CA1312860C - Process for preparation of aloe products - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A process is described for extracting a pharmaceutically active polysaccharidic substance from the aloe plant. The process involves obtaining aloe juice having solubilized matter; adjusting the pH of the aloe juice to a range from about 3 to about 3.5; adding a water soluble, lower aliphatic polar solvent to the aloe juice to precipitate the active chemical substance.
The water soluble, lower aliphatic polar solvent with the solubilized matter is removed and the precipitated active chemical substance is isolated. The pharmaceutically active polysaccharidic substance and its characteristic properties are described.

Description

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~anuary 14, 1988 BACRGROUND OF THE IN~ENTION
; l. Field of the Invention This invention pertains to the field of processing aloe plants and removing portions of said plant for processing same into compositions for topical and internal applications, and compositions of matter comprising said portions of aloe.

2~ Description of the Prior Art, and Other Information Aloe vera is no~ a cactus plant as widely believed, but rather a member of the lily family. There are about 360 species of aloe plants known. Harding, Aloes of the World: A
Checklistc index and code, Excelsa 9: 57 - 94 11979). They seem to thrive in hot arid areas and are widely scattered from the Mediterranean Sea, Middle East, ~frica, China, Japan, Mexico and the southern U.S.A. A few of the important species used for their medicinal properties are Aloe barbadensis Miller (aloe vera), A. arborescens, ~. ~licatilis, A. vahombe, A. saponaria, A. africana, A. ferox and Aloe Perryi.
Reynolds, Aloes_of Tropical Africa and Mada~asar, The Trustees, The Aloe Boo~ Fund, Mbabane Swaziland. ~owever, A. bar-badensis Miller is generally recognized as the ~'true aloe"
; because of its wide use and reportedly most effecti~e h~aling power~ although in Japan, A. arborescens Miller traditionally has been used as a folk remedy for various ailments ranging from gastrointestinal disorders to athlete's foot.
Aloe vera is a perennial plant with turgid green leaves joined at the stem in a rosette pattern. The leaves of a mature plant may be more than 25 inches long with saw-like spikes along their margins.

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RHF:RCB/sm January 14, 1988 ~ 3 ~

Slicing the leaf transversely as shown in Figure~ 1 and 2 reveals the outer walls of the epidermis 3 ~overed with thick cuticles. Beneath the epidermis 3 is the mesophyll which is differentiated into chlorenchyma cells and thinner walled cells known as parenchyma. The parenchyma cells harbor a transparent mucilaginous jelly 1. The vascular bundles 2 with inner bundle sheath cells contain the yellow sap having laxative properties and are sandwiched between the two major cells. Needle shaped crystals of calcium oxalate, produced as a metabolic byproduct in plant cells, are found mostly at the central portion of the leaf.
Aloe vera contains two major liquid sources, a yellow latex (exudate ) and the clear gel (mucilage~. The dried exudate of Aloe barbadensis Miller leaves is referred to as Aloe. The commercial name is Curacao aloe. It is composed mainly of aloin, aloe-emodin and phenols~ Bruce, South African Medical Journal, 41: 984 ~1967); Morrow et al., Arch. _Dermat410gy, 1 : 1064-1065 tl980~; Salek et al., Corrosion Prevention & Control, 9 - 10 ~1983); Mapp et al., Plan a Medica, 18: 361 - 365 (1970); Ranwald, Arch._ _ _ Pharmacology, 315: 477 - 478 (1982). A number of phenolics, including anthraquinones and their glycosides, are k~own to be pharmaceutically active. Brucet Excelsa 5: 57 - 68 ~1975);
Suga et al., Cosmetic and_Toiletries, 980 105 - 108 (1983).
The mucilayinous jelly ~rom the parenchyma cells of the plant is referred to as aloe vera gel. There are gen~rally no anthraquinones to decompose and cause discolora-tion of the gel unless the gel is contaminated by an improper processing technique.

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RHF:RCB/sm ~ J
January 14 J 1988 Aloe vera gel is about 98.5% by weight water. More than 60% of the total solid is made up of polysaccharides of carbohydrate origin. organic acids and inorganic compounds, especially calcium oxalate, account for the remainder of the solid.
Whole leaves, exudates and fresh gels of aloe plants have been used for a variety of human afflictions. Evidence of their use as a medicinal remedy can be traced to the Egyptians of 400 BC. Aloe vera was also used to embalm the dead as well as to protect the embalmers from the death-causing agent. Other early civilizations used aloe vera for skin care, relieving insect stings and bites, treating scratches, wound healing, hair loss, as a purgative and for ulcerated skin. It was the traditional medicine of many c-~ltures as an anthelmintic, cathartic, stomachic, and was used inter alia for- leprosy, burns and allergic conditions.
Cole et al. Archives of Dermatoloqy and Syphilology, 47: 250 (1943); Chopra et al.~ Glossary of Indian Medicinal Plants, Council of Scientific and Industrial Research, New Delhi;
Ship, Journal of American Medical Association, 238: 1770 -1772 t1977) Morton, Atlas of Me~icinal Plants of ~iddle American Bahamas to Yucatan, Charles C. ~homas ED.~ 78 - 80 (1981) Diez-Martinez, La Zabila, Communicado NO. 46 Sobre Recursos B~oti os Potenciales del Pais, INIREB, Mexico tl981);
Dastur, Medicinal Plants of India and Pakis~an; D.B.
Taraporevala Sons & Co., Private Ltd., Bombay 16 - 17 (1962).
Aloe vera has enjoyed a long history of lay accep-tance as possessin~ "curative" or "healing qualities". Over the last few years, numerous books and articles meeting scien-AVCI

RHF:RCB/sm ~ 3 January 14, 1988 tific standards have been written on Aloe vera. organizationssuch as the Aloe Vera Council and recognized medical institu-tions through publications and ca~e histories of physicians, veterinarians and other scientists have given credence to the "aloe phenomenon". Aloe vera has been featured extensively in the area of dermatology, especially for treating radiation-caused skin conditionsO Mackee, X-Rays and ~adium in the Treatment of Diseases of the Skin, 3rd Ed., Lea and Febiger, Philadelphia, 319 - 320 tl938); Rovalti et al., Industrial Medicine and Sur~ery, 28: 364 - 368 (1959); Zawahry et al., Quotations_From Medical Journals on Aloe Research, Ed. Max B.
Skousen, Aloe Vera Research Institute, Cypress, California 18 - 23 (1977); Cera et al., Journal of the American Animal Hospital Association, 18: 633 - 638 (1982). The body of scientific literature documenting medical applications in digestive problems, as a virucidal, bactericidal, and fungici-dal agent and in gynecological condition~ is extensive and has been adequately reviewed by Grlndlay and Reynolds (Journal of Ethnopharmacoloqy, 16: 117 - 151 (1986)).
The importance of chemicals found in aloes is indi-cated by the fact that they have been listed in every known national pharmacopeia. U.S. Pharmacopeia, 20th Rev~sion, The National Formulary, 14th Edition, United States Pharmacopeial Convention, Inc., Rockville, Maryland, July 1, 1980. However, the U.S. Pharmacopeia describes the yellow sap drug portion of aloes but not the mucilage. The fresh unpreserved gel is about 98.5-99.2 percent water. The total solid that remains after the water has been removed ranges from 0.8 to 1.5 per-cent. The major constituents of the solid comprise mucilage, c AYCI
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January 14, 1988 sugars, fiber, proteins, ash, fats, aloin and resin. Robson et al., Journal of Burn Care Rehabilitation, 3: 157 - 163 (1982). Reports of compositions which include enzymes, orga-nic acids, inorganic salts, amino acids and alkaloids have been noted. Rowe et al., Journal of the American Pharmaceutical Association, 30: 262 - 266 ~1941); Roboz et -al., Journal of_the American Chemical Society, 70: 3248 - 3249 (1948); Waller et al., Proceedings_ of Oklahoma Academy of Science, 58: 69 - 76 (1978). Depending on how the leaves are processed, mucilage and sugars ar~ the major components of the dehydrated gel. The sugars found are galactose, glucose, man-nose, rhamnose, xylose and uronic acids. Although conflicting reports have been observed, the mucilage is mainly composed of mannan or glucomannan. Eberendu et al., The Chemical Characterization of Carrisyn~ (in preparation~; Mandal et al., Carbohydrate Research, 87: 249 - 256 (1980b); Roboz et al., Journal of the American Chemical Society, 70: 3248 - 3249 (1948); Gowda et al., Carbohydrate Research, 72: 201 - 205 (1979); Segal et ~1., Lloydia, 31: 423 (1968).
~ resently, the controversy over the identity or the active substance(s~ in aloe vera has not been settled. It is therefore important to clearly distinguish between~ the com-ponents present in the gel and those found in the exudates.
larger part of the gel is a mucilage of mainly polysaccharide nature with minor amounts of various other compounds. It is conceivable that there may be some synergistic action bet~een the polysaccharide base and other components in some of the activities observed. Leun~, Excelsa 8: 65 - 68 (1978); Henry, Cosmetic and Toiletries, 94: 42 - 50 tl979). For example, RHF-RCB/sm ~ 3 January 14, 1988 several workers report that the effective components for wound healing may be traumatic acid (Freytag, Pharmazie, 9: 705 (1954)) and a kind of polysaccharide. Kawashima et al., Chemical Abstracts, 93: 13075 (1979). Mackee, supra, noted that the gel,not the rind or the exudate, was responsible for the ben~ficial effects in ~he treatment of radiation burns, and he s~ressed the importance of using fresh leaves for effective treatment. Polysaccharides degrade with time and certain molecular weight sizes may be necessary to elicit spe-cified pharmacological response. Goto et al., Gann, 63: 371 -374 (1972).
~ here are many examples in the literature of poly-saccharides demonstrating pharmacological and physiological activities without known synergistic help from other com-ponents. Ohno et al., Chem._Pharm. Bull., 33: 2564 - 2568 (1985); Leibovici et al., Chem. Biol. Interactions, 60: 191 -200 (1986); Ukai et al., Chem. Pharm. Bull., 31: 741 - 744 (1983); Leibovici et al., Anticancer Research, 5: 553 - 558 (1985)~ Hyperlipidemia in the general population and espe-cially in familial hypercholesterolemia is associated with coronary heart disease and death. In countries where dietary fiber intake is high, atherosclerosis appears to be~uncommon.
Trowell et al., Editors~ Refined_ Carbohydrate Food~ and Disease, London, Academic Press, 207 (1975). Pectin and guar are reported to lower cholesterol i~ normal and hyperlipidemic patients. Ray et al., American Journal of Clinical ~utrition, 30. 171 - 175 (1977). Locust bean gum, a polysaccharide com--posed of mannose and galactose, decreased the lipoprotein cho-lesterol le~els in cases o~ normal and familial RHF:RCB/sm January 14, 1988 hypercholesterolemic subjects. Zavoral et al., American Journal of Clinical Nutrition, 38: 2~5 - 29~ (1983). Addition of guar gum to carbohydrate meals decreased the postprandial rise of glucose in both normal and diabetic subjects. Jenki~s et al., Lancet, 2: 779 - 780 (1977). Kuhl et al., (Diabetes Carel 6 (2): 152 - 154 (1983)) demonstrated that guar gum exhibited glycemic control of pregnant insulin dependent diabetic patients.
Anti-tumor activity of polysaccharides is widely reported. Polysaccharides prepared from Lentinus cyathiformis are known to increase hosts' defense against tumors. Rethy et al., Annuals of Immunoloqy Hungar~, 21: 285 - 290 (1981).
There are several reports of polysaccharides from mushroom, yeast or bacterium extracts eliciting a high degree of host defense activity against viral and tumor infestations.
Chihara et al., Nature, 222: 687 (1969); Schwartzman, Proc.
Soc. Exper. _ Biol. Med., _: 737 (1932); Rethy, X.
International Congress of Microbioloqy; Moscow, 642 (1966).
Su~uki et al (Journal_ of Pharm.__~yn., 7: 492 - 500 (1984)) also repor~ed anti-tumor activity of a polysaccharide fraction extracted from cultured fruiting bodies of a fungus, Grifola frondosa. The fraction (GF-l) 5howed equivalent levels of higher inhibiting activity when administered intraperitoneally (IP), intravenously ~IV) and intratumorally (IT). H~wever, oral administration (PO) was reported not effective. The GF-l also exhibited anti-tumor action against the solid ~orm of Meth A fibrosarcoma and ~M 46 Carcinoma in mice. Lentinan, which is a 6-branched ~ 3-linked glucan similar to GF-l, was ineffective against Meth A fibrosarcoma. Chihara, The antitu-AVCI

RHY:RCB/sm ~ ~4 January 14, 1988 mor polysaccharide Lentinan: an overview; ~Manipulation ofHost Defense Mechanisms"; ed. by Aoki et al., Excerpta Medica, North ~olland, 1 - 16 (1981); Sasaki et al., Carbohydrate Research, 47: 99 - 104 ~1976). Synthesized branched poly-saccharides were reported to demonstrate bioactivities against tumors. Matsuzaki et al., Makromol. Chem., 186: 449 (1985)~
Matsuzaki et al. (Makromol. Chem., 187: 325 - 331 (1986)) synthesized branched polysaccharides from ivory nut mannan, 4)-D-mannopyranose and ~ 4)-linked glucomannan which showed significant activities. A partially acetylated linear @~ 3)-D-mannan extracted from fruit bodies of Dictyophoria Indusiata Fisch exhibited anti-tumor activity. Hara et al., Carbohydrate Researchv 143: 111 t1982). It appears that anti-tumor action depends on the kind of polymer main chain and its degree of polymerization since ~ 3)-glucan type polymers show higher anti-tumor activity than ~ 4)-g1ucan and hemi-cellulosic polymers. Matsuzaki et al., Makromol. Chem., 187:
325 - 331 (1986). A carboxymethylated derivative of

3)-glucan obtained from bacterial culture filtrate caused severe cell loss from established sarcoma 180 tumors within two hours after the injection of the derivative. Baba et al., J. of_ Immunopharmacolo~, 8: 569 - 57~ (1986). The same author observed a compensatory increase in polymorphonuclear leukocytes due to the injection of the substance.
Incidentally bestatin, a dipeptide known to possess immune-modulating and anti-tumor activity (Ishizuka et al., Journal of Antibiot1cs, 33: 642 - 652 (1980), influenced neither the tumor yield nor the polymorphonuclear leukocyte count. Baba et al., sue~a.

