Aloe vera cell walls contain a unique pectin (Ni etal, Chapter 4, this volume). This low-methoxy pectin contains up to 90% glucuronic acid. As a result it has unusual biological properties, such as the ability to bind and stabilize certain mammalian growth factors. Its presence in crude acemannan preparations may explain, in part, why these preparations can accelerate wound healing under certain circumstances (Tizard etal, 1994).
Other complex carbohydrates may be present in aloe gel extracts in small amounts and some of these may exert significant biological activity. For example Pugh and his colleagues (Pugh etal., 2000) have identified a high molecular weight polysaccharide from A. vera (aloeride) that is a very potent macrophage activating agent. Its molecular weight may be as large as 7 million. It contains glucose, galactose, mannose and arab-inose and it is as potent as bacterial endotoxin at activating nuclear factor (NF)-kB in human macrophages. Aloeride also induces the expression of the mRNAs encoding Il(interleukin)-1P and TNF (tumor necrosis factor)-a to levels equal to those observed in cells maximally activated by bacterial endotoxin. Thus, although aloeride constitutes only 0.015% of dry weight of aloe gel juice, its potency may fully account for the macrophage stimulating activity of this juice. Limited studies have been conducted on the carbohydrates of other Aloe species (Vilkas and Radjabi-Nassab, 1986; Radjabi-Nassab etal, 1984; Yagi etal, 1977). They are discussed in detail in Chapter 4 by Ni etal, in this volume.
While the leaf gel consists primarily of a complex carbohydrate mixture, the green plant rind contains many complex organic compounds such as chromones, flavonoids and anthraquinones. The precise composition varies greatly between aloe species (Viljoen and van Wyk, 1988). Some of these molecules, especially the chromones and flavonoids, can have significant anti-inflammatory activity (Read, 1995) or antiviral activity (Andersen etal, 1991).
The ability of aloe leaf gels to reduce the severity of acute inflammation has been evaluated in many different animal models (Adler etal, 1995; Davis etal, 1989; Beatriz etal, 1996; Davis and Maro, 1989; Davis etal, 1994a; Davis etal, 1994b; Saito etal, 1982; Vazquez etal, 1996). For example, Adler studied inflammation in the hind paw of the experimental rat induced by kaolin, carrageenan, albumin, dextran, gelatin and mustard (Adler etal., 1995). Of the various irritants tested, A. vera was especially active against gelatin-induced and kaolin-induced edema and had, in contrast, minimal activity when tested against dextran-induced edema. Ear swelling induced by croton oil has also been used as an assay (Davis etal., 1987). The swelling induced by croton oil on a mouse ear is significantly reduced by application of an aloe gel. In addition, soluble acemannan-rich extracts administered either orally or by intraperitoneal injection to mice will also reduce this swelling (Bowden, 1995). In another model, the acute pneumonia induced in mouse lungs by inhalation of a bacterial endotoxin solution is significantly reduced by systemic administration of an aloe carbohydrate solution (Bowden, 1995). In both these cases the reduction in inflammation is associated with a significant reduction in tissue infiltration by neutrophils. In general, aloe free of anthraquinones was more effective than aloe with anthraquinone. Some of this anti-inflammatory activity is due to the activities of bradykininases (Fujita etal., 1976; Yagi etal., 1987).
Another model that has been studied is radiation-induced acute inflammation in mouse skin (Roberts and Travis, 1995). Male mice received graded single doses of gamma radiation and aloe gel was applied daily beginning immediately after irradiation and continuing for up to five weeks. The severity of the radiation reaction was scored and dose-response curves were obtained. It was found that the average peak skin reactions of the aloe-treated mice were lower than those of the control mice at all radiation doses tested. Thus the radiation ED50 values for skin reactions of 2.0—2.75 were approximately 7 Gy higher in the gel-treated mice. The average peak skin reactions and the ED50 values for mice treated with lubricating jelly or aloe gel were similar to irradiated control values. Reduction in the percentage of mice with severe skin reactions was greatest in the groups that received aloe gel for at least two weeks beginning immediately after irradiation. There was no effect if gel was applied only before irradiation or beginning one week after irradiation. Aloe gel, but not lubricating jelly, reduced acute radiation-induced skin reactions in C3H mice if applied daily for at least two weeks beginning immediately after irradiation. This experiment can, however, be criticized on the grounds that an inappropriate control substance was used. The acemannan effect should have employed an identical gel lacking acemannan as control (excipient) since there were many other components in the gel in addition to acemannan.
Acetylated mannans from the pulp of A. saponaria (As mannans) (Yagi etal, 1984) have also been shown to be anti-inflammatory. Thus a P1 ^4-linked D-mannopyranose containing 18% acetyl groups inhibited carrageenin-induced hind paw edema at 50mg/kg intraperitoneally in rats. A crude preparation of both As mannans was effective when given intraperitoneally, but not when given orally.
The effects of aqueous, chloroform, and ethanol extracts of A. vera gel on carrageenan-induced edema in the rat paw, and neutrophil migration into the peritoneal cavity stimulated by carrageenan has also been studied (Stuehr and Marletta, 1987), as has the ability of the aqueous aloe extract to inhibit cyclooxygenase activity. The aqueous and chloroform extracts decreased the edema induced in the hind-paw and the number of neutrophils migrating into the peritoneal cavity, whereas the ethanol extract only decreased the number of neutrophils. The aqueous extract inhibited prostaglandin E2 production from [14C] arachidonic acid. These results demonstrated that the extracts of A. vera gel have anti-inflammatory activity and suggested that some of this activity at least was due to an inhibitory action on the arachidonic acid pathway via cyclooxygenase.
