Aloes and the immune system: Specific activities

2015

Anti-inflammatory effects

The ability of aloe leaf gels to reduce the severity of acute inflammation has been evaluated in many different animal models. For example, Adler studied inflammation in the hind paw of the experimental rat induced by kaolin, carrageenan, albumin, dextran, gelatin and mustard. Of the various irritants tested, Aloe 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. 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. 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. 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.

Another model that has been studied is radiation-induced acute inflammation in mouse skin. 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) have also been shown to be anti-inflammatory. Thus a β1→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 Aloe vera gel on carrageenan-induced edema in the rat paw, and neutrophil migration into the peritoneal cavity stimulated by carrageenan has also been studied, 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 Aloe 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 Aloe 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. 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 Aloe 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. 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 Aloe 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. 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). 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. 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 β2 integrins and hence block neutrophil emigration into inflamed tissues. Aloe carbohydrate solutions inhibit swelling in the mouse ear model and reduce the inflammation in a mouse lung endotoxin 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. 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.

Macrophage activation

Aloe-based carbohydrates can activate macrophages. Consequently they stimulate antigen-processing, non-specific immunity, wound healing and resistance to infection and neoplasia. Thus macrophages from normal animals are relatively quiescent, but can be readily activated and acquire the ability to kill tumor cells or certain microorganisms. Macrophage activation can be mediated by several different pathways. For example, one major pathway is through T cells secreting the Th1 cytokines, interferon-γ (IFN-γ) and interleukin-2 (IL-2). IFN-γ 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). 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-γ and bacterial lipopolysaccharide (LPS), suggesting that at least two stimuli are required for complete activation. One of the most marked biological activities of mannans in mammals is the activation of macrophages and stimulation of T cells. It has been shown that each of these molecules interacts with specific high affinity receptors located on the macrophage plasma membrane.

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. acemannan immunostimulant can activate macrophages. This macrophage activating ability is probably responsible for its activity as an adjuvant, its pro-wound-healing activity, as well as its anti-tumor and anti-viral activity.

Calcium flux

The first step in the macrophage activation process involves endocytosis of aloe carbohydrate and a rise in intracellular calcium. Thus acemannan immunostimulant can be observed within the cytoplasm of cultured macrophages as apple green fluorescence within an hour after exposure to fluorescein-labeled carbohydrate solution. When the location of this fluorescence is compared to labels for mitochondria and lysosomes, it shows greater than 98% correlation with lysosomal distribution. A significant increase in intracellular Ca can be detected in macrophages following exposure to 50 µg/ml AI. The Ca flux occurs within seconds of addition of acemannan immunostimulant solution and appears as a single spike followed by a return to basal levels in less than one minute. No Ca stimulatory activity can be detected in response to the pellet derived from centrifuged AI. Since this pellet consists of plant cell wall fragments rich in pectin it is likely that the calcium flux is not simply due to ingestion of cell fragments by phagocytosis. When macrophage cultures are pretreated with the calcium chelating agent EGTA, the AI-induced Ca response is completely abolished suggesting that the response to acemannan immunostimulant requires extracellular Ca. An acemannan immunostimulant solution can increase intracellular Ca levels, not only in macrophages but also in uterine smooth muscle but not in liver epithelial cells. Free intracellular Ca plays a pivotal role as a second messenger involved in signal transduction, and as the initiating step in macrophage activation.

Anti-tumor applications

Given that acemannan immunostimulant is a macrophage stimulating agent, several investigators have examined its anti-tumor effects. For example, using the implanted Norman murine sarcoma as a model, mice treated with acemannan immunostimulant showed diminished tumor growth rates and increased mouse survival. Maximum survival occurred in mice receiving a single injection of 0.5 mg/kg acemannan immunostimulant i.p. at the time of tumor implantation. Tumor growth was apparently normal for 12 days after implantation. Decreased tumor growth was apparent between 12 and 15 days. Thirty-five per cent of treated animals survived by 60 days while all untreated animals were dead by 46 days. The effect of the acemannan immunostimulant was time critical. Thus it was less effective if administered 24 hours before or after implantation and had no significant effect if given 48 hours before or after implantation. Histopathology of tumors from treated animals showed extensive areas of edema, leukocytes (especially mononuclear) infiltration, necrosis and hemorrhage. Eventually the tumors were walled off by significant fibrosis. Animals which recovered from a Norman murine sarcoma transplant rejected subsequent tumor transplants. However, it is unclear how acemannan immunostimulant exerts this wide variety of effects and we believe that some of these effects are mediated through the macrophages. The combined data suggest that AI-stimulated synthesis of monokines resulted in the initiation of immune attack, necrosis, and regression of implanted sarcomas in mice. This experiment may, however, have been influenced by low levels of endotoxin in the acemannan immunostimulant preparation.