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R~F:RCB/sm January 14, 1988 There are numerous reports on the anti-tumor effect of sulfated polysaccharides, including heparain (Jolles et al., Acta Univ.~Int. Cancer, 16: 682 - 685 (1960); Suemasu et al., Gann, 61: 125 - 130 (1970)), sulfated laminaran and dextran. Jolles et al., British Journal of Cancer, 17: 109 -115 (1963). Yamamoto et al. (Japan Journal of Experimental Medicine, 54: 143 - 151 (1984)) reported enhancement of anti-tumor activlty of a fucoidan fraction by further sulfation.
The sulfated product demonstrated acSivity against L-1210 leukemia. The authors postulated that the mechanism of the anti-tumor action might be due partly to inhibition of inva-sive growth of L-1210 cells resulting from electrostatic repulsion between the tumor cell and mesothelial cells.
Yamamoto et al., supra. Polysaccharides with sulfate groups are also reported to be human T cell mitogens and murine polyclonal B cell activators. Sugawara et al., Microbiological_Immunoloqy, 28: 831 - 839 (1984). Generally, homopolysaccharides of high molecular weight with sulfate groups possess these properties. Dorries et al., European Journal of Immunology, 4: (1974); Sugawara et al., Cell Immunolo~y, 74: 162 - 171 ~1982).
It has been reported that glucan extracted from the yeast Saccharomyces cerbisiae is a modulator of cellular and humoral immunity. Wooles et al., Science, 142: 1078 - 1080 (1963). The polysaccharide also stimulated the proliferation of murine pluripotent hematopoietic stem cells, granulocyte-macrophage colony-formin~ cells, and cells forming myeloid and erythroid colonies. Pospisil et al., Experientia, 38: 1232 -1234 (1982); Burgaleta et al., Cancer Research, 37: 1739 -c AVCI

RHF:RCB/sm January 14, 1988 1742 (1977). Maisin et al. (Radiation Research, 105: 276 -281 (1986)) also reported an IV administra~ion of a polysaccharide that induced protection of Murine hematopoietic stem cells against x-ray exposure, thereby decreasing the mor-tality of the mice so exposed.
Seljelid et al., (Experimental Cell Research, 131:
121 (1981)) have observed that insoluble or gelforming glycans activated macrophages in vitro whereas the corresponding soluble glycans did not. They postulated that the way the glycan was presented to the mononuclear phagocyte was decisive for activation. Bogwald et al. (Scand. Journal of Immunoloqy, _ : 355 - 360 (1984)) immobilized glycans which had a stimula-tory effect on the macrophages in vitro. This led the authors to believe that the degree of fixed or steric arrangement of the glycan was decisive for the effect on the macrophages in vitro. A purified polysaccharide isolated from Candida albi-cans induced antibody response in vitro by human ~eripheral blood lymphocytes. Wirz et al., Clinical Immunoloqy and Immunopathology, 33 199 - 209 (1984), There were significant differences between the anti-candida antibodies in sera of normal and candida-infected individuals Wirz et al., suDra.
The antiviral activity of polysaccharides and poly-saccharides linked to peptides has been observed. Suzuki et al., Journal Antibiotics, 32: 1336 - 1345 (1979). Suzuki et al., supra,reported antiviral action of ~eptidomannan (KS-2) extracted from culture mycelia o Lentinus edodes. Both oral (PO) and intraperitoneal ~IP) administration resulted in increased peak serum interferon titer which ~rotected mice against viral infections. This was different from dextran c AYCI ~ 3 ~ 'J' ~5220CIP2 RHF:RCB/sm January 14, 1988 phosphate (DP-40) (Suzuki et al., Proc. Soc. ExP~ Biol. Med., 149: 1069 - 1075 (19751) and 9-methylstreptimidone t9-MS) ~Saito et al., Antimier. Aqent & ChemotheraPY, 10: 14 - 19 (1976~) which induced higher titers of interferon only if administrated intravenously (IV) or intraperitoneally (IP) to mice.
Anti-inflammatory activi~y of aloe vera gel has been widely repoxted by both oral testimonies and respected ~cien-tific journals. Rubel (Cosmetics and Toiletries, 98: 109 -114 (1983)) discussed fully the possible mechanism of the anti-inflammatory effect of aloe gel. Ukai et al., (J.
Pharmacoloqy Dyn., 6: 983 - 990 (1983)) noted anti-inflammatory activity in polysaccharides extracted from fruit bodies of several fungi~ The polysaccharides demonstrated significant inhibitory effect on carrageenan induced edema.
One of the polymers, O-acetylated-D-mannan (T-2-HN), in addi-tion demonstrated more marked inhibitory effect on scald hyperalgesia than phenylbutazone. Ukai et al., supra. The fact that the polysaccharide is said to be free from protein and lipids strongly suggests that the anti-in~lammatory effect is due to the acetylated mannan only~ Other researchers have also reported anti-inflammatory effects of complex poly-saccharides (Saeki et al., Japan J. Pharmacology, 24: 109 -118 (1974)), glycoproteins (Arita et al., J. Pharmacolog~, 24:

861 - 869 (1974)) and sulfated polysaccharides (Rocha et al., Biochemical Pharmacology, 18: 12a5 - 129S (1969))~
Literature reports of polysaccharides demonstrating pharmacological and physiological activities continue to flood the pages of well respected scientific journal~. It is there-A~CI

RHF~RCB/sm ~ n January 14, 1988 1 ~1 ~ O V u fore, not illogical that the mucilaginous gel o the aloevera, which is essentially a polysaccharide, holds the secret to aloe vera's medicinal properties. The discrepancies over whether the polysaccharide is a glucomannan~ mannan, pectin, or some other composition are a result of chemical purifica-tion steps. By processing Aloe according to the present invention, a partially acetylated polymannose has been con-sistently isolated as the major polysaccharide having phar-macological activity. Yagi et al, (Planta Medica, 31: 17 - 20 ~1977)) using a slightly modified extraction method, isolated acetylated mannan (aloe mannan) from Aloe arborescens Miller var. natalensis. Ovodova, (Khim. Prior. Soedin, 83: 93833 (1975)~ however, earlier isolated pectin as the main component of the same aloe species.
SUMMARY OF THE INVENTION
The present invention is directed to a process whereby the active chemical substance in the aloe plant is physically extracted from whole aloe leaves. The chemical substance is substantially non-degradable and can be admi-nistered in a prescribed amount.
The present invention is also directed to the`active chemical substance in the aloe plant in the form produced by the process described above. The active chPmical substance has been found to be a substantially non-degradable lyophi-lized linear polymer of substantially acetylated mannose mono-mers. The mannose monomers are preferably bonded together by

4) bonds. The active chemical substance has been measured, standardized and characterized by analytical chemistry tech-niques.

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B5220CIP2 ~4 ~ Q ~n RHF:RCB/sm ' ~ u January 14, 1988 The term "active chemical substance~ as used herein means the substance which is responsible for the wound healing and other beneficial properties of aloe vera. The term "substantially non-degradable~l as used herein means a product which decreases in molecular weight by less than 5 percent over a period of two years and a product which maintains more than 95 percent of its biological activity over a period of two years. The term "substantially acetylated mannose mono-mers" as used herein means partially or substantially comple-tely acetylated mannose monomers.
The process of the present invention is one for extracting the active chemical substance in the aloe plant from a leaf of th~ aloe plant which process basically compri-ses at least the following steps:
~ a) washing an aloe leaf in a bacteriacidal solution to remove substantially all surface dirt and bacteria;
(b) removing at least a first end portion from said washed leaf;
(c~ draining, preserving and collecting anthra-~uinone rich sap from said cut and washed leaf;
(d) removing rind from said leaf to produce a substantially anthraquinone-free aloe gel fillet;
(e) yrinding and homogenizing said substantially anthraquinone-free aloe gel fillet to produce substantially anthraquinone-free aloe juice having solubilized matter;
(f) adding a water soluble, lower aliphatic polar solvent to the aloe juice to precipitate the active chemical substance and thereby to form a heterogeneous solution;
(g) removing the water soluble, Lower aliphatic AVCI
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January 14, 1988 polar solvent and the solubilized matter from the heteroge-neous solution to isolate the precipitated active chemical substance; and (h) drying the precipitated active chemical substance.
One skilled in the art will appreciate that one may obtain aloe juice having solubilized matter from aloe leaves in whatever manner possible, and then subject this juice to steps (f3, (g) and (h) to extract the active chemical substance.
Indeed one skilled in the art will appreciate that instead of steps (b), (c) and (d), one may instead (b) crush the washed aloe leaves and (c) dialyze the crushed leaves che-mically removing unwanted fractions, i.e., anthraquinones, minerals and acids and the rind to produce a substantially anthraquinone-free gel that may then be subjected to steps (e), (f), (g) and (h) to extract the active chemical substance.
One skilled in the art will also appreciate that instead of steps (b), (c), (d) and (e), one may instead crush the washed aloe leaves and extrude anthraquinone-rich aloe juice having solubilized matter and then subject the aloe juice to steps (f), (g) and (h) to extract the active chemical substance. The active chemical substance is effectively separated from anthraquinones and deleterious ions by this process since the anthraquinones and ions are water soluble and remain in the liquid solvent phase and do not precipitate.
One skilled in the art will also appreciate that AVCI
B5220CIP2 ~ Q g RHF:RCB/sm 1 ~ ~ ~ v v January 14, 1988 instead of steps (b), ~c), (d~ and (e) one may instead grind the whole washed aloe leaves, filter out fibrous material, and homogenize the remainder to produce anthraquinone-rich aloe juice having solubilized matter. The aloe juice can then be subjected to steps (f), (g) and (h) to extract the active che-mical substance. The ac~ive chemical substance is effectively separated from anthraquinones and deleterious ions ~y this process for the reasons noted above.
One skilled in the art will appreciate that an addi-tional process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant comprises the following steps:
a) washing an aloe leaf in a bacteriacidal solution to r~move substantially all surface dirt and bacteria;
b) removing rind from said leaf to produce an aloe gel fillet;
c) grinding and homogenizing said aloe gel fillet to produce aloe juice having solubilized matter;
d) adding a water soluble, lower aliphatic polar solvent to the aloe juice to precipitate the active chemical substance and thereby to form a heterogeneous solution;
e) removing the water soluble, lower aliphatic solvent and the solubilized matter from the hetero~eneous solution to isolate the precipitated active chemical substan-ce; and f) drying the precipitated active chemical substance.
As noted above, the active chemical substance is effectively separated from anthraquinones and deleterious ions AVCI

R~F:RCB/sm t. 1~ 2860 January 14, 1988 1 ~1 by this process since the anthraquinones and ions are water soluble and remain in the liquid solvent phase and do not pre-cipitate.
Removal of "substantially all surface dirt and bac-teria" as used herein means ~1) removal of dirt to the extent that remaining dirt is less than 0.1% by weight of the weight of the leaf and (2) killing such surface bacteria that the remaining surface bacteria are less than 100 count per gram of leaves.
Furthermore, my preferred process may further comprise the step of ultrafiltration in order to osmotically adjust the aloe juice or aloe vera raction, or to reduce even further the levels of anthraquinones to less than 5 ppm, even down to less than 100 parts per billion by weight.
These steps enable the processor to use large or small leaves(even less than one year old)because the polymer size found in the mature leaves can be selected and processed out of smaller, immature leav~s.
One of the advantages of the instant process is that damaged leaves previously considered unusable due to strong winds or poor collection techniques can be processed, and the undesirable contaminants can be dialyzed out. r The ultrafiltration (dialysis) step incorporates membrane technology that allows the selection of filters with different pore sizes, depending on the condition of the cut aloe leaves, that can accomplish any combination of the following:
small pore size filter ~preferably about 100 Daltons) that separates out water and salts from RHF.RCB/sm ' ~ 3 ~ 2 ~ ~ ~
January 14, 1988 the aloe vera gel, if needed.
(2) Larger pore size filters (preferably about 500 Daltons) that can separate out the acids from the aloe vera gel, if needed.
(3) Even larger pore size filters (preferably about 2000 Daltons) that can separate the yellow sap components from the aloe vera gel, if needed.
~4) And even larger pore size fil~ers ~preferably from about 10,000-100,000 Daltons that can size the gel matrix polymers, and divide them out by molecular weight.
A Romicon 4-column (Romicon Co., 100 Cummings Park, Woburn, MA 01801, Model No. HF4 SSS, Membrane Type PM50, Membrane Nos. H526.5 - 43 pm50) as an ultrafiltration device is recommendedO
As an additional preferred embodiment, the washing step of the process may comprise washing the substantially anthraquinone-free aloe gel fillet in a tumbler washer prior to grinding said fillet.
During all of the above-described processes of extracting the active substance from aloe vera gelJ minor amounts of organic and inorganic sub5tances are found to co-precipitate with the product. A large Eraction of the inorga-nic salts comprise calcium oxalate. The presence of inorganic salts such as calcium oxalate should be eliminated or at leas~
minimized, for consistency of product and health reasons It has now been surprisingly found that by adding an effective amount of a mineral acid to adjust the pH of the gel to about 3.0 to about 3.5 prior to addition of alcohol results in a AVCI
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January 14, 1988 product having a substantially lower level of oxalates and other inorganic salts~ Accordingly, it is preferred in all of the above processes for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant, that the pH of the gel be adjusted from about 3.0 to about 3.5 prior to the addition of alcohol.
Preferably in all th~ above processes for extracting the active chemical substance in the aloe plant from a leaf of th~ aloe plant, four volumes of the water-soluble, lower aliphatic polar solvent are added to one volume of aloe juice to precipitate the active chemical substance. Preferred water soluble, lower aliphatic polar solvents are methanol, ethanol and propanol. The most preferred solvent is ethanol. One skilled in the art will recognize that other water soluble, lower aliphatic polar solvents may be substituted for the pre-ferred solvents as long as the active chemical substance will precipitate therefrom.
It is preferred in the above extraction processes that the active chemical substance is allowed to precipitate from the water soluble, lower aliphatic polar solvent and aloe juice mixture for about four hours. It has been determined that allowing the mixture to precipitate for four hours gives the optimum yield of active chemical substance and that after four hours the precipitated active chemical substance begins to degrade. It has also been determined, however, that signi-ficant amounts of active chemical substance have been reco-vered after a precipitation period o~ 24 hours. One skilled in the art will appreciate that the optimum precipitation time period is deper.dent on ambient temperature and pressure as AVCI
522OCIP2 ~ 3 ~ 28~0 January 14, 1988 well as the nature of the water soluble, lower aliphatic polar solvent.
It is ~also preferred in the above extraction pro-cesses that the precipitated active chemical substance be dried by lyophilization rather than by oven drying since heat may aid in the hydrolysis or degradation of the active chemi-cal substance.
In all of the above extraction processes, any fibrous material tcellulose) contained in the aloe juice is also precipitated by the water soluble, lower aliphatic polar solvent- but is precipitated early with the addition of the solvent and is less dense than the active chemical substance.
The fibrous material remains on the surface of the solvent after the active chemical substance has been allowed to settle and can therefore be removed quite easily. One skilled in the art will appreciate that one may instead filter the aloe juice to remove fibrous material prior to the addition of solvent.
More preferably, in all of the above processes, aloe leaves or whole plants may be collected from the field suf-~ ficiently clean to eliminate a washing step.
; The dried, precipitated active ingredient, optionally, may be irradiated by gamma or microwave radiation whereby said active chemical substance is sterilized and pre-served.
Accordingly, the present invention is believed to provide new and improved method9 for the production of aloe vera products.
The present invention furthermore is fur~her believed to provide improved methods for processing the leaf AVCI

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of the aloe vera plant in a manner which avoids the unde-sirable combination or mixture of distinctively characteristic portions of such plant leaf.
The present invention moreover is further believed to provide improved processes for preparing various extracts of the leaf of the aloe vera plant which minimizes the con-centrations of undesirable components in the finished extracts.
The present invention also is believed to provide new and improved processes for preparing extracts of the leaf of the aloe vera plant which maximize the concentration of certain components characteristic of particular portions or segments of the leaf while minimizing or eliminatin~ certain components characteristic of other portions or segments of the leaf.
The present invention also is believed to provide new and improved processes for preparin~ extracts from ,he leaf of the aloe vera plant which are low in concentration of yellow sap of the leaf.
The present inv ntion finally is thought to provide a method for extracting the active chemical substance in aloe vera gel. This chemical substance has utility as a non-toxic immune stimulating compoundO The substance shall hereinafter be referred to as Carrisyn* extract or acemannan. As mentioned aho~
acemannan has been found to be a substantially non-degradable lyophilized linear polymer of substantially acetylated mannose monomers which is standardized and characteri~ea by analytical chemistry techniques.

* trade mark AVCI

RHF:RCB/sm ~ 8 ~ 0 January 14, 1988 1 ~ 1 ~

- DESCRIPTION OF THE FIGURES
Figures 1 and 2 show cut-away por~ions of an aloe vera leaf.
Figure 3 shows a schematic of a preferred leaf washing appara~us used in the process of the present inven-tion.
Figure 4 shows a schematic of a preferred apparatus for sectioning and soaking of aloe vera leaves.
Figure 5 shows a schematic of preferred apparatus for the cutting of aloe sections into fillets and for coarse grinding.
Figure 6 shows a schematic of preferred apparatus for fine homogenization and filtering.
Figure 7 shows a schematic of a preferred use of dialysis equipment for fine separations of processed aloe material.
Figure 8 shows infrared spectra for two samples of Carrisyn~ extract under dfferent pH conditions.
Figure 9 shows a differential thermogram of Carrisyn~ extract.
Figure 10 shows a differential thermogram of Carrisyn~ extract contaminated with calcium oxalate.
Figure 11 shows a schematic for the characterization of Carrisyn~ extract.
Figure 12 shows a size exclusion chromatogra~ of pullulan polysaccharide standards.

