are given in table 4 and tabic :^ respectively. .1 table 4 Inhibitory activity of subfcaction I-Di of an AV extract on the adherence o f P. aeruginosa In the same way the effect of the adherence of an Aloe vera subfraction according to the invention on a Syto-13 labeled strain of Streptococcus mutans anci ow ;i Syto-13 labeled strain oiStreptococcus sanguis was tested on agglutinin-enricheci sal i va coated to the plates. The amount of bacteria bound to the plates was dependent on t!iL: amount of coated MUC-5. The results are given in table 6 and table 7, respectively. table 6 Inhibitory activity of subfraction I-Dj of an AV extract on the adherence of Streptococcus mittans It will be appreciated by the person skilled in the art that the anti adhesive polysaccharides according to the invention are anti infectiva against all microor^^ainsai: which invade the surface of the host tissue which are exemplified by the Helicohacfcr pylori, Pseudomonas aeruginosay Streptomyces mutans and sanguis bacteria as mentioned above. Except of bacteria invasion is a phenotype common to cancer cei!: leukocytes, parasites, bacteria and viruses involving cell-cell adhesion, cell adhesion, proteolysis and motility. These activities are regulated by the cross tal] highly susceptible to morbidity and mortality associated with drug resistant patliogciu;. Inhibition of adhesion is therefore an important property of new anti infectiva. The polysaccharides according to the invention reduce the biofilm load. Thi:; \:. due to a reduction in adhesion of Gram negative and probably also of Gram positive bacteria to the cells. Furthermore, said polysaccharides also interfere with the adliesi v;' processes of viruses, fungi, flagellates and other parasites and can be part of a thera;)v ii; treat or prevent affections and diseases of the whole body of both humans, animals anil possibly plants. Said polysaccharides which consist of simple monosaccharides are lux expected to be toxic both in oral, topical, injectable and systemic applications. Relating to the application of the negatively charged polysaccharides or plant Examples of affections, infections and diseases which can be prevented and treated by the anti adhesive polysaccharides of the present invention are besides th():;e caused by microorganisms which invade the gastro-intestinal tract like the stomach by for instance Helicobacter pylori those of the; skin, caused by — Staphylococcus aureus and Staphylococcus epidermitis which are common pathogens, e.g. in hospitals — viruses such as Kaposi' sarcoma-associated herpes virus, herpes simplex virus — fungi such as Candida sp., Blastomyces dermatidis; adhesion to the skin also includes adhesion to demial microvascular endothehal cells eyes, caused by — Staphylococcus epidermitis which plays an important role in the pathogenesis ()some forms of endophthalmitis occurring after cataract surgery — Moraxella bovis as the source of infectious bovine keratoconjunctivitis ear, nose and throat, caused by — Staphylococcus aureus which adheres to the skin and mucous tissues — bacteria involved in Otitis media and nasopharyngal infections such as Haemophilus influenza. Streptococcus pneumoniae and Moraxella catarrhalis tlie oral cavity, wherein the dental plaque biofilm plays a pivotal role in the progression of dental diseases and polysaccharides are of great importance in the ecology of the dental biofilm, caused by — bacteria involved in caries such as Streptococcus sobrinus as acariogenic strain, Streptococcus miitans. Streptococcus salivarius. Streptococcus gordonii and Actinomyces viscosus, Actinobocillus actinomycetemcomitans — periodontopathogenic bacteria such as Poiphyromonas gingivalis and Streptococcus salivarius, Streptococcus oralis, Fusobacterium nucleatiim and Prevotella intermedia — all oral spirochetes which are classified in the genus Treponema, such as denticola,pectinovoru77ty socranskii and vincentii, — Mycoplasma salivarium — microorganisms involved in nasal polyposis — microorganisms involved in Sinusitis the urogenital tract, caused by . — gram negative Uropathogenic Escherichia