International Immunopharmacology 5 (2005) 271 279 www.elsevier.

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Identification of optimal molecular size of modified Aloe polysaccharides with maximum immunomodulatory activity
Sun-A Ima, Sun-Tack Oha, Sukgil Songa, Mi-Ran Kimb, Dong-Seon Kimb, Sung-Sick Woob, Tae Hyung Job, Young In Parkc, Chong-Kil Leea,*
College of Pharmacy, Chungbuk National University, Cheongju 361-763, South Korea b Uunigen Inc., Cheonan 330-863, South Korea c School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea Received 29 June 2004; received in revised form 26 September 2004; accepted 27 September 2004
a

Abstract Polysaccharides isolated from the gel of Aloe species have been known to have diverse biological activities, including immunomodulatory and antitumor activities. The molecular sizeimmunomodulatory activity relationship of modified Aloe polysaccharide (MAP) was examined in this study. Crude MAP (G2E1) was prepared from the gel of Aloe vera that was partially digested with cellulase. Proteins in crude MAP were removed by passage through a DEAE-Sephacel column, and then the protein-free MAP (G2E1D) was further separated into three fractions, G2E1DS3 molecular weight (MWz400 KDa), G2E1DS2 (5 KDaVMWV400 KDa), G2E1DS1 (MWV5 KDa), by Sephacryl column chromatography and ultrafiltration. Immunomodulatory activities of MAP preparations were examined on a mouse macrophage cell line, RAW 264.7 cells, and in ICR strain of mouse implanted with sarcoma 180 cells. We found that polysaccharides between 400 and 5 KDa exhibit the most potent macrophage-activating activity as determined by increased cytokine production, nitric oxide release, expression of surface molecules, and phagocytic activity. In accordance with the in vitro activity, polysaccharides between 400 and 5 KDa also exhibited the most potent antitumor activity in vivo. D 2004 Elsevier B.V. All rights reserved.
Keywords: Aloe vera; Polysaccharide; Macrophage activation; Antitumor activity

1. Introduction
Abbreviations: Da, dalton; IL, interleukin; LPS, lipopolysaccharide; MAP, modified Aloe polysaccharide; TNF-a, tumor necrosis factor-a; MHC, major histocompatibility complex; MW, molecular weight. * Corresponding author. Tel.: +82 43 261 2826; fax: +82 43 268 2732. E-mail address: cklee@chungbuk.ac.kr (C.-K. Lee). 1567-5769/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2004.09.031

Polysaccharides isolated from the gel of Aloe vera have been known to have diverse immunomodulatory activities in vivo, as well as in vitro (reviewed in Ref. [1]). Acemannan, a mixture of various length polymer chains of h-(1,4)-linked acetylated galactomannan, may be the most well-characterized polysaccharide

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isolated from the gel of A. vera [2]. When administered intraperitoneally to tumor-bearing animals, acemannan was shown to cure completely or reduce the tumor burden significantly [35]. The antitumor activity of acemannan appears to be mediated via activation of the host defense system. Acemannan has been shown to increase lymphocyte responses to alloantigens in vitro [6,7], activate macrophages to produce inflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor-a (TNF-a) [8], increase NO production by macrophages [911], and up-regulate phagocytic and candidicidal activities of macrophages [12]. Acemannan has also been shown to augment hematopoiesis [13] and induce maturation of immature dendritic cells [14]. Total polysaccharide content of fresh native Aloe gel is approximately 10% by dry weight, and the average molecular weight (MW) of native polysaccharides in Aloe gel is thought to be 2 million Da or above. The types and molecular sizes of the polysaccharides isolated from Aloe gel appear very diverse (reviewed in Ref. [1]). Diversity in the type and molecular size of polysaccharide may be due to differences in plant subspecies or different geographical origin. In addition, heterogeneity in molecular size may also result from technical differences used to isolate the polysaccharide, or degradation of polysaccharides by endogenous enzyme activity. Although there is a general consensus on the diverse immunomodulatory activities of the polysaccharide fraction isolated from Aloe gel, the optimal molecular size exhibiting maximum immunomodulatory activity is not clear. Acemannan (Carrisynk), which has been shown to have multiple therapeutic properties, including wound healing and immunomodulating activities, was initially reported as h-(1,4)-acetylated mannan polymers with an average molecular weight of 80 KDa [2]. An injectable form of acemannan, CARN 750, developed from Carrington Laboratories (Irving, TX), consists of acemannan polysaccharide polymers with an average MW of 1 million Da. More recently, another form of high molecular weight polysaccharide, aloeride, was isolated from Aloe gel. Aloeride was between 4 and 7 million Da and was shown to have potent immunostimulatory activity [15]. The same authors claimed that the immunomodulatory activity of acemannan preparation was attributed to