Figure 13 shows a size exclusion chromatogram of Carrisyn@~ extract.
Figure 14 shows an infrared spectrum of non-protease ~ 22 -AVCI
RHF.RCB/sm ' ~ 3 ~ 2 8 ~ ~
January 14, 1988 treated Carrisyn~3 extract.
Figure 15 shows an infrared spectrum of protease treated Carrisyn ~ extract.
Figure 16 shows a differential thermogram sf Carrisyn ~ extract.
Figure 17 shows an HPLC chromatogram o a standard mixture of glucose, galactose, mannose and inositol.
Figure 18 shows an HPLC chromatogram of Carrisyn~extract Figure 19 shows a GLC chromatogram of a standard mixture of rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose and inositol as their glycitol acetates.
Figure 20 shows a GLC chromatogram of Carrisyn~ extract.
Figure 21 3hows a standard curve of mannose/inositol ratio against amount of acemannan~
Figure 22 shows a total ion chromatogram of par-tially methylated and partially acetylated glycitol of Carrisyn~extract.
Figure 23 shows a mass spectrum of the partially acetylated glycitol of Carrisyn~ extract.
Figure 24 shows a schematic for partially methylated mannitol acetates.
Figure 25 shows a schematic for fragment ions of methylated mannitol acetate under mass spectrometry analysis.

AVCI ~ 312 8 ~ ~
RHF:RC~/sm January 14, 1988 DETAILED DESCRIPTION OF THE PREFERRED EM~ODIMENTS
It has been discovered and recognized that sub-portions of the~ yellow sap and internal gel matrix have characteristics which are unique to those sub-portion5, the extracts therefrom therefore having potentially distinct uses from one another. A summary of these distin~t portions, and some of their characteristics and potential uses is as follows:

PORTION SUB-PORTION UTILIrIES

Yellow Sap (1) Sediment laxative, antifungal, antibiological, pest-icidal and sunscreen (2) Supernatant mucosal protective action, sunscreen Internal Gel (1) Mucilage penetrant, hypo-Matrix allergenic, mois-turizer ~2) Gel Fillets ulceroprotective, cell stimulative moisturizer, wound healing (3) Interstitial natural preservative, Fibers hemostat (4) Residual Matrix cell growth stimulant Outer Rind pesticidal insect re-pellent, pape~ pulp fiber Based upon the aforementioned knowledge and recogni-tion, and in order to optimize the quality and concentration of the desired components in the final extract depending upon the intended use thereof, the process of this invention is directed to the initial fractionating of the leaves of the aloe vera plant into the particular distinct portions and sub-portions defined above as ~ell as the separation and isolation B5220CIP2 ~3128~0 RHF:RCB/sm January 14, 1988 of particular components of such sub-portions defined above.
The specific details and features of such processes will become more readily understood and appreciated from the following detailed descriptionO The present invention is also directed to particular components that are isola~ed by the above-mentioned processes.
The products produced by the process of the present invention are preferably obtained fxom the outer, lower leaves of mature, outdoor grown aloe vera plants. A two year old plant is normally mature; however, the broader leaves of a four or five year old plant typically contain larger amounts of the desired extracts and are also easier to handle. Four or five year old leaves of Aloe barbadensis Miller grown in the Rio Grande Valley of Texas are most preferable. These leaves generally weigh about 1~ - 2~ pounds each. Depending upon the particular use or products that are desired, the leaves can be processed immediately after cutting them from the plant or they can be stored under appropriate conditions for varying time periods berore they are proc~ssed.
Additionally, concentration of various components of the leaf is affected by seasonal variations and the environmental con-ditions to which the leaves ar~ subjected, all of which should be taken into consideration depending upon the specific intended use to which the plant extracts are to be directed.
The leaves should be pulled or cut from near the base of the plant, preferably without breaking or damaging any part of the leaf prior to processing. One preferably employs a small knife of less than 6 inches, e.g., a pocket knife and cuts the leaf at the base immediately above the stalk, and AVCI
B5220CIP2 1312 8 6 ~
January 14, 1988 peels the leaf from the stalk in order to prevent leakage of the clear cellular gel or contamination of the gel with the yellow sap. Any breakage or bruising of the leaf can result in the undesirable comingling of the distinct portions, and therefore component characteristics, of the leaf.
After removal from the plant, the leaves are nor-mally cleaned by washing them with a mild scrubbing action or spraying with a suitable detergent solution (such as OLYMPIC
POOL CHLOR 65~, distributed by York Chem Co., Dallas, Texas).
In some instances, the cleaning takes place with the aid of soft brushes. After cleaning, the leaves are rinsed thoroughly in clean water to remove any vestige of any detergent solution.
The bottom white or light colored portion of each leaf and the upper tip portion thereof are removed by cutting carefully with a small sharp knife. These Qortions, essen-tially constituting the ends of the leaf, may be separately processed to obtain the yellow sap therefrom for those appli-cations in which it is desired to produce products having com-ponents with the yellow sap characteristics referred to above.
The remaining portion of each aloe vera leaf is then crosscut into short segments, preferably one-half inch in length, and each segment is placed upright in an aqueous solu-tion (preferably deionized water) which may be hypertonic, isotonic or hypotonic, resulting in the yellow sap draining from the segments. Alternaeively, in those applications in which it is desired to collect the yellow sap ~or use in other preparations having the characteristics noted above, the AVC I
B5 2 2 0 CI P 2 ~ o ~ n RHF:RCB/sm 131~o~u January 14, 1988 segmen~s may be placed upright in a dry collectisn container, preferably of stainless steel with a stainless steel wire mesh bottom to allow ~drainage and for water contact to allow the water to dialyze the leaves.
The segments are permitted to drain for approxima-tely twenty to thirty minutes in this manner. The cut segments will eventually form a seal and cease draining. The yellow sap that is collected will then, upon standing for an appropriate period of time, separate into two sub-portions, namely sediment and supernatant, respectively. The yellow sap is useful for making a good sunscreen on in~act skin (not bro-ken skin) and provides the skin with an olive tan color, and is also useful for the manufactuxe of laxatives.
After completion of the procedures which remove the yellow sap from the cut leaf segments, the segments are then pared to form fillets utilizing any suitable equipment such as a wire ti.e., consumer cheese) slicer or paring blades (e.g., paring knife) to remove the outer rind or skin of the leaf segments and the layer that is immediately below such outer skin. The leaf segments may be frozen to facilitate this skinning procedure. After paring, what remains is the inter-nal gel matrix portion (fillet), and this portion is inspected and hand cleaned to remove any adhering skin or discolored portions to eliminate any residual yellow sap therefrom. One uses a mild water spray, preferably deionized water (and free of alcohol~ or submer~es the gel matrix portion under flowing clean water, to facilitate removal of such yellow sap resi-duum.
The resultant ~illet tinternal gel matrix) can then AVCI

RHF:RCB/sm 1 3128~
January 14, 1988 ~

be drained for approximately an hour. During this draining procedure, a slimy coating usually forms on the surfaces of the gel matrix, this coating b~ing collected by gravity or assisted by appropriate means such as centrifugation. This collected coating is the mucilage sub-portion referred to above.
The remainder of the gel matrix, in the form of gel matrix strips, may then be ground, shredded or blended to break up the interstitial fibers present th~rein, or the gel matrix strips may be forced through a wire mesh or filter screen in order to achieve liquefaction. This resultant substance may then be subsequently homogenized.
Alternatively, the gel matrix strips may be frozen and thawed and subsequently mixed to produce a liquid substance with fibers (such substance constituting the gel fillets sub-portion referred to above). This substance can then be filtered to obtain the interstitial fibers sub-portion, leaving the residual matrix sub-portion referred to above.
The homogeniæed extract thus obtained typically has a pH of approximately a~out 4 to about 5, preferably about 4.
All steps in the process described are performed at about room temperature.
Figures 3-7 disclose in even further detail pre-ferred embodiments of the instant process. Specifically, in Figure 3, there is disclosed apparatus for leaf washing. Aloe vera leaf washing equipment (Thompson Manufacturing Company, Harlington, Texas) A is utilized whereby leaves ~re first ore-soaked in vat 4. The whole leaves ~ are then placed by hand onto a conveyor belt 8 by which they are pulled underneath two RHF: RCB/sm January 14, 1988 brushes 9a and 9b. Conveyor belt ~ is driven via a chain 7 which is rotated on a second end by motor and pulley 6 which extends from a housing 5, also provided by Thompson Manufacturing Company. As the leaves are brushed and washed, upon passing through the second brush 9b the leaves are inspected at the end 10 of conveyor belt 8, by which the leaves are visually inspected and determined whether or not they are sufficiently clean. If the leaves are not suf-ficiently clean, they are placed into vat 12 for further washing; if the leaves are sufficiently clean, they are placed upon an upwardly moving conveyor B having steps 13 for which each individual leaf may be further washed with tap water by sprayers 11. The conveyor B is provided by Dallas Mill Wright Co. of Seagoville, Texas. The rinse sprayers 11 are provided by Key Plumbing, Seagoville, Texas. Stainless steel vat 12 is made of 316 stainless steel and is custom made by the National Sheet Metal Company of Dallas, Texas.
After washing, as indicated in Figure 4, the leaves are sectioned and soaked. After moving up through steps 13 of conveyor B, the clean, raw leaves ~ drop onto a tray 14 which is provided with a hole 15 for removal of trash. Tray 14 is part of a sectioning and soaking apparatus C of 316 stainless steel provided by the National Sheet Metal Company of Dallas, Texas. This equipment is custom fabricated. On tray 14 the leaves are manually sectioned at both ends with the tips and butts disposed through the hole 15 into a trash receptacle (not shown). The cut leaves ~ are then stacked into any one of a number of baskets 16 o~ stainless steel each having a wire mesh bottom made of stainless steel. l`hen these baskets AVCI ~ 3~28fi~

RHF:RCB/sm January 14, 1988 16 are placed in a stainless steel track which forms the top part of a trape~oidal funnel 17 by which yellow sap is drained from the bottom ~of the leaves through the baskets, falling into the bottom portion of the funnel 17. The yellow sap is periodically removed and is kept frozen for storage. The yellow sap draining step takes about 30 minutes.
After this step, the cut leaves ~, still retained in basket 16 are manually moved to water bath 18 at positions closest to trapezoidal funnel 17. In countercurrent flow, water comes into bath 18 through inlet water pipe 19, at a point farthest removed from trapezoidal funnel 17 and is sub-sequently removed through exit water pipe 20 at a position closest to trapezoidal funnel 17. Trays are gradually moved manually through the water bath in a direction away from tra-pezoidal funnel 17, and the washing step whereby the baskets remain in water bath 18 takes approximately one hour.
~ fter washing r the baskets are placed on tray 21 for drying, which lasts for only a few minutes. Again, the entire assembly in Figure 4, pertaining to the baskets, including wire mesh, yellow sap draining and auto washing equipment is made of 316 stainless steel,custom fabricated by the National Sheet Metal Company, Dallas, Texas.
After washing, the cut leaves ~ stacked in basket 16 on tray 21 are then moved to an area for further sectioning into fillets, as shown in Figure 5. Rind 22 is removed from the fillets so that only substantially clear material remains.

The rest of the rind 22 is discarded. Fillets ~! are then placed into trough 23 which feeds to a rough coarse grinder D.
The trough 23 is manufactured of 316 stainLess steel by the AVCI 1 3128~

RHF:RCB/sm January 14, 1988 National Sheet Metal Company of Dallas, Texas. The grinder is a ~odel No. 5150-N-Sink-erator (Watson Food Service Industries, Inc., Dallas, Texas~ After rough coarse grinding through grinder D, the processed material ' emerging from the grinder passes through to a portable tank E of approximately 100 gallon capacity which comprises a vertical single shell tank of 316 stainless steel (Perma-San, Manufacturer, distri-buted through Van Tone, Inc., Dallas, Texas). Coarse ground fillet ~' is agitated in tank E by a Perma-San agitator (Model No. AAPH2~ also distributed through Van Tone, Inc. of Dallas, Texas.
After rough homogenization, material from tank E is taken to a separate staged area for fine homogenization and (optional~ filtering. In Figure 6, material from tank E is pumped through pump 26 (centrifuge pump provided by Crepaco, Inc., Dallas, Texas, Model No. 4V-81, of stainless steel) dri-ven by Reliance Motor Model No. B76Q2139M-VF of the Reliance Electric Company, Cleveland, Ohio into a fine homogenizer F
(Crepaco, Inc., Dallas, Texas, Model No. 3DD13). After fine homogenization the material is transported to a large 1,000 gallon vertical single shell mixing tank G made of 316 stainless steel (Perma-San, Manufacturer, distributed to Van Tone, Inc. of Dallas, Texas~. The finely ground fillet ~ is ~itated by a Perma-San`agitator 26a (Perma-San,~ Model No.
AAPH2, distributed through Van Tone, Inc., Dallas, Texas).
Material _ in tank G may be removed and sent to pump 27a which forms one unit with filter 27b for removal of pulp through discard line 28. Pump 27a and filter 27b form the part of a Lomart diatomite filter (~odel No. 99-2138 distri-AVCI
B5220CIP2 ~ 3128~0 RHF:RCB/sm January 14, 1988 b1~ted by Allen Recreation Company, Dallas, Texas). Filteredmaterial is then pumped into tank H, which like tank E, can be provided with a lid 25.
In Figure 7, finely ground fillet ~ , is partially filtered, i5 s~irred by mixer 29 and pumped through pump 30 into a dialyzer. Mixer 2g is a Perma-San agitator (Model No.
AAPH2) distributed through Van Tone, Inc. Company, Dallas, Texas. Pump 30 is a Superior stainless steel Model SCS45 pro-cess pump (Superior Stainless Company, Delavan, Wisconsin).
Attached to process pump 30 is motor 30a of- 3 horsepower made by Baldor ~otor of 4450 rpm (Cat. No. CM3559T of the Baldor Electric Company~ Ft. Smith Arkansas). Material pumped~~
through pump 30 passes through dialysis unit J, (a Romicon Model ~F4SSS ultrafiltation system made by Romicon Inc., Woburn, Massachusetts) having 4 filters 31 (not shown) each filter housed in filter housing 32. Material passes ver-tically to a point where portions can be removed through separation lines 34 and into separation discharge lines 35.
Other material not separated is recycled through recycle return line 33 back into the dialysis unit, or in the alter-native, through separation return line 36 bac~ into the vat I.
Depending on the fraction of aloa desired and end product sought, the desired material can either be obtained through separation di5charge line 35 after processing or in vat I. For example, if excess water and minerals need to be removed, a small pore size ultrafilter can be used to separate out the water and minerals which are discharged through line 35 and the desired aloe fraction is returned to vat I. This process can be repeated until the desired amount of salt and ~3~28~

water is removed from the product contained in vat I simply by circulating it through the dialysis unit. Thi3 process can include more than one dialysis step. For example, as pre-viously described, the salts, low molecular weight acids and unwanted anthraquinones can be removed in a first dialysis step. The unwanted material is discharged through separation discharge line 35 and the desired ~raction is returned to vat I. This step is accomplished by using ultrafilters obtained from Romicon wi~h 10,000 Dalton pores. Next, the 10,000 Dalton ultrafilters are replaced with 50,000 Dalton ultra-filters also obtained from Romicon and the dialysis process is repeated. The dialysis process now separates the gel matrix polymers into two fractions; a first fraction consists of gel matrix polymers ranging in size from 10,000 to 50,00G Daltons and is discharged through separation discharge line 35, a second fraction consists of gel matrix polymers greater than 50,000 Daltons in size and is returned to vat I. This process can last from minutes to hours depending upon the amount of salt and water that is needed to be removed from a given pro-duct.
In Figure 7, separation return line 36 is made of Tygon tubing (food grade) provided by the Texas Rubber Supply Company, Dallas, Texas. The separation discharge line 35 is a 316 stainless steel pipe distributed by Van Tone, Inc., Dallas, Texas.
The residual matrix sub-portion may then be treated to separate, isolate and purify the active chemical substance, acemannan, in aloe vera gel. To separate acemannan from the residual matrix sub-portion, an excess o~ a water soluble, * Trade-mark AVCI
RHF-RCB/sm ~ 312, 8 ~ ~
January 14, 1988 lower aliphatic polar solvent is added to the residual matrix sub-por~ion. Acemannan then begins to precipitate from this mixture. The solution is allowed to settle for a sufficient period of time to allow as much active ingredient to precipi-tate out of solution as possible but not so long that the acemannan begins to degrade. After this period of time the supernatant is decanted or siphoned off without disturbing the settled precipitate. The precipitate and remaining solution are then placed in an appropriate centrifuge apparatus and the precipitate is collected into a pellet. After centrifugation, the supernatant is decanted and discarded. Optionally, the pellet is washed with a fresh aliquot of the water soluble, lower-aliphatic polar solvent and collected again and the supernatant is again discarded. The pellet is then lyophi-lized to dryness and allowed to dry overniqht. The resulting product is a substantially non-degradable lyophilized form of acemannan. The resulting product may be ground to form a powder. Preferably, a suitable acid is added to Aloe vera gel prior to the addition of the lower aliphatic polar solvent.
The acid is added to solubili2~ calcium oxalate contained in the Aloe vera gel in order to facilitate its removal. The acid is preferably added at a strength sufficient to solubi-lize the calcium oxalate impurity while not degradinq the ace-mannan polymer chain.
An alternate and preferred process for separating and isolating acemannan includes the following steps.
Leaves from mature, outdoor grown aloe vera plants are pulled or cut from near the base of the plant, preferably without breaking or damaging any part of the leaf prior to - 3q -AVCI
B5220CIP2 i ~3128~0 RHF:RCB/sm January 14, 1988 processing. The leaf is preferably cut at the base of the plant immediately above the stalk and is peeled from the stalk, in order to prevent leakage of the clear cellular gel or contamination of the gel with the yellow sap.
After removal from the plant, the butt and tip por-tions of the leaves are removed and the cut leaves are pared to form fillets as described above in the fractionation pro-cess.
The resultant fillet (internal gel matrix) is then ground, shredded or blended to break up the interstitial fibers present therein or the internal gel matrix may be forced through a wire mesh or filter screen in order to achieve liquefaction. The resultant liquefied internal gel matrix is then homogenized. The homogenized extract thus obtained typically has a pH of approximately about 4 to abou~