coli which adhere to the tissues of the 1 urogenital tract — Mycoplasma genitalium — Trichomonas vaginalis — Candida species — Neisseria gonorrhoeae adhesion to oviductal epithelium — Treponema pallidum which is mvolved in perivasculitis, endothelial cell abnormalities that are prominent histopathologial features of syphilis and various cutaneous lesions that are the main clinical features of syphiUs — Escherichia coli — Ciirobacter spQcics the gut, caused by — Salmonella species e.g. Salmonella typherium — Proteus mirabilis — Clostridium species, e.g. difficile^perfringens^ bifermentans — Shigella species, e.g.flexneri — Mycoplasma species, e.g. gallisepticum — Enterococcus species — Bacteroides fragilis — Bacillt^s species — Listeria monocytogenes — Hepatitis A virus — Campilobacter jejuni — Salmonella typhimurium — Yersina enterocolitica and Yersina pseudotuberculosis — Aeromonas veronii biovar sobria — Erwinia chrysanthemi which is a model plant pathogen that has the potential to parasitize mammalian hosts as well as plants the respiratory tract caused by — Pseiidomonas aeruginosa^ a gram-negative facultative pathogen of the bronclui .md the lung as well as cystic in fibrosis patients — Klebsiella pneumoniae — Bordetella sptcies^ pertussis, parapertussis and bronchiseptica — bacteria of the genus Legionella are intracellular parasites and major human pathogens — the respiratory syncytical virus (RSV) which causes potentially lower respLratury tract infection in children — Mycoplasma pnetanoniae — Rhinovirus which potentiates induction of proasthmatic changes — Cryptococcus neoformans which usually occurs in the lungs, and is involved in interactions between yeasts and alveolar epithelial cells — Streptococcus species such zs pyogenes or gordonii — Escherichia pneumoniae^ an important respiratory pathogen — the Burkholderia cepacia complex which consists of at least five well-documciVLCi! bacterial genomovars, each of which has been isolated from the sputum of dilTcr.ni patients with cystic fibrosis — Mannheimia (Pasteurella) haemolytica which is one of the most important respiratory pathogens of domestic ruminants and causes serious outbreaks of aciiio pneumonia in neonatal, weaned and growing lambs, calves and goats. It is also an important cause of pneumonia in adult animals — Rhinotracheitis virus, parainfluenza-S virus or bovine respiratory syncytial virus which predispose animals to M. haemolytica infection the organs, blood, lymph, bloodvessels and the lymphatic system, caused by — Staphylococcus aureus in bacterial endocarditis, — Streptococcus sanguis in bacterial endocarditis, — Stcphylococcus epidermidis in bacterial endocarditis — Gram-positive and Gram-negative bacteria, such as S. aureus and E, call in intra vascular infection — Coxsackievirus — Rotavirus — Murine cytomegalovirus — Adenovirus — Neisseria meningitides — Chlamydia pneumoniae — Wolbachia bacteria related to Gram-negative Rickettsiales, in Onchocerca volvnius-infected persons — the Lyme disease spirochete Borrelia burgdorferi — Coxiella bwmetii^ the agent of Q fever — Acholeplasma laidlawii — intracellular invasion is an important aspect of Carrion's disease caused by Barti^ndhi Bacilliformis, Both the hematic and tissue phases of the disease involve tlie initial attachment of the organism to erythrocytes and endotlielial cells. — Paracoccidioides BrasiJiensis, a dimorphic fungus known to produce invasive systemic disease in humans. Therefore, accordmg to the invention a composition of matter comprising negatively charged polysaccharides, optionally present in an plant or animal NAG-2 5 extract or in an Aloe ultra filter extract according to the invention, which can effecti vrl y be applied for the prevention and treatment of infections with microorganisms, presumably by prevention of the adhesion of said microorganisms. Said composition i M NAG-25 extract or in on Aloe ultra filter extract can be applied as supplement of fooc: and in dietary food, in personal care and in cosmetic use, and in pharmaceutical use. The present invention will be exemplified further by the following