trace amounts of aloeride [16]. In contrast, much smaller form of highly acetylated polysaccharide, termed as modified Aloe polysaccharide (MAP), was isolated from cellulase-treated Aloe gel and was reported to have much stronger immunomodulatory activities than native 2 million Da polysaccharides [16]. MAP consists of polysaccharides with an average MW of 80 KDa. Other active polysaccharides isolated from Aloe gel include a 70 KDa polysaccharide [17] and polysaccharides between 420 and 520 KDa [18]. To determine optimal molecular size of MAP exhibiting maximum immunomodulatory activity, polysaccharides were isolated from the gel of A. vera that was partially digested with cellulase, and then fractionated according to their molecular size. Examination of macrophage-activating activity in vitro and antitumor activity in vivo showed that polysaccharides between 400 and 5 KDa exhibit the most potent immunomodulatory activity.

2. Materials and methods 2.1. Isolation and characterization of MAP The lyophilized cellulase-treated Aloe gel used in this study was dA. vera gel 200:1 concentrateT, prepared in 2002 from mature leaves of A. vera on an industrial scale by Aloecorp (Harlingen, TX, USA). The basic processing methodology of the dA. vera gel 200:1 concentrateT, which involves incubation of Aloe gel with cellulase, termination of the reaction by heating, and then passage through a charcoal column to remove anthraquinones and other colored substances, was described in detail in an earlier report [16]. Crude MAP was prepared from the lyophilized Aloe gel as described previously with minor modifications [16]. Briefly, the lyophilized Aloe gel was extracted with 80% ethanol for 1 h. The extract was then centrifuged to collect precipitates, hereafter referred to as G2E1. The freeze-dried G2E1 was dissolved in 20 mM TrisHCl (pH 7.4), applied to a DEAE-Sephacel column (Amersham Pharmacia Biotech), and then eluted with the same buffer. The unbound polysaccharide (G2E1D) was concentrated at 55 8C in vacuum, and then dried by a freeze-dryer. Total protein contents

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in G2E1 and G2E1D were determined by microbicinchoninic acid assay kit (Pierce, Rockford, IL) according to the manufacturers instructions, using bovine serum albumin as standard protein and were 3.65% and 0.21%, respectively. For further fractionation, G2E1D was dissolved in distilled water and then applied to a Sephacryl S400 HR column (Amersham Bioscience) with distilled water as eluent. Molecular weight of the polysaccharides in fractions obtained from S400 HR column chromatography was determined by comparing their retention time with that of broad dextran standards, MW 7.2, 16.23, 230, and 520 KD (Phenomenex, Torrance, CA), in Biosep-SEC columns (S4000 and S3000, Phenomenex). Polysaccharides or dextran standards in the column were developed with distilled water and then detected with a refractive index detector. The fractions from S400 HR column were pooled into two fractions, polysaccharides with MW larger than 400 KDa (G2E1DS3) and polysaccharides with MW smaller than 400 KDa. The polysaccharide fraction with MW smaller than 400 KDa was further fractioned into two subfractions, polysaccharides between 400 and 5 KDa (G2E1DS2) and polysaccharides smaller than 5 KDa (G2E1DS1) using an ultrafiltration cell (Millipore). Polysaccharide fractions separated according to their molecular size were concentrated at 55 8C in vacuum and then freezedried. Endotoxin levels in these fractions were determined at a concentration of 1000 Ag/ml by a limulus amoebocyte assay (sensitivity 0.125 ng/ml; Associates of Cape Cod, Woods Hole, MA, USA) and found to contain less than 0.125 ng/ml. The yield of each fraction was as follows: G2E1, 50.3%; G2E1D, 39.7%; G2E1DS1, 5.6%; G2E1DS2, 2.6%; G2E1DS3, 12.5%. 2.2. Cell culture RAW 264.7 cells were cultured in Dulbeccos modified Eagles medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone), 100 U/ml penicillin, and 100 Ag streptomycin (Invitrogen), and 50 AM 2-mercptoethanol (Invitrogen) at 37 8C, 5% CO2 condition. 2.3. Proliferation assay RAW 264.7 cells were cultured in 96-well plates (2104 cells/well) in the presence or absence of MAP