5, preferably about 4. The homogenized extract is then filtered to remove the interstitial fibers. The homogenizea and filtered extract may then be treated in the identical manner as the residual matrix sub-portion, referred to imme-diately above, to separate and isolate acemannan.
~An additional alternate and preferred process for ;~separating and isolating acemannan includes the following steps.
Leaves from mature, outdoor grown aloe vera plants are pulled or cut from near the base of the plant, preferably without breaking or damaging an~ part of the leaf prior to processing. The leaf is preferably cut at the base of the plant immediately above the stalk and is peeled from the stalk, in order to prevent leakage of the clear cellular gel AVCI 13 1 2 8 ~ O

RHF:RCB/sm January 14, 1988 or contamination of the gel with the yellow sap~
The leaves may then be crushed by suitable means, for example a ~nThompson Aloe Extruder" made by Thompson ~anufacturing Company, Harlingen, Texas, to extrude aloe ~uice. The extruded aloe juice may then be treated in the identical manner as the residual matrix sub-portion, referred to above r to separate and isolate acemannan.
All steps in the processes described are performed at about room temperature except for the lyophilization step which is preferably performed at about -50C.
Various modifications of the disclosed processes and compositions of the inventiont as well as alternative modifi-cations, variations and equivalents will become apparent to persons skilled in the art upon reading the above general description. The following examples are illustrative only and are not intended to limit the scope of the appended claims, which cover any such modifications, equivalents or v~riations.
Example 1 Process for Separating and Isolating acemannan.
A. PRELIMINARY WORK:
1. Previously cleaned tanks, mixers and fittings were sanitized with 50% isopropyl alcohol (IPA) solution and rinsed free of IPA with hot deionized water.
2. Pumps and attached hoses were drained with 5~

"HTH" chlorine swimming pool solutions, then flushed with water.
3. The pumps and attached hoses were sanitized with 50~ isopropyl alcohol solution. Pumps and attached hoses ~ 36 -AVCI ' ~ 31 28~

RH~:RCB/sm January 14, lg88 were flushed with hot deionized water until free of isopropyl alcohol.
4~ A homogenizer and attached hoses and pumps were sanitized with 50% isopropyl alcohol solution. The homoge-nizer and attached hoses were flushed with hot deionized water until free of isopropyl alcohol.
Aloe barbadensis Miller leaves collected from the Rio Grande Valley were transferred in a refrigerated truck at 40 to 45F within eight hours after harvest and stored under refrigeration at 40 to 45F until processed to reduce degrada-tion~
~ wenty to sixty pounds of stored leaves were then placed in a prewash bath of an aqueous solution of calcium hypochlorite at room temperature substantially to remove sur-face dirt from the leaves and kill surface bacteria on the leaves. The aqueous solution of calcium hypochlorite was pre-pared by adding approximately 0.125 grams of 98% calcium hypochlorite to one liter of water to produce a solution con-taining 50 ppm of free chlorine. The leaves remained in the prewash bath for a period of approximately five minutes ~ ext, the substantially dirt and bacteria free leave5 were placed on the horizontal conveyor belt of a "Thompson Aloe Washer" made by Thompson Manufacturing Company, ~arlingen, Texas. The "Thompson Aloe Washer" washed the leaves with room temperature water to remove the surface dirt and aqueous solution of calcium hypochlorite rom the leaves.

Again, the leaves were visually inspected and hand scrubbed as necessary to remove any s~lrface dirt remaining on the leaves.
Such leaves were then rinsed with room temperature water c B5220CIP2 1 3~28~
RHFORCB/sm January 14, 1988 The tip and bu~t por~ion vas then removed from each leaf and the leaves were placed in stainless steel basket-type containers, placed t~g~ther on top of ~ funnel shaped s~ainless steel collector, each container having a mesh bot-tom. Yellow sap was allowed to drain from the leaves for approximately 30 minu~es. The yellow sap passed through the mesh bottom of the stainless steel basket and was collected in a funnel shaped collector.
The stainless steel basket ~ype containers con-taining the aloe leaves were removed from the collector, and were then submerged in a second stainless steel vessel comprising a room temperature water bath of continuously hori-zontally flowing rinse water moving counter-current to the containers which are slowly moved by hand from one end of the vessel to the other, for approximately thirty minutes to one hour. This allows the yellow sap to drain further from the leaves. The leaves were allowed to soak in this solution for 30 minutes~
The leaves were then removed from this solution and the rind was removed from each leaf ~ith a sharp knife or wire cheese slicer to produce an aloe gel fillet from each leaf portion. The aloe gel fillets were visually inspected and any contaminated aloe gel fillets or fillet part detected by a characteristic ~ellowish discoloration were discarded. The total mass of uncontaminated aloe gel fillets was 20 to 60 percent of the starting leaf mass depending on the leaf size and condition.
The uncontaminated aloe gel fillets were then placea in a 750 seat restaurant set stainless steel garbage disposal c AVCI

RHF:RCB/sm January 14, 1988 ~ U

unit which coarse ground ~he fillets to an average particle size of the consistency of a thick, but freely flowing (coarse homogenized) liquid. The stainless s~eel garbage disposal unit was made by the N-sink-erator Division of Emerson Electric Co., Racine, WI, ~odel ~o. SS-150-13, Serial No.
115132.
The coarse ground aloe gel fillets then passed to a 100 gallon stainless steel holding vat. The holding vat was made by Process Equipment Corp. of Belding, Michigan, Model No. 100 gallon OVC, Serial No. 40865-3.
From the holding vat, the coarse ground aloe gel fillet solution was pumped to a homogenizer. The homogenizer was made by Crepaco Food Equipment and Refrigeration, Inc., of Chicago, Illinois, Serial No. 04-03. The homogenizer was of a type typically used in dairy processes for the homogenization of milk. The coarse ground aloe gel fillet solution was finely homogeni2ed under a pressure of about 1,500 psi.
From the homogenizer the finely homogenized aloe gel fillet solution was pumped to a stainless steel storage tank.
The storage tank was made by Process Equipment Corp. of Belding, Michigan, Model No. 1000 gallon OVC, Serial ~o.
40866-2. The total mass of the homogenized aloe gel fillet solution was 20 to 60 percent of the starting leaf mass.
Then, if necessary, the homogenized product was dialyzed using ultrafiltration.
The finely homogenized aloe gel fillet solution was then filtered to remove the interstitial fibers using a Leslie's Diatomaceous Earth Filter Model DE-48. The in~ersti-tial fibers themselves inxtead of diatomaceous earth were used AVCI
~5220CIP2 ~ 312 8 ~ O
January 14, 1988 as the filter media, the fibers being supported by a nylon mesh cloth filter support. The gel was pu~ped through the filter for several minutes before opening the exit port so that a sufficient amount of fibers could build up to serve as the filter media.
Twenty gallons of the filtered aloe gel fillet solu-tion was then pumped into a 100 gallon tank and 80 gallons of 190 proof undenatured ethanol (Ethyl Alcohol, 190 proof, .S.P., punctilious, 54 gal. ~a~ch I.D. #CT185JO4 available through U.S. Industrial Chemicals, Co., P. O. Box 218, Tuscola, Illinois 61953) was added to the aloe gel fillet solution. The solution was stirred for 20 to 30 minutes using a Perma-San propeller agitator model #AAPGF-4A, Process Equipment Corp., Belding, Michigan.
The alcohol-gel solutions were then immediately transferred to several 11 quart pans 10~" diameter and 8" high of 18-8 stainless steel (Bloomfield Industries Inc., Chicago, Illinois, available through Watson Food Service Equipment and Supplies, 3712 Hagger Way, Dallas, Texas).
The alcohol-gel solution~ were then allowed to settle for approximately four hours.
The clear liquid supernatant was then decanted or siphoned off usinq care not to disturb the precipitate that had settled on the bottom of the pans. The precipitate and remaining solutions were then transferred to four 1 pint stainless steel centrifuge buckets with about 500 g. of preci-pitate and remaining soLution being transferred to each bucket. The buckets were then SQUn at 2000 x g for about 10 minutes using an IEC Centra-7 Centri~uge ~International G

AYCI 13 ~ 2 8 ~ ~
RHF:RCB/sm JanUary 14, 1988 Equipment Co., 300 2nd Avenue, Needham Heights, Massachusetts 02194 available through American Scientific Products, P.O.
Box 1048, Grand Prairie, Texas 75050).
After centrifugation the supernatants were decanted and discarded~ The pellets were then washed with fresh 190 proof, undenatured ethanol and collected again at 2000 x g for about 10 minutes. After spinning again the supernatant was discarded.
The pellet was then transferred to several 600 ml.
~IRTIS lyophilization jars and swirled in liquid nitrogen until frozen. The lyophilization jars were then attached to a lyophilization apparatus consisting of a Welch Duo-seal Vacuum Pump ~Model No. 1402, available from Sargeant-Welch, P.O. Box 35445, Dallas, Texas 75235), a Virtis Immersion Coil Cooler (Model No. 6205-4350 Cooler in acetone bath) and a Virtis 18 Port Vacuum Drum Manifold (Model No. 6~11-0350). A11 Virtis equipment is available from American Scientific Products, P.O.
Box 1048, Grand Prairie, Texas 75050O The lyophili~ation drum was filled with acetone that was maintained at -50C.
The samples were lyophilized to dryness overnight and were then weighed on a Mettler AE 163 balance. The samples remaining consisted of substantially non-degradable lyophilized Carrisyn~9extract. The yield from 20 gallons of aloe vera gel was approximately 145-155 g. of Carrisyn ~ extract.

AVCI
B5220CIP2 ~ 8 ~ ~
RHF:RCB/sm January 14, 1988 EX~MPLE 2 Process for Separating and Isolating acemannan.
Aloe barbadensis Miller leaves collected from the Rio Grande Valley were transferred in a refrigerated truck at 40 to 45F within eight hours after harvest and stored under refrigeration at 40to 45F until processed to reduce degrada-tion.
The tip and bu~t por~ion was removed from each leaf.
The rind was then removed from each leaf with a sharp knife or wire cheese slicer to produce an aloe gel fillet from each leaf portion.
The aloe gel fillets were then placed in a 750 seat restaurant set stainless steel garb~ge disposal unit which coarse ground the fillets to an average particle size of the consistency of a thick, but freely flowing ~coarse homogeni-zed) liquid. The stainless steel garbage disposal unit was made by the N-sink-erator Division of Emerson Electric Co., Racine, WI, Model `~o. S~ 1-13, Serial No. 115132.
~; The coarse ground aloe gel fillets then passed to a 100 gallon stainless steel holding vat. The holding vat was made by Process ~quipment Corp. of Belding, Michigan, Model No. 100 gallon OVC, Serial No. 40865-3.
From the holding vat, the coarse ground aloe gel fillet solution was pumped to a homogenizer. The homogenizer was made by Crepaco Food Equipment and Refrigeration, Inc., or Chicago, Illinois, Serial No. 04-03. The homogenizer was of a type typically used in dairy processes for the homogenization of milkO The coarse ground aloe gel fillet solution was finely homogenized under a pressure of 1,500 psi.

B5220CIP2 ~3128~0 RHF:RCB/sm January 14, 1988 From the homogenizer the finely homogenized aloe gel fillet solution was pumped to a stainless steel storage tank.
The storage tan~ was madQ by Process Equipment Corp. of Belding, Michigan, Model No. 1000 gallon OVC, Serial No.
40866-2. The total mass of the homogenized aloe gel fillet solution was 20 to 60 percent of the starting leaf mass.
Then, if necessary, the homogenized product was dialyzed using ultrafiltration.
The homogenized gel was then filtered to remove the interstitial fibers using a Leslis's Diatomaceous Earth Filter Model DE-48. The interstitial fibers themselves instead of diatomaceous earth were used as the filter media, the fibers being supported by a nylon mesh cloth filter support. The gel was then pumped through the filter for several minutes before opening the exit port so that a sufficient amount of fibers could build up to serve as the filter media.
Twenty gallons of the filtered gel was then pumped into a 109 gallon tank and 80 gallons of 190 proof undenatured ethanol tEthyl Alcohol, 190 proof, U.5.P., punctilious, 5~
gal. Batch I.D. #CT18SJ04 available through U.S. Industrial Chemicals, Co., P.O. Box 218, Tuscola, Illinois 61953) was added to the aloe gel fillet solution. The solution was stirred for 20 to 30 minutes using a Perma-San propeller agi-tator model # ~APGF-4A, Process Equipment Corp., Belding, Michigan.
The alcohol-gel solutions were then immediately transferred to everal 11 quart pans 10~" diameter and 8" high of 18-8 stainless steel tBloomfield Industrie~ Inc., Chicago, Illinois, available through Watson Food Servlce Equipment and ( G

B5220CIP2 ~3i28~
RHF:RCB/sm January 14, 1988 Supplies, 3712 Hagger Way, Dallas, Texas).
The alcohol-gel solutions were then allowed to settle for approximately four hours.
The clear liquid supernatant was then decanted or siphoned off using care not to disturb the precipitate that had settled on the bottom of the pans. The precipitate and remaining solutions were then transerred to four 1 pint stainless steel centrifuge buckets with about 500 g. of preci-pitate and remaining solution being transferred to each bucket. The buckets were then spun at 2000 x g for about 10 minutes using an IEC Centra-7 Centrifuge (International Equipment Co., 300 2nd Avenue, Needham Heights, Massachusetts 02194 available through: American Scientific Products, P. O.
Box 1048, Grand Prairie, Texas 75050).
After centrifugation the supernatants were decanted and discarded. The pellets were then washed with fresh 190 proof, undenatured ethanol and spun down again at 2000 x g for about 10 minutes. After spinning again the supernatant was discarded.
The pellet was then transferred to several 600 ml.
VIRTIS lyophilization jar5 and swirled in liquid nitrogen until frozen. The lyophilization iars were then attached to a lyophilization apparatus consisting of a Welch Duo-seal Vacuum Pump (Model No. 1402, available from Sargeant-Welch, P.O. Box 35445, Dallas, Texas 75235), a Virtis Immersion Coil Cooler (Model No. 6205-4350 Cooler in acetone bath) and a Virtis 18 Port Vacuum Dru~l Manifold (L~odel No. 6211-0350). All Virtis equipment is available from ~merican Scientific Products, P O.
Box 1048, Grand Prairie, Texa~ 75050. The lyophilization AVCI
~5220CIP2 ~ 8 ~ ~
RHF:RCB/sm 1 a January 14, 1988 drum was filled with acetone that was maintained at -50C.
The samples were allowed to dry overnight and were then weighed on a Mettler AE 163 balance. The samples remaining consisted of substantially non-degradable lyophi-lized Carrisyn ~ extract. The yield from 20 gallons of aloe vera gel was approximately 145-155 g. of Carrisyn ~ extract.

Standard Laboratory Scale Process for Separating and Isolating Carrisynt~ extract Approximately 50 pounds of Aloe barbadensis Miller leaves were washed with water and brushed to remove dirt, dried latex and other contaminants. The outer cuticle of each leaf was then removed and the whole fillets were placed in a large beaker ~on ice).
In 1.5 liter batches a Waring blender was loaded with the whole aloe fillets. The fillets were blended at high speed twice for two minutes at room temperature. The blended fillets were then cooled at 4C to allow foam generated during blending to settle.
The blended aloe juice was then filteLed through four layers of cotton (Cleveland Cotton) to remove any fibrous cellulosic pulp. The filtrate was then passed through six layers of cotton and approximately 4 liters of aloe juice was collected.