example;; which are not to be considered as restricting the scope of the invention in any way. Materials and Methods Materials: Disposable polystyrene columns with maximal bed volumes of 2 ml, were obtained firom Pierce, Rockford, Ireland. Sephadex G-25 Fine, DEAE-Sepharosc, ]a:;i flow 5-ml desalting columns and Superose 200 HJRlO/30 and 1-ml MonoQ HR 5/5 columns were pmrchased &om Amersham Pharmacia Biotech, Uppsala, Sweden. Filtration units of 10 ml and 50 ml as well as a range of ultra filtration membranes w er: obtained from Amicon Corp., Lexington, USA. and Millipore, Bedford, USA. Ccirho; ii TM MAI and PAl analytical columns (4x250 rmn) in combination, with a Carbop:u; 1 :^ 1 Aminotrap Guard column (10x32mm) and a HPAEC-PAD system were obtained fiurn Dionex, Suimydale, CA, USA. Fluorotrac 600 high binding flat-bottom 96 wells microtiter plates were obtained from Greiaer, Frickenhausen, Germany. High-mole i:.-; weight human salivary mucin as well as mouse anti-human monoclonal antibodies (MabF2) against salivary mucin were kindly provided by Dr. E. Veerman, Department (> i Oral Biochemistry, ACTA, Amsterdam. Agglutinin-enriched hiunan saliva was a kind gift of Dr. A. J, M. Ligtenberg, Department Oral Biochemistry, ACTA, Arasterdan). Horseradish peroxidase labeled goat anti-mouse IgG and IgM were obtained from American Qualex, San Clemente, CA, U.S.A.. Fluorescein 5-isothiocyanate (FldXT) \ .xi;: obtained from Sigma, St Louis, MO, U.S.A. Syto-13 green fluorescent nucleic acid stain was obtained from Molecular probe:; (Leiden, The Netherlands) as a 5 mM solution in dimethylsulfoxide. Standard surai s used for carbohydrate analysis were from commercial sources and of anal}1:ical grade. Aloe vera extracts: Aloe vera extracts (AV-1 to AV-7, AV-15, AV-16 and AV-A to AV-F) were provided by Bioclin B.V. (Delft, the Netherlands) and originated from various commercial sources. AV-A, AV-3 and AV-4 comprise Aloe vera extract and gel in a ratio of 1 : 1, AV-B and AV-D are concentrates of commercial sources with a fecior M) and 10, respectively. AV-2 extract contains 813 and 325 ^g of glucose and manno.e respectively. AV-16 was prepared by ultra filtration of the filtered sap of the Aloe vci\ i irmer gel fillet product as described further, with a cross flow method over a hollow il; i membrane with a cut-off of 30 kD, followed by 10 x concentration. AV-5, AV-6, A \ ■ 7 and AV-E were received as lyophilized powders, AV-F and AV-17 as a spray-dried powder. All these products were Aloe iimer gel fillet products. These gel fillets were prepared as described in CA patent No. 1305475. The processes of lyophilizing aiici spray-drying are known to the skilled in the art; the details differ for the various source;_ Extracts and powders were stored frozen directly after receipt; in between experiments resolubilized powders and extracts were kept at 4 'C for no longer {hun ^ ii -month. The extracts were obtained from the leaves taken from Aloe barbendemis M i! K r A 2 % mixture of stabilisation components, consisting of ascorbic acid, sodium benzoate, potassium sorbate, tocopherol, ethyl alcohol, citric acid and sorbitol, v/as added directly after harvesting on the plantation. Some preparations were received ae lyophilized powders which were reconstituted by the addition of milliQ-watcr to th.e desired volume. A crude preparation of Acemannan was kindly provided by dr. R. Zarzycki, Carrington Laboratories Inc. (Irving, TX). Subfractionation of Aloe vera extracts: 50-150 ml of Aloe vera extracts and reconstituted powders were centrifuged ibr 45 minat 15,000Xgat 15 "C. The pellet was discarded and the supernatant was filtered over a 0.2 |am membrane. In the routine, the resulting clear solution was optionally filtered over a small bed volume (1 ml per 5 ml) of Sephadex G-25 to remove Aloe vera components that had affmity for this material (Fraction IE, also indicated as Aloe vcrd NAG-25 extract, see hereunder). Fraction I (apparent mol. wt > ± 5 kD) and U (ap|):ir rw mol. wt water. Fraction I-DQ was concentrated to 0.1 of the original volume of the Aloe vera extract by ultra filtration, under nitrogen pressure, through a PMIO membrane using n 10- or 50-inl filtration unit Fraction I-Dj was desalted by the automated procedure described above and subsequently was concentrated to 0.1 of the original volume of ili ■ Aloe vera extract by ultrafiltration over a PM-10 filter. In some experiments, subfi*actions with apparent molecular weights of > 300,100-300, 50-100 and 10~5() k I) were prepared from Fractions I-DQ and I-Dj by sequential ultra filtration, respective!