A similar experiment has been conducted using an A. vera extract treated with 50% ethanol. The resulting supernatant and precipitate were tested for anti-inflammatory activity using the croton oil-induced ear-swelling assay (Davis etal., 1991). The supernatant decreased inflammation, when applied topically, by 29.2%, while the precipitate decreased inflammation by 12.1%.
The mechanisms by which aloe extracts exert anti-inflammatory effects are multiple, and several distinct pathways have been described. For example, some evidence suggests that the activity is due to gibberellins. Thus the anti-inflammatory activities of A. vera and gibberellin were measured in streptozotocin-induced diabetic mice by measuring the inhibition of polymorphonuclear leukocyte infiltration into a site of gelatin-induced inflammation (Davis and Maro, 1989). Both aloe and gibberellin similarly inhibited inflammation in a dose-response manner. These data were interpreted to suggest that gibberellin or a gibberellin-like substance is an active anti-inflammatory component in A. vera. A second possible mechanism is due to antibradykinin activity. Thus a fraction with antibradykinin activity has been partially purified from the pulp of A. saponaria by gel chromatography (Yagi etal, 1982). The antibradykinin-active material was probably a glycoprotein that cleaved the Gly4-Phe5 and Pro7-Phe8 bonds of the bradykinin molecule. A third possible mechanism may be due to complement depletion ('t Hart etal., 1988). Thus an aqueous extract of Aloe vera gel was fractionated into high (h-Mr) and low (l-Mr) molecular weight fractions by dialysis. Subsequent fractionation generated two fractions with molecular weights of 320 and 200 kDa. Preincubation of human pooled serum with these fractions resulted in a depletion of classical and alternative pathway complement activity. The inhibition appeared to be due to alternative pathway activation, resulting in consumption of C3 ('t Hart etal., 1989). The active fractions were mannose-rich polysaccharides.
A fourth possible mechanism may relate to the fact that mannose-rich carbohydrate solutions inhibit the activity of certain P2 integrins and hence block neutrophil emigration into inflamed tissues (Bowden, 1995). Aloe carbohydrate solutions inhibit swelling in the mouse ear model and reduce the inflammation in a mouse lung endo-toxin model. Histological staining and tissue myeloperoxidase assays show that treated tissues contain significantly fewer neutrophils than untreated control tissues. Static neutrophil adherence assays demonstrate that acemannan enriched fractions can inhibit adherence of human neutrophils to human endothelial cells. Flow adherence assays have demonstrated that this solution has no effect on leukocyte rolling (a selectin-mediated phenomenon) but does inhibit complete adherence and transmigration (mediated by integrins). By using recombinant endothelial cell lines it can be shown that the acemannan solution has no effect on selectin-mediated adherence but can inhibit adherence to the integrins MAC(macrophage)-1 (CD11b) and leucocyte function-associated antigen (LFA)-1 (CD11a). It inhibits LFA-1-mediated adherence at concentrations at least 50-fold less than required to inhibit MAC-1 mediated adherence. These reactions are not a result of neutrophil activation.
Aloe gels also contain low molecular weight components (dialysates) that can inhibit the release of reactive oxygen and hydrogen peroxide by stimulated human neutrophils ('t Hart etal, 1990). The compounds inhibited the oxygen-dependent extracellular effects of neutrophils, such as lysis of red blood cells, but did not affect the ability of the neutrophils to phagocytose and kill microorganisms. The inhibitory activity of these compounds was most pronounced on the PMA(phorbol 12-myristate 17-acetate)-induced oxygen production, but this was antagonized by a Ca-ionophore, suggesting that the effect was mediated by reduced intracellular free calcium.
Aloe-based carbohydrates can activate macrophages. Consequently they stimulate antigen-processing, non-specific immunity, wound healing and resistance to infection and neo-plasia. Thus macrophages from normal animals are relatively quiescent, but can be readily activated and acquire the ability to kill tumor cells or certain microorganisms (Tizard etal, 1989; Tizard etal, 1994). Macrophage activation can be mediated by several different pathways. For example, one major pathway is through T cells secreting the Th1 cytokines, interferon-y (IFN-y) and interleukin-2 (IL-2) (Bielefeldt-Ohmann and Babiuk, 1986). IFN-y is a potent macrophage-activating agent and it is especially effective when supplemented by exposure to microbial products such as endotoxins, muramyl dipeptide or cell wall carbohydrates (glucans, mannans) (Adams and Hamilton, 1984; Lackovic etal., 1970.). Thus, activation is a multi-stage process. For example, inflammatory macrophages may first be primed by interferon. In a second step bacterial products or complex carbohydrates can activate these primed macrophages. Macrophages can destroy some tumor cells only after treatment with both recombinant IFN-y and bacterial lipopolysaccharide (LPS), suggesting that at least two stimuli are required for complete activation (Drysdale etal, 1988). One of the most marked biological activities of mannans in mammals is the activation of macrophages and stimulation of T cells (Inoue etal, 1983; Inoue etal, 1983; Tizard etal, 1989). It has been shown that each of these molecules interacts with specific high affinity receptors located on the macrophage plasma membrane (Lorsbach etal, 1993).
Acemannan immunostimulant (AI) is a commercially available, partially purified carbohydrate preparation containing about 60% acetylated mannan together with other carbohydrates, especially pectins and hemicelluloses. It should not be confused with the complex carbohydrate acemannan. AI can activate macrophages (Zhang and Tizard, 1996; Merriam etal., 1996; Chinnah, 1990). This macrophage activating ability is probably responsible for its activity as an adjuvant (Chinnah etal, 1992; Chinnah, 1990), its pro-wound-healing activity (Tizard etal., 1994), as well as its anti-tumor (Peng etal, 1991) and anti-viral activity (Chinnah, 1990; Kahlon etal, 1991; Sheets etal, 1991; Yates etal, 1992).
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