The aloe juice was then placed in a large five gallon stainless steel container; To the filtered juice was added 16 liters of chilled ethanol (Fisher Ethanol reagent grade, Cat. No. A995). The ethanol was added slowly while stirring the aloe juice. A flocculent precipitate formed and G

B5220CIP2 ~.3128~
RHF:RCB/sm January 14, 1988 the mixture was stirred for 15 to 30 minutes and was allowed to settle at room temperature for about two hours.
The supernatant was then decanted off and the pellet was placed in a small blender to which one liter of deionized water was added. This mixture was blended for several minutes at low speed to wash the pellet and was then placed in an eight liter nalgene container. To this mixture was added four more liters of ethanol and the mixture was stirred for 30 minutesO The precipitant that formed was allowed to settle for about two hours.
The majority of the supernatant was then decanted off and the resultant pellet was centrifuged at 2000 x g for 20 minutes at room temperature to pellet the precipitant for easy decanting of the remaining solvent.
The pellet was then placed in a lyophilization flask and lyophilized overnight in a Virtis lyophilizer.
The lyophilized powder weighed 10.9 g. The percent yield was 0.273% or 2.73 x 10-3 g/mX.

The Reduction Of Calcium Oxalate Contaminant In Aloe Vera Extract - Acemannan During the process of extraction of acemannan from aloe vera gel by alcohol, minor amounts of organic and inorga-nic substance are found to co-precipitate with the product. A
large fraction of the inorganic salts and indeed the major contaminant in the alcohol extraction of aloe vera ~el to ace-mannan is calcium oxalate. The presence of calcium oxalate is confirmed by optical microscopy, infrared spectroscopy and thermogravimetric analysis. Although the quantity of calcium AVCI

RHF:RCB/sm Januaxy 14, 19~8 ~2~

oxalate may vary from batch to batch, calcium oxalates a~counting for about 30% of the total extract by weight have been observed. The purpose of the process described in ~xample 4 is to reduce the oxalate content in acemannan.
Calcium oxalate is very insoluble in water and alcohol. By treating aloe gel with alcohol, the oxalate in the Carrisyn extract precipitat~ is concentrated. This concentration of oxalate is demonstrat~d by the strong infrared absorption of Carrisyn ~ extract between 1600-1587 cm~1 due to the carbonyl ass~mmetric stretch of the oxalate and the high ash content of Carrisyn ~ extract from thermogravimetric analysis. Sinc2 the acemannan is the active substance in Carrisyn ~ extract, inorganic salts such a~ calcium oxalate should be eliminated or at least minimized for consistency of product quality and for health reasons.
One procedure for separating ~he oxalates and other inorganic salts would be by membrane dialysis. However, this method has many drawbacks and disadvantages. The process is very time consuming since the crude Carrisyn ~ extract must be rehydrated, re-extracted in alcohol and freeze dried again. Most importantly, unless the product is preserved during the dialysis step, microbial degradation and spoilage will result in very inactive Carrisyn ~ extract.

Generally, carboxylic acid salts are converted to their corresponding acids when treated with dilute mineral acids. By treating calcium oxalate with hydrochloric acid, oxalic acid and calcium salts or oxalic acid would be produced according to the following mechani~m:

CaC24 + 2H+ + Cl- ~ C2H204 + Ca++ + 2Cl-(calcium oxalate) (oxalic acid) Oxalic acid is highly soluble in water and alcohol and is therefore preferentially extracted into the c ~ ~3128~0 AVCI

RHF:RCB/sm January 14, 1988 water/alcohol mixture. The result is that the Carrisyn@~extract has a much lower level of oxalates and other inorg~nic salt-~ bout -2 liters of aloe vera gel previo~lsly homoge-nized at 500 psig and Eiltered through a new swimming pool filtex was collected for this ~xample. An initial pH of the gel was taken aftPr stirring. Gel samples of known weight were placed into several 600 mL beakers and the pH adjusted as appropriate with a suitable acid, here a dilute mineral acid (preferably 6N hydrochloric acid) or concentrated sodium hydroxide solution, as follows:

Sample # 0 1 2 3 4 5 6 7 8 Weight(gel) 100. 100. 99.3 100. 100. 99.7 100. 99.9 99.5 pH 4.56 4.07 2.45 3~35 5.09 10.6 6.16 7.0 8.58 After the pH of each sample was adjusted, four t4) volumes oE SDA-3A ethanol were added as soon as possible to minimize the time the gel would be under the stipulated pH
condition. After stirring the mixture for less than 2 minu-tes, each mixture was allowed to stand for about 3-4 hours.
Each extract was then centrifuged, freeze dried and weighed to determine the yield under the various pH conaitions, The infrared spectra of the nine solid Carrisyn ~ extract samples were obtained from a disc made by mixing an appropriate amount of the substance in potassium bromide.

Each disc was scanned from 4000 to 400 cm~l (wave numbers) on an IBM FT-IR spectrometer. The spectra of the Carrisyn~
extract samples at different pH conditions were compared qualitatively.
Thermoqravime~ric Analysis:
The thermal weight loss oE Carrisyn~ extract has a definite finger print profile. About 10 milligrams of each sample was B5220CIP2 13128~9 RHF:RCB/sm January 14, 1988 heated in a Mettler thermoanalyzer from 25C to 780C a~ a rate of 20C/min. Nitrogen gas atmosphere was used until a temperature of -600C was reached, followed by oxidizing environment to 780C and then each sample was allowed to remain at this temperature for 2 minutes. From the weight loss thermogram, the moisture content, the carbohydrate, oxi-dizable carbon skeleton, oxalate and ash contents were deter-mined.
Results and Discussion:
The most important factor with an acid treatment of Carrisyn extract is whether the physiochemical properties of the substance have been adversely affected by the process. A
suitable acid must be selected: (i) an acid capable of achieving the proper pH range (from about 3.0 to about 3.5) in reasonable volumes, (ii) will not react adversely with benefi-cial components (polydisperse acemannan), the solvent mixture (ethanol) and the holding vessels and equipment; and (iii) additionally, chosen in such a concentration as not to degrade the acemannan chain. Many dilute mineral acids and organic acids in higher concentrations are suitable, although a non-oxygenated mineral acid is most uitable as esterification and degradation are minimlzed. With the selection of a suitable acid, e.g., 6N hydrochloric acid, rather than having an *

adverse affect,the ~ua~ity of Carrisyn extract appearsto improve appreciably at lower pH conditions. For example, the same concentration ~w/v) of Carrisyn extract gave a more viscous, rehydrated solution when acid treated A ViaCOUS solution signifies good product. Size exclusion chromatographic separation of the solution demonstrated the same chroma-* trade mark.

~ ~3~2860 tographic profile as an untreated Carrisyn* extract. Therefore, under the conditions applied, degradation was not observed.
The following results demonstrate that the yield of Carrisyn* extract as represented by total solid in a unit volume of gel is reduced after acid treatment:

Sample # 0 1 2 3 4 5 6 7 8 pH 4.56 4.07 2.45 3.35 5.09 10.56 6.16 7.0 8.68 Yield% 0.26 0.22 0.12 0.14 0O27 0.23 0.25 0.26 0.24 However, subsequent analyses of the products by infrared spectroscopy and thermogravimetric analysis rev al that the high yield clearly correlated to oxalate and ash con-tent.
Infrared spec~roscopy may be used extensively to characterize Carrlsyn* extract both qualitatively and quantita-tively. The increase in the absorption peak of the acetyl group located about 1740 cm~l and the decrease of the oxalate peak about 1950 cm~l of Carrisyn* extract at low~r pH demonstrat~ a reduction of the oxalate content (Figure 8).
Thermogravimetric analysis gives a characteristic weight loss profile with temperature. Generally, pure Carrisyn*
extract gives 3 major peaXs on the differential thermogram namely: (a) moisture 30-100C, ~b) carbohydrate (acemannan) 200-400C, and (c) oxidizable carbon skeleton 600-630C (Figure 9). On ~the other hand, Carrisyn* extract contaminated with calcium oxalate for example exhibits 2 more peaks located between 456-500C and 650-720C with high ash content (Figure 10).
Tables lA - E below present thermogravimetric analyses of Carrisyn* extract at various pH conditions.

* trade mark c ~ 3~286~
AVCI

RHF:RCB/sm January 14, 1988 TABLE lA
THERMOGRAVIMETRIC ANALYSIS
OF CARRISYN EXTRACT pH 4.56 _ _ _ OXALATE
S~MPLE WT % CARBO- 425 631 ID(mg) ~H20 RESIDUE HYDRATE 540 C 750 C
703029.7220 6~912217.3949 49.9485 10.3680 12.14 703078.8020 6.680318.0750 51.1364 9.7364 11.64 7031110.1150 6.772118.9030 51~3980 8.6604 11.36 703129.1790 6.634718.4330 52.4889 8.7700 10.89 703138.9810 5.2667-18.53g0 53.0124 8.9522 10.85 703149.4530 4.728718.5130 52.0994 9.4785 12.1g 703169.6000 5.427117.7810 52.5517 9.1771 12.19 703209.9120 5.609417.0600 54.7322 8.6259 10.~2 AVERAGE 9.4705 6.0039 18.0873 52.1709 9.2211 11.49 STD DEV 0.4570 0.8391 0.6325 1.4205 0.6087 0.65 N = 8 TABLE lB
HERMOGRAVIMETRIC ANALYSIS
OF CARRISYN EXTRACT pH 3.60 . OXALATE
S~MPLE WT % CARBO- 425 631 ID(mg) ~H2ORESIDUE HYDRATE 540 C 750 C
__ 70315 9.21006.9164 7.7959 72.4104 5.6243 4.082 70321 10.71806.5777 9.0035 70.1716 6.~751 4.879 70323 9.30706.0277 1.9295 73.5680 5.1467 3.900 70324 9.09195.2690 6.9739 74.6780 5.0820 3.707 70326~ 9.6g507.0397 10.108~ 70.0355 6.4879 5.796 70327 9.149~5.7493 9.5639 69.4942 6.6455 5.2~4 70328 9.4~805.5450 8~3333 68.8317 9.5902 4.393 70329 9.31406.4204 8.6859 71.6130 5.5937 4.402 AVERAGE 9.49406.1939 8.5491 71.3503 6.3307 4.548 ¦ STD DEV ¦ 0.530 ¦0.6478 ¦ 1.0118 2.0730 ¦ 1.4527 * trade mark AVCI ~ ~ ~ ~
B5220CIP2 ~ U
RHF:RCB/sm January 14 ~ 1988 : TABLE lC
THERMOGRAVIMETRIC ANALYSIS
OF CARRISYN* EXTRACT pH 3.20 _ . OXALATE
SAMPLEWT ~CARBO- 425 631 ID (mg) %H20 RESIDUE HYDRATE 540 C 750 C
_ _ 70330 9 ~ 3260 3 ~ 5814 6 ~ 2621 77 ~ 1819 5.7152 3 ~ 506 7~331 9~330 4~6273 4~5764~0~7280 4~9425 1~871 70334 10 o8360 6 ~1831 5~038877 ~6395 4~8819 2~685 70401 8.10~0 7 ~ 8196 ~ ~ 8618 80 ~ 3070 4 ~ 9975 2 ~ 023 70402 9 ~ 0650 4 ~ 0927 4 ~ 6222 79 ~ 4380 6 ~ 828~ 1 ~ 522 70403 9 ~ 9490 4 ~ 2~14 4 ~ 3220 80 ~ 1990 4 ~ 7945 2 ~ 472 70405 8 ~ 8930 5 ~ 0497 4 ~ 8353 79.1636 4 ~ 4305 2 ~ 069 70406 9 ~ 8350 4 ~ 3213 4 ~ 0874 80 ~ 9156 4 ~ 7788 1 ~ 9420 70419 9 ~ 0080 5 ~ 3619 2 ~ 1758 81 ~ 8822 4 ~ 7069 1 ~ 7651 70423 9.8510 6~6998 3~928579 ~8803 4 ~7102 2~0506 70426 9 ~ 7880 5 ~ 5885 4 ~ 1479 78 ~ 8307 3~ 9947 1.2873 70427 1~ ~ 1010 ~ ~ 7815 3 O 2670 79 ~ 5866 4 ~ 5144 2.0190 70428 10.7790 3 ~ 6181 2 ~ 6069 80 ~ 1190 4 ~ 4717 2 ~ 7368 70431 9 ~ 9030 5 ~ 9~81 4.1099 7~ ~ 4407 4 ~ 5542 2 ~ 3831 70433 g ~ 39~ 6 ~ 2277 3 ~ 5127 79 ~ 5073 4 ~ 5844 2 ~ 2982 70434 9 ~ 7360 7 ~ 1385 4 ~ 6188 77 ~ 1878 4 ~ 5604 2 ~ 5781 70440 10.0850 5~3148 3~173081~4331 4~4522 1~9534 70442 9 ~ 9470 6 ~ 2933 3 ~ 3779 81 ~ 0805 4 ~ 4234 1.2567 70504 9 ~ 9740 6 ~ 2933 4 ~ 7925 77 ~ 1410 4 ~ 7323 2 ~ 8173 70505 9.1440 5 ~ 9165 3 ~ 63~8 80 ~ 5337 4 ~ 4182 1.4545 70506 10.2590 5 ~ 5659 3 ~ 4214 78 ~ 8573 5 ~ 9460 1.9300 70514 10.2060 7 ~ 7406 5 ~ 5164 74 ~ 4~61 5 ~ 2714 4 ~ 0956 AVERAGE 9. 6827 5 ~ 7172 3 ~ 8581 79 ~ 2956 4 ~ 8504 2 ~ 2145 (X) STD DEV 0. 6851 1.2332 1 ~ 3386 1 ~ 7546 0.6198 0.6831 N = 22 . _ * trade mark ~ 52 ~

E~5220CIP2 ~ ~ 31 28~
- RHF:RCB/sm January 14, 1988 TABLE lD
THERMoGRAvIMETRIc ANALYSIS
OF P~ECYCLED CARRISYI~* EXTRACT
20 MINS AGIT~TION

OXALATE
SAMPLEWT % CAR80-- 425 631 ID (mg) %H2ORESIDUE HYDRATE 540 C750 C

705159.8270 6.9401 9.4739 68.5359 6.0141 6.41~9 705189.9530 5.5059 5.3753 76.3485 5.0638 2.9941 7051910.4880 3.4134 8.1998 68.3159 5.9497 6.483~
705239.5570 3.2855 6.8013 66.6212 5.9642 7.3663 705269.8~50 5.8321 8.1629 74.7279 4.8651 3.1959 AVERAGE9.9300 4.9954 7.6026 70.9099 5.5714 5.2902 STD DEV0.3437 1.5944 I 561l 4.3276 0.5990 2.0401 THERMOGRAVI~fETRIC ANALYSIS
OF R~CYCLED CARRISYN* EXTRACT
4CI ~tllN::j . AGlq~'l'lON

OXALATE
SAMPLE WT 9~ CAR~O-- 425 631 ID (mg) %H2ORESIDUE HYDRATE 540 C 750 C
. _ _ _ 705289.6900 5.7069 6.1197 77.7195 4.7678 2.633 705299.2960 7.0998 4~9699 75.871~ 4.5611 3.~01 705359.7970 6.5428 4.3483 79.3609 4.2462 1.194 AVERAGE 9.5943 6.4498 5.1460 77.6506 4.5250 2.1~2 STD DEV O.2638 0.701l [).8987 1,7457 O.2627 o,922 The tables reveal that:
1. Carrisyn* extract manufactured from a starting gel having a pH in excess of 4.56 demonstrated average total carbohyclrate and residue tash) contents of 52.296 and 18.1% respectively.
2. With adjustecl pH of starting gel ~rom about ~.56 * trade mark G

AVCI 131286~

RHF:RCB/sm January 14, 1988 to 3.60, the carbohydrate and ash contents were 71.4~ and 8.55~ respectively. This is an impro-vement of 36.8% and 52.8~ for each measureA
parameter.
3. However, with a pH of 3.20, the average car-bohydrate content jumped to 79.3% while ash con-tent decreased to 3.86~ signifying a carbohydrate increase of about 51.2~ and ash decrease of 78.7%.
: 4. Preliminary data demonstrates tha Carrisyn ~ extract batches with increased carbohydrate content and lower ash value demonstrated better antiviral activity; it is therefore recommended that pro-cessing be conducted at a pH between 3.00 and 3.50.