}, over a XM-300, XM-100, XM-50 and finally a PM-10 membrane. Alternatively, comparable subfractions were prepared by preparative FPLC over a Superose 200 I! I-10/30. Each subfraction was washed 3 times by adding milliQ water to 10 times tlie liiu; I volume obtained; the first wash was added to the subsequent fraction prior to filkiit'u >ii over the next filter. All fractions were stored in aliquots at -20 *^C until further u-se. Aloe vera NAG-25 extract: 10 gram spray dried^/^ ve?'a spray dried extract originating from 2 liter Aloe vt^ra extract was solubilized in 200 ml Milli-Q water and passed over a Sephadex G-25 column (5 cm wide and 10 cm high; prepared in Milli-Q water; flow rate 7.5 ral/min) u ^ remove materials that have aJ05nity for the Sephadex G-25 matrix and reduce tlic content of low molecular weight molecules. The column is subsequently washed with Milli-O water and the Aloe vera NAG-25 extract is collected as the 60 - 310 ml eluate. Bacteria and bacterial extracts: Wildtype H. pylori (ATCC 43504) was grown under micro aerophilic conditions on blood agar DENT plates as described by F, Namavar et al.. Infection Immunity; u^ 444-447 (1998)]. Helicobacter pylori extracts containing the adhesins of the outer membrane, the so-called S-layer, were prepared from confluent bacterial cultures froi -i two or more agar plates. The bacteria were suspended in 0.15 M NaCl, vortexed t^or ! mill, and centrifliged for 30 min at SOOOXg. The supernatant containing the bacterial extract was stored at -SO^'C after detemiination of the protein concentration by IIIL- BCA protein assay (Pierce, Rockford, USA). FITC-labelled H pylori were prepared by incubating bacteria in 1 ml 0.2 M carbonate buffer (pH 8.0) containig 0.1 mg/mJ Fir (? for 15 min in the dark at albumin ( PBST-BSA), the cells were suspended in the sani • buffer at adensity on 0.13-0.20 Agoonm ^^its and were stored in 0.1-ml aliquots until used. Bacterial cultures of Psiiedomohas aeruginosa strains PA025 and PA 14 wei -obtained from the Department of Medical Microbiology, VU medical centre and o; Streptococcus mtitca^s and Streptococcus sanguis were obtained from the Departmcri-Oral Biochemistry, ACTA, Amsterdam. In case the bacteria were used in a fluorcsLci^i inhibition assays, the bacteria were suspended and diluted in 100 mM sodium aceii! j (pH 5), containing 0.5 % Tween-20 to a final absorbance at 700 nm of 0.1. The bad. lia were fluorescent labelled by the addition of Syto-13 (1:500 v/v). Fluorescent inhibition assay. Fluotrac 600 plates were coated with a dilution range of salivary mucin (lor /.' pylori and Pseiidomonas aeruginosa) or agglutinin-enriched human saliva (for Streptococcus mutans and Streptococcus sanguis) in coating buffer (0.1 M sodium carbonate (pH 9.6)). The plates were incubated overnight at 4 T and subsequenri> washed 4 times with PBS-0.1 % Tween-20 (washing buffer). Syto-13 labelled bactt-n;! (50 |lL) were added to the wells followed by 50 p-L of a dilution of an Aloe vera M) sample or water (positive control). Wells without coated mucin served as a negali\ c control. After incubation for 1 h at 37 "C the plates were washed with washing buficr. The fluorescence was measured with the Fluostar Galaxy, excitation and emission wavelength were respectively 485 and 520 run. Monosaccharide analysis: Analysis of monosaccharides was performed by high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on a Carbopav;!; TM MAI column after hydrolysis of the fractions for 4 h in 2 M tri fluoroacetic acid ai 100 °C. The column was eluted with 0.2 M NaOH at a flow rate of 0.4 ml/niin and \\ i;; calibrated with a mixture of