for 2 days. DNA synthesis was measured by [3H]thymidine (Du Pont, 0.5 ACi/well) incorporation for the final 6 h of the culture period. 2.4. Phenotypic analysis Cells were stained with monoclonal antibodies recognizing murine cell surface markers as described previously [14], and flow cytometric analysis was performed on a FACS Caliver (Becton-Dickinson). The monoclonal antibodies, anti-CD40 (clone 3/23), anti-I-Ab (clone AF6-120.1), anti-B7-1(clone 1610A1), anti-B7-2 (clone GL1), and isotype-matched control antibodies were purchased from Pharmingen (San Diego, CA). Dead cells were gated out by their low forward angle light scatter intensity. 2.5. Phagocytic activity RAW 264.7 cells were cultured in 6-well plates (2106 cells/well) in the presence or absence of MAP for 2 days, and then added with biodegradable microspheres (average diameter, 300 nm) containing both ovalbumin (OVA) and fluorescein isothiocyanate (FITC). After 2 h, unphagocytozed microspheres were removed by washing with prewarmed PBS. Cells were then harvested by pipetting after cooling on ice for 20 min, fixed in 1% paraformaldehyde in PBS, and flow cytometric analysis was performed on a FACS Caliver. Microspheres containing both OVA and FITC were prepared by a solvent-evaporation method [19]. Briefly, OVA was dissolved in 3% polyvinyl alcohol (final, 4 mg/ml), and poly(l-lactic acid) (PLG, final, 5%), and FITC (final, 5 mg/ml) were dissolved in a mixture of acetone and ethanol (9:1). These two solutions, 150 ml of OVA solution and 30 ml of PLG solution, were mixed slowly and emulsified by a continuous stirring for overnight at room temperature. The hardened microspheres were collected by centrifugation at 300g, and washed twice with PBS. 2.6. Cytokines and nitric oxide production RAW 264.7 cells were cultured in 24-well plates (5105 cells/well) in the presence or absence of MAP for 2 days. The amounts of IL-1h and TNF-a in the culture supernatants were measured using commercially available ELISA kits (R&D System). The

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amount of nitrite was measured using Griess reagent (1% sulfanilamide/0.1% naphthylenediamine dihydrochloride/2.5% H3PO4). The standard curve for nitrite was generated using known concentrations of sodium nitrite. 2.7. Antitumor activity MAP was injected into peritoneum of ICR strain of mouse (1 mg/mouse) once a day for six consecutive days. On day 4 from the initiation of MAP injection, sarcoma 180 cells that were maintained in the peritoneal cavity of ICR mouse were collected, washed, and then injected into peritoneum of MAPtreated mouse (4105 cells/mouse). Four days after the injection of sarcoma 180 cells, mouse was sacrificed, and then all the cells in the peritoneum were collected by washing the peritoneum with 5 ml of ice-cold PBS three times. The harvested cells were washed with PBS, treated with ACK lysis buffer for 3 min to remove contaminating red blood cells, and then stained with FITC-conjugated anti-CD45 monoclonal antibody (Pharmingen). Flow cytometric analysis was performed on a FACS Caliver (Becton-Dickinson). 2.8. Removal of endotoxin Contaminants of endotoxin possibly associated with the MAP preparations were removed using Affi-Prep Polymyxin Matrix (BIO-RAD). Briefly, 1 ml of AffiPrep Polymyxin Matrix was packed in a Bio-spin column (BIO-RAD), centrifuged for 1 min at 30g, and then added with 0.5 ml of MAP (10 mg/ml). The column was incubated overnight at 4 8C, and then MAP was recovered from the Bio-spin column by centrifugation at the same condition. To ensure the performance of the affinity column, lipopolysaccharide (LPS; 10 mg/ml) was treated with Affi-Prep Polymyxin Matrix column by the same methods, and then added to cultures of RAW 264.7 cells at a concentration of 100 ng/ml as calculated from the initial concentration. 3. Results 3.1. Activation of macrophage by MAP Crude MAP (G2E1) was prepared from cellulasetreated A. vera gel by extraction with 80% ethanol,