Effects of pH Quality on Carris~n ~ extract (TGA METHOD) pH 2.45 3.35 4.07 4.56 5.09 6.16 7.00 8.68 Moisture 5.6762 6.2433 6.9750 8.4035 8.5905 8.8~31 8.9819 3.7702 Acemannan 73.925 69.930 46.929 38.651 37.649 36.796 3~.593 36.316 Carbon 17.812 15.455 12.840 12.846 12.969 12.508 11.485 10.769 Skeleton Calcium 0.4990 4.3160 9.7965 10.670 10.251 10.347 11.725 10.148 Oxalate Ash 1.9644 3.9494 13.811 15.999 16.847 17.908 17.~92 17.198 Yield % 0.12 0.14 0.22 0.26 0.27 0.25 0.26 0.24 Carrisyn ~ extract(total solid) yield, calcium oxalate, ash content and moi~ture appear to increase with increased pH up to 4 - 5. 8ut acemannan and the corresponding carbon skeleton increase with decreased pH. Therefore a pH greater than 4.0 ,.
- 5~ -AVCI ~3128~

RHF:RCB/sm January 14, 1988 is not recommended if ash and calcium oxalate contents must be decreased. A yield of Carrisyn* extract greater than 0.2% must be analyzed for calcium oxalate contamination.
By ~reating aloe vera gel with a suitable acid, e.g., a dilute mineral acid, preferably hydrochloric acid, to adjust the pH of the gel to between 3.0 to 3~5 followed by ethanol extraction effected a significant reduction in the amount of oxalates By this step, both calcium oxalate and ash content may be reduced by more than 80%. The treatment al50 concentrated the amount of the active Carrisyn* extract without degrading the polymer as demonstrated by physiochemical analytical methods.

Two batches of Carrisyn* extract were manufactured as described in Example 1, but with the additional step of acidi-fication to pH 3.6 prior to ethanol precipitation. In Lot ~
70315, concentrated nitric acid (61 ml) was added to 20 gallons of homogenized aloe vera gel. In Lot ~ 70321, con-centrated hydrochloric acid (171 ml) was added to 45 gallons of homog~nized aloe vera gel. The yields were 55.6g (0.07~) and 186~6g (0.11~), respectively. Chemical analysis by IR and TGA showed both batches to have similar quality and reduced calcium oxalate. The analyses of these two batches are included in Table lB.
It should become apparent to one skilled in the art, that nitric acid can be used solely for acidifiration, as well as hydrochloric acid or any other suitable acids.

*trade mark B5220CIP2 ' 131 286~
RHF:RCB/sm January 14, 1988 CHARACTERIZATION OF CARRISYN* EYTRACT
Using pharmaceutical screening techniques a poly-saccharide extracted from aloe vera has now been found to be the active chemical substance in aloe vera. This poly-saccharide will be hereinaftex referred to as acemannan.
Acemannan is an ordered linear polymer of substantially acety-lat d mannose monomers. Other ingredients, such as proteins, organic acids, an~hraquinones, vitamins and amino acids make up less than one percent of Carrisyn* extract. The concentration of Carrisyn* extract in aloe vera has been found to be approxi-mately 0.05 to 0.3 wei~ht-percent of the-aloe Jera jUiC2 . The yield or concentration of Carrisyn* e~tract in the leaves depends on leaf maturity.
The pharmacological data that evidences that aceman-nan is the active chemical substance in aloe vera can be sum-marized as follows:
1. The dose-response of acemannan was the same as alOe vera juice with an equivalent amount of acemannan.
~2. Acemannan was effective in the ulceroprotection - model by different routes of administration,namely intrave-nously, intraperitoneally and orally.
3. Asemannan accounted~ for 100 percent of the effects in both pharmacologica~ models.
4. The chemical substance, glucomannan a substance similar to acemannan from a completely different source, the Konjac plant, provided some pharmacological response.
Carrisyn* extract has been shown in laboratory studies to increase up to 300% in 48 hours the replication of fibroblasts in tissue culture which are known to be responsible for * trade mark c BS220CIP2 i 3 12 8 ~ O
RHF:RCB/sm January 14, 1988 healing burns, ulcers and other wounds of the skin and of the gastrointestinal lining.
Carrisyn* extract has also been shown to increase DNA
synthesis in the nucleus of fibroblasts. The increase in DNA
synthesis in turn increases the rate of metabolic activity and cell replication which are fundamental steps to the healing process Carrisyn* extract has been shown in controlled studies to increase the rate of healing in animals.
Carrisyn* extract has also been shown to be an effective treatment for gastric ulcers in animal studies. Over a three year period laboratory rats, the stomachs of wnich react simi-larly to that of humans, were tested. Carrisyn* extract was found to be equivalent to or superior to cuxrent medications used for the treatment of gastric ulcers. Most such products act to inhi-bit hydrochloric acid in the stomach. Carrisyn* extract works on a different principle and does not alter the natural flow of digestive acids.
As noted above, Carrisyn* extract can be precipitated out of liquidified aloe vera gel by the addition of a water soluble, lower aliphatic polar solvent, preferably ethyl alcohol.
Carrisyn* extract powder can then be prepared by lyophilization anc optionally the lyophilization product can be ground into a powder with a grinding apparatus such as a Moulinex coffee-grinder (available from Dillard's, Dallas, Texas). Carrisyn*
extract powder is highly electrostatic and is an off-white to pinkish-purplish amorphous powder depending on the oxidization stat of any anthraquinone contaminant. Carrisyn extract is stabilized and becomes substantially non-degradable by freeze drying or * trade mark ~3128~0 lyophilization which removes the water which causes hydrolysis.
Freeze dried aloe vera gel with a given amount of Carrisyn*
extr-act has maintained its effectiveness for two years. It is believed that Carrisyn* extract in freeze dried form will be stable for up to ten years.
Heat and time have been found to be important factors in the production of powdered Carrisyn* extract. Heat can aid in the hydrolysis or degradation of Carrisyn* extract and the longer it takes tD process out the Carrisyn* extract at a given temperature/ the more is the degradation. Accordingly, it is preferred that the process which allows for the quickest extraction of Carrisyn* extract from whole aloe vera leaves be used when high molecular weight Carrisyn* extract powder is desired and it is preferred that the process which allows for the slowest extraction of Carrisyn* extract from whole aloe vera leaves be used when low molecular weight Carrisyn* extract powder is desired.
Rehydration of Carrisyn* extract powder at a weightjvolume concentration of 0.2 to 1 percent resulted in the reformation of a viscous "gel" like fresh aloe vera. The gel-like consistency which returns upon rehydration of Carrisyn*
extract powder is indicative of the high molecular weight polysaccharidic nature of Carrisyn* extract. Generally, as polysaccharides are degraded or hydrolyzed their viscosity is lowered. Accordingly, the viscosity of rehydrated Carrisyn*
extract powder gives a good indication of quality and may be used as a parameter for quality assurance.
Carrisyn* extract produced according to the process of the present invention may be characterized as a substantially non-degradable lyophilized linear polymer of substantially acety-* trade mark-58-~ 3~28~
AVCI

RHF:RCB/sm January 14, 1988 lated mannose monomers, pre~era~ly bonded ~ogether by4) bonds.
Various modifications o~ the disclosed composi~ions of the invention, as well as alternative modifications, variations and equivalents will become apparent to persons skilled in the art upon reading the abov2 general description.
The following Examples ~Examples 5 - 8) were conducted in order to further characterize and identify Carrisyn* extract. The following examples are illustrative only and are not intended to limit the scope of the appended claims, which cover any such modifications, equivalents or variations.

Isolation. Purification And Characterization Of Carrisyn* Extract A. Isolation of Carrisyn* Extract An aloe leaf was washed, sliced open and filleted.
The clean inner gel was retained while the green rind and latex materials were discardedO The filleted material ~as homogenized and extensively filtered with a Finisher ~odel 75 (FMC, Chicago, IL), to remove most of the pulp. The clear, viscous gel was acidified to a pH of approximately 3.20 with dilute ~Cl to solubilize the oxalates and lactates of calcium and magnesium that are usually present to their corresponding water soluble acids. The acid treated gel was then extracted for 4-5 hours with four volumes of 95% ethanol at ambient tem-perature. Floating fibers were removPd, then the alcohol/-water mixture was siphoned off while the solid precipitate was collected by centrifugation. Most alcohol/water solubla substances such as organic acids, oligosaccharides, monosu-* trade mark 13128~
AVCI

RHF:RCB/sm January 14, 1988 gars, anthraquinones and inorganic salts were eliminated in the process. The solid was then washed with fresh alcohol, centrifuged, freeze dried, and ground to a white amorphous powder.
B. Purification .
Carrisyn* extract at this stage is generally contamina~ed with proteins, monosugars, oligosaccharides and inorganic salts. These contaminants do not affect the bioactivity of the product hence further purification is not necessary for manufactured bulk material. However, as a further step in the characterization of Carrisyn* extract, more purification steps were needed. The above-mentionecl contaminants were removed by redissolving the bulk powder in phosphate buffer and treating it with non-specific protease ~Sigma Lot #5147) followed by extensive dialysis. The non-filterable product, which is mainly acetylated polymannose, was freeze dried and charac-terized using a number of analytical methods which include IR
spectroscopy, thermogravimetric analysis (TGA~, ~PLC, GLC and GC/MS as depicted in Figure 11.
For the standardization of aloe vera products HPLC
and GC are recommended. The gas liquid chromatographic tGLC) method is used to separate and ~uantitate mannose in aloe vera g~l extract. In order to prevent the spiking of products with mannose to make a product appear to have a higher aloe gel content, a dialysis ~tep may be added (MW cutoff 12,000 -14,000). In addition, spiking with locust bean gum, guar gum or glucomannan could be detected by a high galactose or glu-cose to mannose ratio.

* trade mark 13~2~0 AVCI

RHF:RCB/sm January 14, 19~8 C. Molecular Weiqht_Determination Introduction:
~cemannan (AM) is a plant extract polysaccharide.
It is polydispersed which means that it is comprised of more than one molecular weight size.
Objective:
The object of the study is to determine the molecu-lar weight distribution of acemannan by size exclusion chroma-tography.
Reagents and Chemicals:
0.05% sodium azide.
Standards-0.2% (w/v) of each standard, Pullulan 853K, 100K and 12.1X daltons in 0.05% sodium azide. (Shodex P-82, Showa Denko, K.K.) Instrumentation:
High Performance Liquid Chromatograph, model 590 (Waters Associates, Milford, MA) Differential refractometer, model 1770 (Bio-rad) Integrator SP 4290 (Spectra-Physics~
Sam~e Preparation:
-Weigh 20 mg of Carrisyn* extract into a glass vial (105x25mm) with a teflon lined cap.
-Add 10 mL of 0.05~ sodium azide and dissolve by shaking (4 hours) in a Junior orbit shaker ~Lab-Line Instruments, Mel Rose Park, IL).
-Filter through a 1.2 um membrane, (Acrodisc(R), Gelman Sciences). Save the filtrate for an injection into the HPLC.

* trade mark 1312~
AVCI

RHF:RCB/sm January 14, 1988 Hiqh Performance Liquid Chromatoqraphic (HPLC) Conditions Column Spherogel TSK 5000 PWHR (Beckman Instruments) Detector: Differential refractometer (Bio-rad) Mobile Phase: 0.05~ sodium azide Flow rate: 1 mL/min at 40C
Vol. inj.: 50 uL
Results:
Acemannan is a polydisersed polysaccharide with at least 73% of the polymer greater than 10,000 daltons as deter-mined by size exclusion chromatography (SEC) and pullulan poly-saccharide as the standard. Figure 12, represents the SEC
chromatogram of pullulan standard of known molecular weights;
853K, 100K and 12.1K daltons respectively. The corresponding chromatogram of Carrisyn* extract is shown in Figure 13. The chromatogram highlights three (3) main peak functions, and C. Peak A represents the acemannan fraction ~reater than 100,000 daltons and peak B is the fraction greater than 10,000 but less than 100,000 daltons. Peak C represents lower mole-cular weight constituents. The sum of peaks A and B consti-tute the active fraction.
D. Infrared ~IR) Spectrosco~ic Analysis Introduction:
The functional groups of the acemannan, the active product of Carrisyn* e~tract, absorb at characteristic infrared frequencies. Infrared spectroscopy therefore is an important method to characterize this material Objective:
The objective of the study is to ~urther charac-* trade mark ~3~28~
AVCI
~5220CIP2 RHF:RCB/sm January 14, 1988 terize Carrisyn* extract by an infrared s ectroscopic method.
Reaqents and Chemicals:
Infrared grade potassium bromide (KBr) powd~r (Mallinckrodt Inc., Paris, Kentucky).
Instrumentation:
IBM Fourier Transform infrared (Ft-Ir) spectrometer model ~32 equipped with an IBM 90t)0 computer and a printer/plotter.
Sample Preparation~
-Carrisyn* extract is preground to fine powder by usinq Wiley Mill ~Thomas Scientific Co.) and a screen which allows particles smaller than 60 mesh size.
-A 5 mg preground sample is mixed with 495 mg of dry KBr to a total of 500 mg mixture.
-The mixture is reground by hand using agate mortar and pestle to a homogeneous material.
-A representative sample (80-lOOmg) is pressed into a transparent disc using a hydraulic jack (WalkerR) at a pressure of 40,000 psi.
-The disc is scanned from 4000 cm 1 to 400 cm~l. A
multiple scan (30 scans) is performed with a 4 cm~l resolution to improve signal to noise ratio.
Results:
Analysis of the spectrum depicted in Figure 14, highlights the following characteristic absorption peak fre-quencies tcm~~

Wavenumber(cm~l; Assignment 1066.8 C-O stretch of pyranose ring structure 3422.1 O-H stretch 1250.0 Ç-O-C stretch ~acetyl group) ~ 63 - * trade mark 13128~
AVCI

RHF:RCB/sm January 14, 1988 1740.0 C=O stretch (acetyl) 1377.3 C-~ bending 1649~3 ~ C=O stretch (Amide I) 530.5 Internal rotation modes etc.
599.9 Internal rotation modes etc.
2928.3 C-H stretch 1541.3 N-H deformation (Amide II) 806.3 ---897.0 Axial H on ring at C
773.6 ring breathing It i9 noted that the spectrum of Carrisyn* extract demonstra--tes a strong absorption due to O-~ stretching about 3422 cm~l.
The carbonyl and C-O-C stretches of acetyl group are located near 1740 and 1250 cm~l respectively. The strong single band of C-O-C system is indicative of O-acetyl group bonded equatorially to the monomer unit. The amide carbonyl stretch (amide I) at about 1649 cm~l superimposed on the moisture absorption peak and the N-H deformation (amide II) about 1541 cm~l are due to protein/proteoglycan impurities.
The absorption bands between 960-730 cm~l can be correlated with certain stereochemical features of acemannan.
For example, the absence of a band near B44 cm~l due to equatorial Cl-H and the presence of axial Cl H near 897 cm~l, demonstrate that the acemannan is B-linked. The ring vibra-tion (955 cm~l shoulder) and the ring breathing located about 773 cm~l indicate a D-mannan with beta-glycosidic linkage.