standard sugars. Example 1 Inhibition of adherence of i?. pylori to salivary mucin Inhibition of the binding ofH. pylori adhesin to human salivary mucin by AInc vera extracts or subfractions was studied by an established ELISA (see F. Navainar c' al.. indicated above), in which microtiter plates coated with a S-layer preparation of Helicobacter pylori (100 jal/well; 10-20 |ig protein/ml; 16 h at 4°C; washing buficr Pi^S (pH 7.5)-0.1 % Tween-20 (v/v) (PBST)) were incubated in duplicate with human salivary mucin in the presence and absence of dilution ranges of Aloe vera extracts oi fractions in for 2 h at 37 °C. The total volume of the incubation mixtures was 100 pi. which was composed of 50 fil of salivary mucin (0.2-0.5 fig/ml) and 50 |il of a diiutiv )n of Aloe vera sample both in 50 mM sodium acetate-150 mM NaCl -0.5 % Tween-20 i i>l 1 5.0). The monoclonal antibody F2, recognizing the sulfo-Lewis^groups expressed on ihc salivary mucin, and peroxidase-labeled goat-ante mouse antibodies were used for detection of the amount of bound mucin after being washed with PBST as described earlier, for instance by E. Veerman et al., Glycobiology 7,737 (1997). Samples were tested ia duplicates in a 2-fold dilution range. The inliibitory activity was expressed as the percentage decrease of A490njn relative to control wells containing only mucin after correction for the reagent blank. The results arc given in the figures 2 and 3 and in table 1. As described above the inhibitory activities of the DH/Vl' binding fractions are much higher than of the non-binding fractions. From this DEA1> binding fraction the 100 - 300 kD subfraction expresses the highest inhibitory activii} (82%). Example 2 Inhibition of adherence ofH. pylori to gastric mucosa Adherence of FITC-Iabeled bacteria to gastric antrum sections was detected according to Boren et al, see above. Sbc-jim sequential slices of human gastric antrum, dereved from normal tissue and from patients with slightly and moderately inflmiied ;ti id metaplastic tissue were provided by the Department of Pathology. Sequential slices \-. e; c de paraffinized in xylene (10 min, 3 times rinsing), followed by washing 3 times for 5 min m ethanol, rehydration in slowly running miUiQ water and washing 3 times for 5 minutes in PBS. A circle was drawn around the slices with a PAP pen PA03 (Diagnostics BV, Uithoom, The Netherlands) followed by incubation with 0.1 ml PI/>:s 1 -BSA under humid conditions, for at least 1 hr at 4°C. Finally, the buffer was replaced i v, 0.1 ml of FITC-labelled bacteria plus or minus (positive control) a dilution range of tl AV-5 extract or subfraction in PBST-BSA. The slides were incubated for 1 hi in the dark. Unbound bacteria were removed by washing 6 times with PBST-BSA on a rotaiii;: table. Finally, PBS in glycerol (1:1 v/v) was appHed to the sections before sealing ilu-i ,i with a cover glass for fluorescence microscopy using a Nikon Eclipse microscope (Uvikon, Bunnik, The Netherlands), with a Nikon digital camera DxM 1200 and the Nikon ACT-1 camera control program. The results are given in figure 4. Controls vdihout Aloe vera subfractions wer: identical to plate c (not shown). As described above the total weight fraction I of an A .' Example 3 Inhibition of adherence of//, pylori on MUC5-labelled multi-well plates In the fluorescent inhibition assay life bacteria, of which the DNA has been labelled with the fluorescent dye Syto-13, are incubated in the presence or absence o T an A, vera I-D| preparation in a 96-well plate coated with the indicated dilutions of salivary mucin MUC-5 or agglutinin-enriched saliva. In each well the same amount of Syto- i ) labelled bacteria were present. Per experiment, all wells contained the same amount oi the A. vera preparation in case of co-incubation with bacteria. All assays were peril ti 111,- i in duplicate. Typical experiments are shown in tables 2 and 3. The extent of binding of lii ■ Helicobacter pylori to iixQ wells v/^s clearly dependent of the amount of coated Ml K -: present on the wells. Co-incubation with the I-Dj-preparation inhibited the binding o\ Helicobacter