and then proteins in crude MAP were removed by an anion exchange resin to obtain protein-free MAP (G2E1D). Immunomodulatory activity of G2E1 and G2E1D was examined using a mouse macrophage cell line, RAW 264.7 cell. As shown in Fig. 1a, G2E1D inhibited the growth of RAW 264.7 cells dosedependently. The growth-inhibitory activity of G2E1D was not due to cytotoxicity of MAP on RAW 264.7 cells. Instead, G2E1D appeared to induce further differentiation/activation of slightly adherent RAW 264.7 cells into strongly adherent mature macrophages with spread cytoplasm. It has been known that mature macrophages are end-stage cells in myeloid differentiation and do not proliferate in response to antigenic or mitogenic stimulus. Thus, it is reasonable to guess that the growth-inhibitory activity of G2E1D is due to differentiation-inducing activity on RAW 264.7 cells, although this point is not the focus of this study. Stimulatory activity of G1E2D on macrophages was further demonstrated by dose-dependent increase in TNF-a production (Fig. 1b), IL-1h production (Fig. 1c), and nitric oxide release (Fig. 1d) by RAW 267.4 cells cultured with G2E1D. It is noteworthy that macrophage-activating activity of crude MAP preparation, G2E1, was much weaker than that of protein-free MAP, G2E1D, suggesting that there were impurities inhibiting macrophageactivation in G2E1. The stimulatory activity of G2E1D on macrophage was not due to LPS contamination. As shown in a representative result in Fig. 1e, incubation of G2E1D with polymyxin Baffinity matrix did not reduce the macrophageactivating activity. The ability of polymyxin Baffinity matrix in removing of LPS was tested with 10 mg/ml of LPS solution, and the result was shown in the left panel of Fig. 1e. Because increased expression of costimulatory molecules could be a marker of macrophage activation, we examined the effect of MAP on the expression of costimulatory molecules on RAW 264.7 cells. As shown in Fig. 2, G2E1D at a concentration of 100 Ag/ml increased the expression of B7-1 and CD40 significantly. Crude MAP preparation, G2E1, exhibited only marginal increases of B7-1 and CD40. One of the most distinguished features of macrophage activation would be an increase in phagocytic

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Fig. 1. Immunomodulatory activity of MAP on RAW 264.7 cells. RAW 264.7 cells were cultured in the presence of G2E1 or G2E1D for 2 days, and then proliferation of RAW 264.7 cells was measured by 3H-thymidine incorporation for the final 6 h (a). The culture supernatants collected after 2 days of culture were assayed for TNF-a (b), IL-1h (c), and nitric oxide (d). The final concentration of G2E1 and G2E1D in (a) through (d) was 0.8, 4, 20, 100, 500, and 1000 Ag/ml, respectively. G2E1 and G2E1D were treated with polymyxin B-affinity column to remove possible contaminants of endotoxin and then added to cultures of RAW 264.7 cell (100 Ag/ml). The culture supernatants collected after 2 days of culture were assayed for TNF-a-inducing activity (e). LPS was used as a control to ensure the performance of polymyxin B-affinity column.

activity. Effects of MAP on phagocytic activity were examined after culturing RAW 264.7 cells with MAP for 2 days. Thus, in the experiment, cultured cells were allowed to phagocytize FITC-labelled microspheres (average diameter, 300 nm) for 2 h, washed, and then the amount of phagocytized microspheres was analyzed by flow cytometry. We found that G2E1D, but not G2E1, increased phagocytic activity strongly (Fig. 3).