Table 3 below depicts the peak absorption f requen-cies with corresponding absorbances arranged according to intensity:

* trade mar~

- 6~ -~312~
AVCI

RHF:RCB/sm January 14, 1988 PEAK ABSORBANCE FREOUENCIES ~OR
NON-PROTEASE TREATED CARRISYN* EXTRACT
Peak ~ Peak Peak StartPeak End Abs 1 1066.8 1201.8 1049.~ .5~6 2 3422.1 3433.7 3~10.6 .474 3 1250.0 1348.4 1201.8 .439 4 1740.0 1821.0 1699.5 .433 1377.3 1410.1 1350.3 .343 1649.3 1697.6 1585.7 .32~

7 530.5 582.6 493.8 .313 : ~ 599.9 634.7 586.4 .28~
9 2928.3 2995.8 2899.4 .242 1541.3 15~ 1527.8 .226 11 806.3 835.3 7~9.0 .224 12 ~97.0 920.2 843.0 .210 :~ 13 773.6 787.1 750.4 .207 E. Infrared Spectroscopy of Protease Treated Carrisyn* Extract The infrared spectrum of bulk Carrisyn* extract revealed traces of protein or proteoglycan impurties as demonstrated by ~ the presence of amide I and amide II peaks. Further purifica-; tion of the bulk material with non-specific protease which hydrolyzes the protein or proteoglycan followed by extensive dialysis, eliminated these impurities.
The spectrum of the pxotease treated sample is shown in Figure 15. Analysis of the spectrum shows some differences when compared to the non-protease treated Carrisyn* extract of Figure 14. For example, the amide I and II peaks located about 1649 and 1541 cm~l in Figure 14 are absent from the protease treated sample. In addition, the moisture absorption peak located about 1639.7 is clearly resolved. Table 4 represents the peak absorption frequencies with corresponding absorbances arranged according to intensities for protease treated Carrisyn* extract as follows:

PEAK ABSORBANCE FREQUENCIES FOR
PROTEASE TREATED CARRISYN* EXTRACT

- 65 - * trade mar~

13~ 2~
AVCI

RH~:RCB/sm January 14, 1988 Peak #PeakPeak StartPeak End Abs 1 1064.8 1093.8 1049.4 .969 2 1032.0 1045.5 970.3 .966 3 34Z2.1 3435.6 340~.8 .814 4 1248.1 1348.4 1201.8 .729 1740.0 1838.4 16g5.6 .679 6 1377.3 1406.3 1350.3 .593 7 530.5 545.9 493.8 .575

8 598.0 677.1 586.4 .532

9 1639.7 1695~6 1554.8 .491

10 2928.3 2990.~ 2903.2 .428

11 897.0 918.2 887.4 .409

12 ~06.3 835.3 790.9 .409

13 771.6 789.0 748.5 .387 The absorbance values are not comparable in intensities with the non-protease treated Carrisyn* extract of Figure 14 since it is qualitative and no attempt was made to use the same concentration in making the sample discs.
On the basis of infrared spectroscopy alone, Carrisyn*
extract is a polysaccharide of essentially ~-linked D-mannose with O-acetyl group side chains. The presence of N-acetyl groups may be due to protein/proteoglycan impurities.
F. Thermogravimetric Analysis (TGA) of Carrisyn* Extract Introdlction:
Thermogravimetric analysis (TGA) is an important analytical method for studying polymers. The mass loss on decomposition of a polymer as a result of temperature change is characteristic of that polymer. Moreover, TGA aids in the determination of moisture (H2O) and ash content of powdered substances.
Objectlve:

It is the objective of this study to use TGA to further characterize Carrisyn* extract.
Reagents and Chem~cals:
None.

* trade mark ~ ~3l2~6a AVCI

RHF:RCB/sm January 14, 1988 . .

Instrumentation-.
1. Mettler Thermoanalyzer, TA 3500 system featuring a TC lOA Evaluation and Control computer, as well as a TG 50 furnace with M3-03 microbalance.
2. Prin~er/plotter model MP3 (Print Swiss Matrix) 3. IBM PC for file storage.
Sample Pree~ration and Analysis:
-A lO mg sample in a 70 mL alumina crucible is weighed in a microbalance accurate to +l ug.
-The crucible with its contents is heated in the TG
50 furnace at a temperature program rate of 20C/min. Lhis rate of heating is standard and adequate for the material to be analyzed.
-Heating is performed in a nitrogen gas atmosphere from 25C to 600C and then from 6~1C to 780C in air (oxidant) atmosphere.
-The temperature is held at this final temperature for an additional (2) two minutes. (The maximum temperature of 780C is chosen because both organic and inorganic substan-ces decompose be~ore this temperature is reached.) Results:

The decomposition profile of Carrisyn ~ extract is characteristic. Figure 16 depicts the real time plot and the corresponding first derivative plot of the decomposition profile of Carrisyn ~ extract. The acemannan (the active substance of Carrisyn ~ extract) decomposition pattern is different from those of other polysaccharides. For example, under identical operating conditions, the temperature at which acemannan exhibits major decomposition is different from those of cellulose, dextran AVCI ~3~2~

RHF:RCB/sm January 14, 1988 or amylan. The weight 10s5 associated with acemannan i5 located between 200C to 630C. The bulk of Carrisyn ~ extract's decomposition occurs between 200C to 540C and 600C to 630C.
The acemannan fraction is determined by the contributions of these two temperature regions, whilP ash and moisture together contribute about 10% weight. Table 5 below represents a replicate analysis of Carrisyn ~ extract with TGA.

AVCI 1 31 2 8 ~ O
RHF:RCB/sm JanUary 14, L988 -- TGA ANALYSIS OF CARRISYN* EXTRACT
Sample # Weight H20 Ash* AM CaOx (mg) % % % %
, 1 9.4390 7.3631 3.8034 82.2119 2.5532 2 9.5860 7.36~9 ~.0163 81.8381 2.7540 3 9.5210 7.4992 4.2012 81.7568 2.9514 4 9.453Q 7.7118 4.1786 81.2010 2.9620 9.4090 7.7691 3,8793 81.6072 2.9546 9.5960 6.8674 3.9912 82.0969 3.1576 7 9.4740 7.6209 4.1904 81.6865 2.8605 9.5730 7.3436 4.2411 81.0719 3.3218 9 9~5550 7.9435 4.1130 80.9652 3.2444 9.5220 7.7715 4.3373 80.8023 3.1611 ~ve.**
(X) 9.5128 7.5255 4.0952 81.5238 2.9920 Std Dev. 0.0626 0.3087 0.1688 0.4864 0.2353 *The a~h consists of the oxides of Ca (1.51), Si (0.1), Na (.55), Mg (.37), Fe (.02) and Al (.00).
**Average value will not total 100% because the process does not account for peaks less than 2% of the base peak.

This method of analysis is qualitative as well as semi-~uantitative. For example, the percent acemannan may be determined as follows: -1. Percent (%) AM = mg (200-630C) x 100 mg (sample) or 2. Percent (%) AM = mg (200-630C) sample x 100 ' mg (200-630C) ref. std.
The moisture and ash contents are important parame-ters in powdered drug substances. These parameters are easily determined using the Thermogravimetric method.

* trade mark .

RHF:RCB/sm January 14, 1988 G. Constituent Sugar Determination of Carrisyn* Extract By ~cid HvdrolYsis an~ H1gh Performance Liquid ChromatograPhy (HPLC) Introduction: ~
_ Polysaccharides are hydrolyzed to their constituent monomers by acid or enzymatic hydrolysis. A 2 M trifluoroace-tic acid (TFA) is chosen for the hydrolysis because it is strong enough to hydrolyze the glycosdic bonds but unlike sulfuric acid it is gentl~ enough that the mononeric sugar residues are not destroyed.
Objective:
The objective is to determine the monomeric sugar constituents of the acemannan fraction of Carrisyn* extract.
Reaqents and Chemicals:
1. 2 M TFA containing 0.5 mg/mL inositol as the internal standard.
2. Isopropanol Instrumentation:
1. Hewlett Packard HPLC model 1084B equipped with an autosampler HP79842A and auto injector HP79841A.
2. Bio-rad differential refractometer model 1770.
Sample Preparation:

Wei~h accurately 2.0 mg of Carrisyn* extract on weighing paper and transfer quantitatively to a ~13x100 mm3 disposable culture tube with a teflon lined cap.

-Add one milliliter (mL) of 2M TFA containing 0.5 mg/mL inositol a~ an internal standard.
-Place the tube in a heating block ~Hycel Thermal Block) at 120C for approximately 1 hour.
-Evaporate the hydrolysate mixture to dryness with * trade mark AVCI 1312~69 RHF:RCB/sm January 14, 1988 dry air.
-Redisperse the solid in 1 mL of isopropanol and evaporate to dryness with dry air.
-Dissolve the solid in 1 mL of deionized water in preparation for injection into the HPLC column.
HPLC Conditions:
Column: Aminex Carbohydrate ~PX-87P
(Bio-rad Labs., Richmond, CA) Mobile phase: Deionized water at 80C
Flow rate: 0.6 mL/min Oven temp: 40C
Chart speed: 0.2 cm/min Detector: Refractive index (Bio-rad #1770) Attn: 2 ~esults:
Figure 17 represents the chromatogram of a standard mixture, comprising glucose, galactose, mannose 1 mg/ml each and 0.5 mg/ml inositol as the internal standard. Figure 18, represents the chromatogram of Carrisyn* extract under identical conditions. It is noted that mannose is the major component of the polymer signifying that the polysaccharide is essen-tially composed of mannose sugar units.

H. Gas Liquid Chromatographic Separation and Quantitation of CarrisYn* Extract .. _ . .... . _ _ . .. _ Introduction-The polysaccharide of Carrisyn* extract is mainly neutral in nature. Neutral polysaccharides are better analyzed by Gas Liquid Chromatography (GLC) as their glycitol acetates.
Objective:
The objective of this study is to further charac-*-trade mark ~312~
AVCI

RHF:RCB/sm January 14, 1988 terize Carrisyn* extract by separating and quantitating the sugafmonomers as their glycitol acetates. By this procedure, the glycitol acetates are separated with a capillary column and detected by a flame ionization method.
Reaqents and Chemicals:
l. A 2M TFA containing 0~2 mg/mL inositol as the internal standard 2. Isopropanol 3. lM NH40H containing 10 mg/mL sodium boro-deuteride (NaB2H4) or sodium borohydride (NaBH4) 4. Glacial acetic acid 5. Methanol 6. Pyridine 7. Acetic anhydride 8. Toluene and dichloromethane 9. Gas chromatography (G.C.) grade acetone Instrumentation:
l. Gas chromatograph Vista 6000 (Varian Instrument Group, Palo Alto, CA) 2. Integrator SP4290 (Spectra-Physics, San Jose, CA) Sam~le Pre~aration:
-Weigh accurately 2 mg of sample and transfer into a culture tube (13xlO0 mm) with a teflon lined cap.
Hydrolysis:
-Add 500 uL trifluoroacetic acid TFA-~2Mr con-taining 200 mg/mL inogitol), heat at 120C (Hycel Thermal Block) for about one hour.
-Remove TFA (water bath at 40C) under flow of air * trade mark AVCI 131286~

RHF:RCB/sm January 14, 198B

and wash residue with isopropanol to remove residual acid.
Reduction:
-Dissolve residue in 1 M NH40H (300 mL) containing 10 mg/mL sodium borodeuteride ~NaB2H4) and leave at ~oom temperature for 1 hour.
-Destroy excess reductant with glacial acetic acid (few drops until no effervescence), remove solvents under air and wash residue with methanol containing 10% acetic acid (3x300 uL~ and finally methanol t3x300 uL).
Acetylation:
-To the dry residue add 200 uL pyridine and 200 uL
acetic anhyride, heat for 20 min at 120C.
-To the cooled solution add toluene 500 uL, remove solvent under air, dissolve residue in water and extract into dichloromethane.
-Transfer the organic phase to a clean tube, remove the organic solvent under air and dissolve residue in acetone (100 uL), prior to GLC.
Gas Liquid Chromato~raphic Conditions (GLC) Column: SP 2330, 15 M or 30M, 0.25 mm I.D., 0.25 um liguid phase thickness ~Supelco, Inc.) Carrier gas: Helium Oven temp.: 235C (isothermal) Inj. temp: 250C
I~j. vol.: 0.5 - 1 uL (spiit) Results:

Figure 19, represents the GLC chromatogram o a standard mixture of rhamnose, fucose, arabinose, xylose, man-nose, galactose, glucose, and inositol as their glycitol ace-c AVCI 13128~0 RHF:RCB/sm January 14, 1988 tates. The chromatogram of Carrisyn* extract is shown in Figure 20. The major peak in the figure, corresponds to mannose. There are traces of ~galactose, glucose, arabinose, xylose, and fucose. These are from cell wall contaminants. ~h~ last peaX
is inositol acetate which is the internal standard.
Since mannitol acetate is the major sugar peak in the hydrolysed and acetylated material, Carrisyn * extract is essentially a polymannose pol~saccharide.
The mannitol acetate peak can be quantitated with the help of inositol as the internal standard. Figure 21, xepresents a standard curve generated by plotting mannose/
inositol area ratio against the known weights of acemannan.
With the standard curve, the amount of acemannan in a given sample of Carrisyn* extract can be calculated.

I. Gas Chromatoqraphy/Mass Spectrometry and Glycosidic Linkage Analysis of Carrisyn* Extract Introductlon:
Polysaccharides are characterized by determining the manner in which the sugar units are linked to one another.
The chemical, physical, and biological activities of poly-saccharides depend on where the sugars are linked in the five possible positions for the hexoses.
O~jective:

It is the purpose of this study to determine the manner in which the sugar units of Carrisyn* extract polysaccharide are linked.
Reagents and Chemicals:
1. Dry dimethylsulfoxide (DMSO) 2. Sodium dimethylsulphinyl anion (2M) 3. Methyl iodide * trade mark - 7~ -f c AVCI ~3128~
, B5220CIP2 RHF:RCB/sm January 14, 1988 4. 2M TFA

5. Isopropanol 6. NH40~ with 50~ methanol, lO mg/mL ~NaB2H

Glacial acetic acld 8. 10% acetic acid in methanol 9. Acetic anhydride lO. Sodium bicarbonate 11. Dichloromethane 12. GC grade acetone strumentation:

Gas chromatograph/mass spectrometer MSD (HP 5970).
'.~
GLC/MS and Glycosidic Linkage Analysis ; The overall characterization of Carrisyn* extract includes the linkage analysis of the polymer. Carrisyn* extract is per-methylated by the method described by Darvil et al., (Plant Physioloqy, 62: 418 - 422 (1978)), the disclosure of which iâ
hereby specifically incorporated herein by reference, hydro-lyzed to the monomers and converted to methylated alditol ace-tates. The volatile derivatives are analyzed on ~ewlett-Packard GC/MS system on a Supelco SP 2330 capillary column (30 m x .25~mm id). Fragmentation of the methylated glycitol acetate of the monomer5 is achieved by electron impact (EI) method. The total ion chromatogram (TIC) of the derivati~ed Carrisyn* extract as the partially methylated and partially acetylated glycitol is shown in Figure 22. The mass spectrum of the p~rtially acetylated mannitol is demonstrated by Figure 23.
The complete procedure is as follows:
(l) The sample (l mg) is placed in a tube with a * trade~ Mar~

AVCI
B5220CIP2 ~ ~ o e n RHF:RCB/sm !~ ~3 L~ O~
- January 14, 1988 teflon lined cap and dried in a vacuum oven at 50C.
(2) To the sample is added dry DMSO (250 uL) and the sample stirred tteflon stir-bar) until it dissolves (sonication may help~. Purge tube with argon (or nitrogen).
(3) To the polysaccharide (in DMSO) is added 250 uL
of sodium dimethylsulphinyl anion (2M) and left stirring for a minimum of 4 hours (reaction should be performed under argon). It may be more convenient to leave the sample oYernight in the anion solution.
(4) The anion solution is cooled in ice, methyl iodide 200 uL is slowly added and the solution stirred for about 1 hour.
(5) To the solution, add about 2.5 mL water and remove excess methyl iodide with argon.
(6) Transfer the mixture to dialysis tubing, 13.3 x 2.1 cm in diameter (M.W. cutoff 12,000

14,000, Spectrum Medical, Inc. Los Angeles, CA).
(7) Concentrate the non-dialysable fraction under a flow of air 150C).
(8) To the dry residue add 250 uL o~ 2M TFA and heat for 1 hour at 120~C.
(9) Remove TFA under air, wash residue with isopro-panol (2 x 250 uL).
(10) Dissolve residue in aq. NH40H 50% methanol (250 uL) containing 10 mg/mL NaB2H4 leave l hour at RHF:RCB/sm January 14, 1988 room temperature.
(ll~ Destroy excess reductant with glacial acetic acid (few drops), concentrate to dryness and wash residue with 10% acetic acid in methanol (3 x 250 uLl.
(12) To the dry residue, add acetic anhydride (100 uL) and heat sample at 120C for 3 hours. To : the cooled sample add water (about 1.5 mL) and then sodium bicarbonate until effervescence stops. Extract the derivative into dich-loromethane.
(13) Concentrate organic phase to dryness, dissolve residue in 100 uL acetone prior to- GLC and GLC/MS.
GLC conditions:
Column: SP 2330 fused silica column (30 m x 0.25 mm).
Temp.: 3 min at 170C followed by 4C/min to 240C hold for 10 min.
Detector~ FID
GLC/MS conditions:
Column: SP 2330 (30m x 0.25 mm) Temp.: 2 min at 80C, increased to 170C at 30C/min then 4/min to 240C hold for 10 min MS: Hewlett Packard MSD.