pylori to MUC-5 m a concentration-dependent way. The inhibition increased when the amount of MUC-5 decreased (Experiment 1) and also when more 1 - Di was added (Experiment 2). The amount of material in 0.01 ml I-Dj present in the wells was derived firom 0.02 g of AV-17 powder (corresponding to 10-20 ml original A. vera gel). Example 4 Inhibition of adherence of Syto-13 labelled Pseudomonas aeruginosa on MUCS-lalx lied multi-well plates The effects of an AV-161-D, preparation were tested on two strains of P. aeruginosa, P. aeruginosa TAOIS soa^d P. ^e/-wgzno^aPA14. The amount of material in 0.01 ml AV-161-D, fraction was derived from 2.5 ml AV-16. The amount of bactcri;i bound to the plates was dependent on the amount of coated MUC-5. The results weiL-given in table 4 and table 5, respectively. Co-incubation with 0.01 ml of AV-16 I- D, resulted in a strong inhibition of the binding of the bacteria. The pilot study suggests IIM! the inhibition is concentration dependent since the inhibition increased when the amouni of coated MUC-5 decreased. Example 5 Inhibition of adherence of Syto-13 labelled S. mutans andS. sanguis on agglutinin - enriched saliva coated to the plates The effects of an AV-161-D, preparation were tested on two strains of Streptococcus mutans and Sti'eptococcus sanguis. The amount of bacteria bound to the plates was dependent on the amount of agglutinin-enriched saliva coated to the plates. Co-incubation with 0.01 ml of AV-16-D1 resulted in a strong inhibition of the binding of the bacteria. The amount of material in 0.01 ml AV-16-D1 was derived from 2.5 ml. Aloe vera 16. Claims 1. A composition of matter in isolated form comprising polysaccharides derivable from Aloe vera with the following characteristics: a) the polysaccharides comprise 60-100 % D-mannose, 40 - 0 % D-glucose and 0-10 % other monosaccharides b) the polysaccharides are negatively charged c) the polysaccharides bind to a positively charged column. 2. A composition of matter in isolated form according to claim 1 comprising polysaccharides derivable from Aloe vera with the following characteristics: a) the polysaccharides comprise 70 - 90 % D-mannose, 30 -10 % D-glucose and 0-10 % other monosaccharides b) the polysaccharides are negatively charged c) the polysaccharides bind to a positively charged column. 3. A composition of matter according to claim 1 or 2 wherem said polysaccharides have an average molecular weight of about 100 - 300 kD. 4. A composition of matter according to according to any one of the claims 1 - 3 wherein said polysaccharides comprise D-mannose and D-glucose in a ratio of a range of about 5 to 20. 5. Process to prepare a composition of matter according to any one of the claims 1 - 4 characterized by the following process steps: a) subfractionation of an Aloe vera extract in two fractions, one with an apparent molecular weight of > ± 5 kD, named subfraction I and one with an apparent molecular weight of b) passing of subfraction I over a positively charged column c) eluting the part of subfraction I bound to said column with a salt solution, resulting in subfraction I-D, d) desalting and ultrafiltration of I-D| e) optionally preparation of subfractions of I-Dj with desired apparent molecular weights of > 300 kD, 100 - 300 kD, 50 - 100 kD and 10 - 50 kD. 6. Process according to claim 5 characterized by the application of a pre piuification step before process step a over a Sephadex G-25 column to remove materials with affinity for said colimm. 7. Process according to claim 5 or 6 characterized by the application of a DEAE-Sephadex or DEAE-Sepharose column during process step b. 8. Process according to anyone of the claims 5-7 characterized by the application of sequential ultra filtration or preparative FPLC over a Superose column. 9. Plant or animal NAG-25 extract comprising a composition of matter as defined in any one of the claims 1- 4. 10. Plant NAG-25 extract according to claim 9 with the plant being Aloe, 11. Plant NAG-25 extract according to claim 10 with the Aloe being Aloe vera. ) 12. Process to prepare a plant or animal NAG-25 extract as defined in any one of the claims 9-11 characterized by the application of a purification step of a corresponding untreated extract over a Sephadex G-25 column to remove materials with affinity for said colunm. 