3.2. Molecular sizemacrophage activating activity relationship To determine optimum molecular size of MAP exhibiting maximum macrophage-activating activity, MAP was separated into three fractions, G2E1DS3 (MWz400 KDa), G2E1DS2 (5 KDaVMWV400 KDa), G2E1DS1 (MWV5 KDa), by Sephacryl column chromatography and ultrafiltration, and then added to

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Fig. 2. Phenotypic analysis of RAW 264.7 cells cultured with MAP. RAW 264.7 cells were cultured in the presence of G2E1 (100 Ag/ml) or G2E1D (100 Ag/ml) for 2 days. The cells were collected, washed, and then used for immunophenotypic analysis. Levels of expression (thin line) were illustrated in comparison to isotype control (dotted line).

cultures of RAW 264.6 cells. As parameters of macrophage activation, we examined growth-inhibitory activity, TNF-a production, and nitric oxide production

from RAW 264.7 cells cultured with MAP. As shown in Fig. 4, G1E2DS2 exhibited the most prominent macrophage-activating activity. Macrophage-activating

Fig. 3. Phagocytic activity of RAW 264.7 cells stimulated with MAP. RAW 264.7 cells were cultured in the presence of G2E1 (100 Ag/ml) or G2E1D (100 Ag/ml) for 2 days and then added with microspheres containing fluorescein isothiocyanate (FITC). After 2 h, unphagocytozed microspheres were removed by washing. Cells were harvested, fixed, and analyzed in a flow cytometry. Thin line histograms represent the phagocytic activity of RAW 264.7 cells stimulated with MAP, and shaded histograms represent the phagocytic activity of untreated RAW 264.7 cells.

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Fig. 4. Molecular sizemacrophage activating activity relationship of MAP. Protein-free MAP (G2E1D) was further separated into three fractions, G2E1DS1, G2E1DS2, and G2E1DS3, based on their molecular size. The growth-inhibitory activity on RAW 264.7 cells (a), effects on the production of TNF-a (b), and nitric oxide (c) from RAW 264.7 cells were examined as described in Fig. 1. The final concentration of G2E1DS1, G2E1DS2, and G2E1DS3 in (a) through (c) was 0.8, 4, 20, 100, 500, and 1000 Ag/ml, respectively.

activity of G1E2DS2 was nearly comparable to that of G1E2D, while the other fractions, G1E2DS1 and G1E2DS3, exhibited much lower activity than G1E2DS2.

3.3. Antitumor activity of MAP Antitumor activity of MAP was examined in mice implanted with sarcoma 180 cells. In this experi-

Fig. 5. Flow cytometric analysis of antitumor activity. G2E1 and G2E1D were injected into ICR strain of mouse (i.p., 1 mg/mouse) once a day for six consecutive days. On day 4 from the initiation of MAP injection, sarcoma 180 cells (4105 cells/mouse) were injected into peritoneum of MAP-treated mouse. Peritoneal exudate cells (PECs) were collected 4 days after the injection of sarcoma 180 cells and stained with FITCconjugated anti-CD45 monoclonal antibody. CD45-negative histogram (M1) represents sarcoma 180 cells, and CD45-positive histogram represents host cells of hematopoietic origin.

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Table 1 Antitumor activity of MAP on sarcoma 180 cells Dose (mg/ Sarcoma 180 cells PEC mouse/ Number % Number % day) (1105) inhibitiona (1105) increaseb Control G2E1c G2E1D G2E1DS1 G2E1DS2 G2E1DS3
a

1 1 1 1 1

3.90 3.59 0.51 3.32 0.33 3.28

7.94 86.92 14.87 91.53 15.89

1.10 1.41 4.49 1.68 4.67 1.72

128 408 152 424 156

Percent inhibition was calculated as follows: {(CN TN )/ CN }100, where CN and TN stand for the number of sarcoma 180 cells of the control group and the treated group, respectively. b Percent increase was calculated as follows: {(TN CN )/ CN }100, where CN and TN stand for the number of PEC of the control group and the treated group, respectively. c Antitumor activity was examined as described in Fig. 5.