Determination of the Major GlYcosidic Linka~es of Samples by Methylation Analysis In this procedure all the free hydroxyl groups o the polysaccharide are converted to methyl ethers (step. 4).

AVCI ~ 312860 RHF:RCB/sm January 14, 1988 The methylated polysaccharide is hydrolyzed to the constituent monosaccharides (step 8), converted to the methylated alditols (step 9) and acetylated (step 10)~ These volatile derivatives are analyzed by ~LC/MS (step 13) and from the fragmentation patterns, the positions of the ~-methyl and 0-acetyl groups on the alditols are determined.

Illustration Consider a glycosyl (mannosyl) residue linked through positions 1 and 4 (ie a (1 ~ 4~ - linked mannan). The hydroxyl groups at positions 2, 3 and 6 are free and can be converted to methyl ethers. On hydrolysis and reduction the hydroxyl groups at positions 1, 4 and 5 are exposed and can be acetylated to yield the derivative 1, 4, 5-tri-O-acetyl-2, 3, 6-tri-O-methylhexitol (Figure 24). When examined by mass spectrometric technique, the dominant primary fragment ions are produced by cleavage between adjacent O-methyl groups or between O methyl and O-acetyl groups. The secondary fragment ions are produced by loss of acetic acid, methanol, etc.
~Figure 25). Figures 22 and 23 demonstrate the total ion chromatogram of Carrisyn* extract and the mass spectrum of 4-linked mannan as the partially acetylated mannitol respectively.
Based on the total ion chromatogram (TIC) of the Carrisyn* extract and the linkage analysis, it is deduced that Carrisyn* extract is mainly a (1 t 4)-linked linear polymer of mannose.

* trade mark RHF:RC~/sm January 14, 1988 A 32 year old patient, was presented with a history of ulcerative colitis for "many years". During an active epi-sode, she had been unresponsive to a daily regimen of 40mg Prednisone, 3 grams Asulfidine, 50 mg 6-mercaptopurine, and FlagylO She continued to have a pai~ful abdomen and 4-8 bloody bowel movements per day. She was placed on hyperali-mentation. Endoscopic findings revealed severe ascending colon ulcerations with mild hepatic to transverse ulcerations.
The patient was placed on 50 mg o Carrisyn* extract q.i.d. in addition to her other medications and sent home. In one week, her symptoms were virtually gone. The abdomen was mildly tender and endoscopy revealed a healed and mildly congested mucosa.
The patient was slowly taken off other medications and the clinical picture continued to improve. The patient iâ
currently maintained on Carrisyn* extract as the sole medication -at this time. Physical exam and symptom~ are recorded as totally normal.
Five additional cases with similar responses to ulcerative colitis and Crohn's disease have been seen. One patient ran out of Carrisyn* extract capsules. In four weeks, milc symptoms began to recur (there was increased stool with mild abdominal discomfort~, and she returned for a supply of medi-cation. In three days she was back to totally normal bowel symptomatology.

* trade mark - 7g -c A~CI 13~ 286~

RHF:RCB/sm January 14, 1988 A number of AIDS patients have received prolonged high doses of Carrisyn* extract without toxicity or side-efects.
rise in T-4 and T-8 lymphocyte ratios and an increase in abso-lute T-4 counts was seen in these AIDS patients with a reduc-tion and elimination of clinical symptoms, as well as a reduction in opportunistic infections. It is suggested that Carrisyn * extract had an anti-viral or immune modulation effect ir.
patients.
A stimulation to the lymphocytes of these patients has been ohserved which suggests that Carrisyn* extract may be involved in imm~ne modulation.

EXA~PLE 8 The Effect of Alcohol Concentration on Carrisyn* Extract Yield PROCEDURE:
- Hilltop leaves tl5.9 ibs.) were washed, filleted and ground in a Waring blender, then filtered through eight layers of cotton cloth. The gel was then transferred to four 11 quart, stainless steel pans, and cold USP grade ethyl alcohol was added to each in two, three, four and five to one ratios by volume. The amounts can be summarized as follows: ~

Ratio ~ethanol:aloe yel) Amt. of Gel Amt. of Ethyl Alcohol 2:1 500 ml 1000 ml 3:1 500 ml 1500 ml 4:1 1670 ml 66~0 ml 5-1 500 ml ~500 ml The precipitateS were allowed to settle out for four hours, then the remaining alcohol-gel solutiolls were carefully * ~rade mark c RHF:RCB/sm January 14, 1988 decanted and saved in separate containers. The precipitates were centrifuged for 10 minu~es at 2800 rpm using a IEC
Centra-7 centrifuge, washed with alcohol, then centrifuged again under the same conditions. The pellets were transferred to 600 ml jars, frozen in liquid nitrogen, and lyophilized overnight An additional volume of alcohol was added to the supernatant from ~he 2:1 ratio and allowed to settle overnight at room temperature. The remaining supernatants were also left at room temperature and allowed to settle out overnight.
The following day, the precipitates were collected from the supernates as previously described with the exception of the pellet from the 2:1 ratio that had been precipitated with an additional volume of alcohol. In this case, approxi-mately 5-10 ml of water was added as the pellet was trans-ferred to the lyophilization jar.
RESULTS
The results of the initial, four hour alcohol preci-pitations can be summarized as follows:

Ratlo (ethanol:aloe qel) yield(ql %Yield(q. Carrisvn*Extract/a.qel?
2:1 .0518 .010 3-1 .3847 .077 4:1 1.945 .11~
5:1 .6675 .134 After addition of another volume of ethyl alcohol, ; the 2:1 supernate produced another 178 mg of Carrisyn* extract.
Just by settling overnight, the 3:1 and 4:1 ratio supernates yielded another 89 and 105 mg, respectively. The 5:1 ratio * trade mark RHF,RCB/sm January 14, 1988 yielded only negligible precipitation after the initi~l isola-tion, and was thus not reharvested.
In the case of the second precipitation from the 2:1 ratio ~3:1), 5-10 ml of water were used to rinse out the centrifuge bucket bef~re lyophilization. This produced a white, fluffy Carrisyn ~ extract o~ low d~nsity that differed greatly from the denser, gray colored Carrisyn ~ extract samples that the other samples produced.

SUMM~RY
Carrisyn ~ extract is a purified white amorphous powder extracted from aloe vera mucilage. The polymer is essentially made up of linear ~ 1 ~ 4~-D-mannosyl units. It is a long chain polymer interspersed randomly with acetyl groups linked to the polymer through an oxygen atom. The degree of acety-lation is approximately 0.8 acetyl group/monomer as determined by the alkaline hydroxamate method (Hestrin, S.: J. Biol.
Chem., 180: 240 (1949)) the disclosure of which is hereby speciflcally incorporated herein by reference. Neutral su~ars linkage analysis indicates that attached to the chain, pro-bably through an ~ (2 - 6) linkage, is a ~-galactopyranose residue in the ratio of approximately one for every seventy sugars. The ratio of mannose to galactose of 20:1 indicates that galactose units are also linked together, primarily by ~~
(1~4) glycosidic bonds.
The chemical structure developed by utilizinq modern techniques of polysaccharide characterization is as follows:

AVCI
B5220CIP2 ~ 3 ~ 2 8 ~ a January 14, 1988 OAc -~ -D-Manp-(l ~ 4)-~-D-Manp-(1 ~ 4)-~-D-Manp-(1 ~ 4)-~-D-Manp-2 OAc -~-D-galp~ 4)-~-D-galp-Carrisyn* extract is polydispersed with at least 70% of the material having a molecular weight greater than 10,000 daltons.
Physical and Chemical Properties:
(a) Solubility Carrisyn* extract is a white to off-white amorphous powder which dissolves slowly in water to a highly viscous colloidal solution (0.4% w/v). With vigorous shaking for several hours, a 1~ (w/v) thick gel may be obtained. Carrisyn* extract is practically insoluble in common organic solvents including propylene glycol. However, Carrisyn* extract dissolves in 20 water and B0~ propylene glycol to a smooth, thick gel which remains sta~le indefinitely.
(b) pH:
A 0.2% (w/v) solution of Carrisyn in water has a pH
of approximately 6.31 + .33.
(c) Optical Rotation:

The specific rotation of a 0.2% (wJv~aqueous solu-tion of Carrizyn clarified by passing through a .45 um membrane filter (Uniflo~, Schleicher & Scheull Inc., Keene, NH) is:

[ ~ ]20 = -20 signifying a B-linkage. Deioni~ed water was used as the blank.
(d) Alkali Treatment:

* trade mark AVCI 13128~

RHF:RCB/sm January 14, 1988 Alkali treatment of Carrisyn causes deacetylationwhich destroys its ability to form a mucilaginous jelly, this indicates that the O-acetyla~ion of Carrisyn influences i~5 viscosity. The product of deacetylation does not dissolve in water apparently due to increased strong hydrogen bonding.
(e) Infrared Spectrosco~y:
The functional groups in Carrisyn are indentified by infrared spectroscopy (Fig~ 14 & lS). The strong IR absorp-tion bands near 1740 cm~l and 1250 cm~l signify acetylation.
Other absorptions located a~out 3422 cm~l, 1066 cm~l, 1639 cm~l, 897 cm~l, 806 cm~l and 773 cm~l are characteristic of polysaccharides with B-mannosyl linkages. Weak amide I and amide II absorptions are located about 1649 cm~l and 1541 cm~l respectively, (Fig. 14) these are due to protein impurities since when the Carrisyn~ is treated with protease and dialyzed, these peaks are absent (Fig. 15).
~f) Molecular Weight Distribution:
Carrisyn is polydispersed with at least 73~ of the material greater than 10 r 000 daltons as determined by size exclusion chromatography. An example, of a high pressure liquid chromatogram ~Fi~ure 13) demonstrates three fractions labeled A, B and C. Peak A represents Carrisyn greater than 100,000 daltons and peak B represents Carrisyn ~reater than 10 r 000 daltons but less than 100,000 daltons. Peak C repre-sents the molecular weight constituents o~ Carrisyn. The area of peaks B and C increase as Carrisyn decomposes which in turn causes a decrease in the area of Peak A.

It will be readily apparent to those in the art from the broad teaching of the invention and the illustrative 13128~

examples that there is the possibility of the substitution of unnamed chemicals and steps for the preferred enumerated che-micals and process steps; the lack of mention of such unnamed chemicals and steps does not indicate that they are not within the scope of the invention; but are omitted only because of the rPquirement that this teaching be concise a~d exact.

~ '

Claims (32)

AVCI

RHF:RCB/sm January 14, 1988

1. A process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant, comprising the steps of:
a) obtaining aloe juice having solubilized matter;
b) adjusting the pH or said aloe juice of from about 3.00 to about 3.50;
c) adding a water soluble, lower aliphatic polar solvent to the aloe juice to precipitate the active chemical substance and thereby to form a heterogeneous solution;
d) removing the water soluble, lower aliphatic polar solvent and the solubilized matter from the heteroge-neous solution to isolate the precipitated active chemical substance; and e) drying the precipitated active chemical substance.

AVCI

RHF:RCB/sm January 14, 1988

2. The process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant according to claim 1, wherein said aloe juice is obtained by:
a) washing an aloe leaf in a bacteriacidal solu-tion to remove substantially all surface dirt and bacteria;
b) removing at least a first end portion from said washed leaf;
c) draining, preserving and collecting anthra-quinone rich sap from said cut and washed leaf;
d) removing rind from said leaf to produce a substantially anthraquinone-free gel fillet; and e) grinding and homogenizing said substantially anthraquinone-free aloe gel fillet to produce substantially anthraquinone-free aloe juice having solubilized matter.

3. The process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant, according to Claim 1, wherein said aloe juice is obtained by:
a) washing an aloe leaf in a bacteriacidal solu-tion to remove substantially all surface dirt and bacteria;
b) crushing the washed aloe leaf; and c) dialyzing the crushed leaf chemically to remove and separate a substantially anthraquinone-free gel from remaining fractions of the crushed leaf.

AVCI

RHF:RCB/sm January 14, 1988

4. The process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant, according to Claim 1, wherein said aloe juice is obtained by:
a) washing an aloe leaf in a bacteriacidal solu-tion to remove substantially all surface dirt and bacteria;
b) crushing the washed aloe leaf; and c) grinding and homogenizing said crushed aloe leaf to produce aloe juice having solubilized matter,

5. The process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant, according to Claim 1, wherein said aloe juice is obtained by:
a) washing an aloe leaf in a bacteriacidal solu-tion substantially to remove surface dir. and bacteria;
b) grinding said washed aloe leaf;
c) filtering said ground aloe leaf to remove fibrous material; and d) homogenizing said ground aloe leaf to produce an anthraquinone-rich aloe juice having solubilized matter,

6. The process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant, according to Claim 1, wherein said aloe juice is obtained by:
a) crushing a leaf of an aloe plant to extrude aloe juice having solubilized matter.

AVCI

RHF:RCB/sm January 14, 1988

7. The process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant, according to Claim 1, wherein said aloe juice is obtained by:
a) washing an aloe leaf in a bacteriacidal solu-tion to remove substantially all surface dirt and bacteria;
b) removing rind from said leaf to produce an aloe gel fillet; and c) grinding and homogenizing said aloe gel fillet to produce aloe juice having solubilized matter.

8. The process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant, according to Claim 1, wherein said precipitate is irradiated, whereby said active chemical substance is sterilized and pre-served.

9. The process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant, according to Claim 1, wherein four volumes of said water soluble, lower aliphatic polar solvent are added to one volume of aloe juice to precipitate said active chemical substance.

10. The process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant, according to Claim 1, wherein said water soluble, lower aliphatic polar solvent is selected from the group consisting of methanol, ethanol and propanol.

AVCI

RHF:RCB/sm January 14, 1988

11. The process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant, according to Claim 10, wherein said water soluble, lower aliphatic polar solvent is ethanol.

12. The process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant, according to Claim 1, wherein said active chemical substance is precipitated from said water soluble, lower aliphatic polar solvent and aloe juice for approximately four hours before said water soluble, lower aliphatic polar solvent is removed.

13. The process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant, according to Claim 1, wherein said precipitated active comma-cal substance is dried by lyophilization.

14. The process for extracting the active chemical substance in the aloe plant from a leaf of the aloe plant, according to Claim 1, further comprising the steps of:
f) redissolving the precipitated active chemical substance in phosphate buffer, g) treating the redissolved precipated active che-mical substance with non-specific protease followed by exten-sive dialysis; and h) lyophilizing the non-filterable product.

15. A product produced by the process of Claim 1.

AVCI

RHF:RCB/sm January 14, 1988

16. A product produced by the process of Claim 2.

17. A product produced by the process of Claim 3.

18. A product produced by the process of Claim 4.

19. A product produced by the process of Claim 5.

20. A product produced by the process of Claim 6.

21. A product produced by the process of Claim 7.

22. A product produced by the process of Claim 8.

23. A product produced by the process of Claim 9.

24. A product produced by the process of Claim 10.

25. A product produced by the process of Claim 11.

26. A product produced by the process of Claim 12.

27. A product produced by the process of Claim 13.

; 28. A product produced by the process of Claim 14.

AVCI

RHF:RCB/sm January 14, 1988

29. Composition of matter, comprising:
a substantially non-degradable lyophilized polymer of linear .beta.-(1 ? 4)-D-mannosyl units wherein randomly interspersed acetyl groups are linked to the polymer through an oxygen atom and wherein D-galactopyranose is linked to the polymer through an .alpha.2-6) linkage at a ratio of about one D-galactopyranose residue per seventy monomer units.

30. Composition of matter according to Claim 29 wherein said polymer is irradiated with gamma or microwave radiation.

31. Composition of matter according to Claim 29, wherein the degree of acetylation is about 0.8 acetyl groups per monomer.

AVCI

RHF:RCB/sm January 14, 1988

32. Composition of matter, comprising:
a substantially non-degradable lyophilized linear polymer having a repeating monomer comprising:
wherein the degree of acetylation is 0.8 acetyl groups per polymer monomer; wherein the galactopyranose units are attached to the polymer at a ratio of approximately one per seventy monomer units; and wherein the mannose to galac-tose ratio is approximately 20:1.

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