13. Aloe ultra filter extract comprising a composition of matter as defined in any one of the claims 1-4. 14. Process to prepare an Aloe ultra filter extract as defined in claim 13 characterized by the application of ultra filtration on the corresponding Aloe extract. 15. Composition of matter elected from one or more of the following ones: composition of matter as defined in any one of the claims 1 - 4 or plant or an animal NAG-25 extract as defined in any one of the claims 9 -11 or an Aloe ultra filter extract as defined in claim 12 for use as food supplement or in dietary foods. 16. Composition of matter elected from one or more of the following ones: composition of matter as defined in any one of the claims 1 - 3 or plant or an animal NAG-25 extract as defined in any one of the claims 8-10 or an Aloe ultra filter extract as defined in claim 12 for use in personal care or in cosmetics. 17. Composition of matter elected from one or more of the following ones: composition of matter as defined in any one of the claims 1 - 3 or plant or an animal NAG-25 extract as defined in any one of the claims 8 - 10 or an Aloe ultra filter extract as defined in claim 13 in pharmaceutical use. 18. Composition of matter according to any one of the claims 15 -17 for one or more of the following applications: as anti-bacterial, anti-viral and anti-inflammatory means. 19. Composition of matter according to claim 18 to prevent or heal an infection with any one of the microorganisms bacteria, viruses and fungi. 20. Composition of matter according to claim 19 to prevent an infection with one or more of the bacteria selected from the group consisting of Helicobacter pylori, Pseudomonas aeruginosa, Streptococcus mutans and Streptococcus sanguis. 21. Use of a composition of matter elected from one or more of the following ones: composition of matter as defined in any one of the claims 1 - 4 or plant or an animal NAG-25 extract as defined in any one of the claims 9 - 11 or an Aloe ultra filter extract as defined in claim 13 a as a medicament in prevention or healing of an infection with infectious microorganisms or in prevention and healing of inflammations. 22. Oral dosage form as a tablet, capsule or syrup comprising a composition of matter elected from one or more of the following ones: composition of matter as defined in any one of the claims 1 - 4 or plant or an animal NAG-25 extract as defined in any one of the claims 9-11 or an Aloe ultra filter extract as defined in claim 13 and optionally excipients. 23. Topical dosage form as a cream or gel comprising a composition of matter elected from one or moire of the following ones: composition of matter as defined in any one of the claims 1 - 4 or plant or an animal NAG-25 extract as defined in any one of the claims 9 -11 or an Aloe ultra filter extract as defined in claim 13 and optionally excipients. 24. Injectable dosage form as an injection liquid comprising a composition of matter elected from one or more of the following ones: composition of matter as defined in any one of the claims 1 - 4 or plant or an animal NAG-25 extract as defined in any one of the claims 9 - 11 or an Aloe ultra filter extract as defined in claim 13 and optionally excipients. 25. A method of inhibiting, preventing or reversing adherence of a micro organism to a cell membrane in a living organism comprising administering to said organism a dosage form according to anyone of the claims 22 - 24. 26. Use of a dosage form according to anyone of the claims 22 - 24 for the manufacture of a medicament for inhibiting, preventing or reversing adherence of a micro organism to a cell membrane in a living organism. 27. An aloe ultra filter extract substantially as herein described with reference to the accompanying drawings. 28. An injectable dosage substantially as herein described with reference to the accompanying drawings.

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