ment, MAP was injected into the peritoneum of the ICR strain of mouse at a dose of 1 mg/mouse once a day for six consecutive days. On day 4 from the initiation of MAP injection, sarcoma 180 cells were injected into peritoneum of MAP-treated mouse. Four days after the injection of sarcoma 180 cells, all the cells in the peritoneum were collected and then stained with anti-CD45 monoclonal antibody. Representative histograms showing the composition of peritoneal exudate cells are shown in Fig. 5. Because CD45 is expressed all cells of hematopoietic origin except red blood cell, CD45-negative cells in Fig. 5 are sarcoma 180 cells. Antitumor activity of MAP-treated mouse was calculated as a percent decrease in total number of sarcoma 180 cells compared with that of untreated group. As shown in Table 1, G2E1D exhibited strong antitumor activity (88.2% inhibition), and most of the antitumor activity of G2E1D was due to G2E1DS2 (92.4% inhibition). These results confirm that the macrophage-activating activity of MAP shown in vitro is correlated with the antitumor activity in vivo.

4. Discussion This study confirms that MAP isolated from cellulase-treated gel of A. vera has immunomodulatory activity, and extends that the optimal molecular size exhibiting maximum immunomodulatory activity is between 5 and 400 KDa. MAP smaller than 5 KDa

or larger than 400 KDa exhibited only marginal immunomodulatory activity. Fresh Aloe gel contains about 10% polysaccharides by dry weight with an average MW of 2 million and thus is very viscous. Treatment of Aloe gel with cellulase reduces the viscosity, making it easy to process. Because treatment with cellulase also lowers the molecular size of Aloe polysaccharides, questions arise about the changes in the biological activities. In an earlier study, MAP with an average MW of 80 KDa was isolated from cellulase-treated Aloe gel and shown to have diverse immunomodulatory activities including UVB-protective activity [16]. Because the average molecular size of Aloe polysaccharides lowers in proportion to the extent of cellulase treatment, determination of optimal molecular size exhibiting maximum biological activity is critical in an industrial point of view. In addition, although there are numerous reports demonstrating that Aloe polysaccharides exhibit diverse immunodulatory activities [2 16], molecular sizeactivity relationship is not clear. In this study, we examined molecular sizeactivity relationship of Aloe polysaccharides isolated from cellulase-treated Aloe gel, and showed that polysaccharides larger than 400 KDa have only marginal immunomodulatory activity, while polysaccharides smaller than 400 KDa have potent immunomodulatory activity. Further digestion of the polysaccharides to less than 5 KDa resulted in loss of the activity. The fact that MAP smaller than 400 KDa exhibits potent immunomodulatory activity has important implications. For in vivo application, smaller MW molecules are usually preferred to higher MW molecules due to bioavailability issues. MAP between 5 and 400 KDa is quite a smaller molecule compared with a few million dalton polysaccharide isolated from native Aloe gel. In addition, the fact that MAP smaller than 400 KDa exhibited much more potent immunomodulatory activity than MAP larger than 400 KDa suggests that partial digestion of native Aloe polysaccharide even increases the immunomodulatory activity. We did not compare the immunomodulatory activity of MAP preparations with that of high MW polysaccharides isolated from native Aloe gel in this study. However, this point has already been addressed in an earlier report [16]. According to the previous report, immunomodulatory activity of MAP with an average MW of 80 KDa was much stronger

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than that of high MW polysaccharide isolated from native Aloe gel. The reason for the augmentation of immunomodulatory activity by partial digestion is not elucidated yet. One obvious finding related to this point was that partial digestion of Aloe polysaccharide was not simply a reduction of a molecular size in homogenously organized polymers. Comparison of the structure between MAP and native Aloe polysaccharide showed that the average molar ratio of mannose, galactose, and glucose was significantly different from each other, although major type of linkage appeared to be identical [16]. Further fractionation and examination of immunomodulatory activity of 5400 KD Aloe polysaccharides to narrow down the most active molecular size is under study.

Acknowledgements We thank Dr. Redi for comments on this manuscript. This work was supported by the program of Research Center for Bioresources and Health, KOSEF, Korea.

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