The effect of Aloe vera administration was studied on a pleural tumor in rat. The growth of Yoshida ascites hepatoma (AH)-130 cells injected (2 ×10(5) in 0.1 ml) into pleura of male inbred Fisher rats was evaluated at different times (7th and 14th days). Winters and his colleagues have demonstrated that lectins from Aloe vera and A. saponaria were cytotoxic for both normal and tumor cells in vitro.

AI is employed clinically for the treatment of fibrosarcomas in dogs and cats. Studies in vitro indicate that acemannan immunostimulant has limited anti-viral activity against herpes viruses, measles, and human immunodeficiency virus. It is an immunostimulant and is licensed by the United States Department of Agriculture for the treatment of fibrosarcoma in dogs and cats. In a pilot study, acemannan immunostimulant was administered intralesionally and intraperitoneally to 43 dogs and cats suffering from a variety of spontaneous neoplasms. Of seven animals with fibrosarcomas, five showed some sign of clinical improvement such as tumor shrinkage, tumor necrosis or both following acemannan immunostimulant treatment. In an additional study four dogs and six cats with recurrent fibrosarcoma that had failed previous treatment were treated with AI. Tumors in two of the animals shrank and disappeared. In eight animals, the tumors became edematous and enlarged rapidly. Necrosis and lymphocyte infiltration was seen in all of them. Survival times ranged from 57 to greater than 623 days with a mean tumor-free interval of 229 days. In another study, dogs and cats with histopathologically confirmed fibrosarcomas were treated with acemannan immunostimulant in combination with surgery and radiation therapy. Following four to seven weekly injections of AI, tumor shrinkage occurred in four of these animals. On histology there was a notable increase in necrosis and inflammation. Following surgery and radiation and monthly acemannan immunostimulant treatment, seven of 13 animals remained alive and tumor free for 440 to 603 days. These results suggested that treatment with acemannan immunostimulant was an effective and useful adjunct to surgery in these animals.

Wound Healing

AI has been reported to promote healing of aphthous ulcers in humans and to accelerated wound healing of biopsy-punch wounds in rats. Acemannan immunostimulant enhances wound healing in rats. Macrophages play a crucial role in wound healing, as it is involved in both the inflammatory and debridement phases. Macrophage-derived nitric oxide modulates angiogenesis. Macrophages are a source of growth factors and play the most important role in removing damaged tissues. Animals that lack macrophages fail to heal. In general, it is difficult to accelerate the healing of uninfected wounds in healthy young animals, given that their healing process is fully functional and probably proceeding at maximal speed. However, that is not the case in old animals. Wound healing slows steadily as an individual animal ages and takes approximately 50% longer, three as opposed to two weeks, in rats over two years of age. Using this model and administering acemannan immunostimulant by local injection it is possible to accelerate this time course so that treated wounds will heal in two weeks. It is possible therefore that this effect is due to macrophage activation. Activated macrophages influence fibroblast function and promote the deposition of collagen in wounds. On the other hand, it is equally likely that the effect is due to growth factor stabilization by the pectins in the mixture.

The effects of Aloe vahombe (sic)

In an extensive series of papers published in the local literature, Ralamboranto and his colleagues have cataloged a remarkable series of effects of A. vahombe (=A. vaombe Decorse et Poiss.), a plant endemic in the south of Madagascar. Because it is not generally available to other investigators, it is difficult to determine the significance of this plant. For example, a partially purified extract of leaves of A. vahombe, administered intravenously to mice, protects them against infection of the bacteria Listeria monocytogenes, Yersinia pestis, Plasmodium berghei and the yeast Candida albicans (). The protective fraction must be administered two days before inoculation of the pathogenic agent. In addition, when the mice were injected with an unrefined extract from A. vahombe, they were protected against Klebsiella septicaemia (). Neither bactericidal nor bacteriostatic activity has been detected in this aloe extract. Nevertheless, the anti-infectious activity was proportional to the dose of extract injected, and the protective activity was the greatest when the mice were treated with aloe two or three days prior to infection.

A fraction extracted from A. vahombe, was studied for its effect on experimental fibro-sarcomas and melanomas in mice. ‘Cures’ were observed only in the case of the McC3-1 tumor but the rate of growth of tumors in animals which were treated was slower than in untreated animals. The active fraction was identified as a water soluble, thermostable, polysaccharide with a molecular weight of more than 30kDa.

 

Selections from the book: “Aloes. The genus Aloe”. Edited by Tom Reynolds. Series: “Medicinal and Aromatic Plants — Industrial Profiles”. 2004.