International Immunopharmacology 4 (2004) 1745 1755 www.elsevier.

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Review

Isolation and characterization of structural components of Aloe vera L. leaf pulp

Y. Nia,*, D. Turnerb, K.M. Yatesa, I. Tizardb
a

DelSite Biotechnologies lab. c/o Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas 77843, United States b Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas 77843, United States

Abstract The clear pulp, also known as inner gel, of Aloe vera L. leaf is widely used in various medical, cosmetic and nutraceutical applications. Many beneficial effects of this plant have been attributed to the polysaccharides present in the pulp. However, discrepancies exist regarding the composition of pulp polysaccharide species and an understanding of pulp structure in relation to its chemical composition has been lacking. Thus, we examined pulp structure, isolated structural components and determined their carbohydrate compositions along with analyzing a partially purified pulp-based product (Acemannan hydrogelk) used to make CarrisynR hydrogel wound dressing. Light and electron microscopy showed that the pulp consisted of large clear mesophyll cells with a diameter as large as 1000 Am. These cells were composed of cell walls and cell membranes along with a very limited number of degenerated cellular organelles. No intact cellular organelles were found in mesophyll cells. Following disruption of pulp by homogenization, three components were isolated by sequential centrifugation. They were thin clear sheets, microparticles and a viscous liquid gel, which corresponded to cell wall, degenerated cellular organelles and liquid content of mesophyll cells based on morphological and chemical analysis. These three components accounted for 16.2% (F3.8), 0.70% (F0) and 83.1% of the pulp on a dry weight basis. The carbohydrate composition of each component was distinct; liquid gel contained mannan, microparticles contained galactose-rich polysaccharide(s) and cell walls contained an unusually high level of galacturonic acid (34%, w/w; Gal A). The same three components were also found in Acemannan Hydrogelk with mannan as the predominant component. Thus, different pulp structural components are associated with different polysaccharides and thus may potentially be different functionally. These findings may help lay a basis for further studies and development of better controlled processing methods and applications for this well-accepted medicinal plant. D 2004 Elsevier B.V. All rights reserved.
Keywords: Aloe vera L.; Polysaccharides; Leaf pulp

* Corresponding author. Tel.: +1-979-845-5599; fax: +1-979-862-2320. E-mail address: yni@delsite.com (Y. Ni). 1567-5769/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2004.07.006

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1. Introduction Aloe vera L. (Aloe barbadensis Miller) is a member of liliaceae family. Of over 300 Aloe species, A. vera L. is most widely accepted and used for various medical, cosmetic and nutraceutical purposes. The plant is made of elongated and pointed leaves. Each leaf consists of two parts, an outer green rind and an inner clear pulp. The pulp, the major part of the leaf by volume, appears to be clear and mucilaginous. It is this part of plant that has been most widely used for therapeutic purposes. Acemannan Immunostimulantk, a pulp extract rich in mannan, has been licensed for treatment of fibrosarcoma in cats and dogs. The pulp has been described using several other terms including inner gel, mucilaginous gel, mucilaginous jelly and leaf parachyma. Here, we will continue to use the term bpulpQ since it denotes a soft moist mass or the soft juicy part of a fruit or vegetable, which most closely describes the inner clear part of the A. vera L. leaves. A. vera L. is a succulent plant. Succulents are xerophytes, which are adapted to living in areas of low water availability and are characterized by possessing a large water storage tissue. Although not clearly indicated in the literature, the pulp from A. vera L. is likely the water storage tissue. Another feature of succulents is the use of crassulacean acid metabolism (CAM), an additional photosynthetic pathway involving malic acid [13]. Many biological activities, including anti-viral, anti-bacterial, laxative, protection against radiation, anti-inflammation and immunostimulation have been attributed to this plant, in particular, its polysaccharides. Acemannan Immunostimulantk has been shown to activate macrophages and accelerate wound healing [4,5]. The use of and research on this plant have been well described in two well-referenced reviews [6,7]. Currently, the plant is most widely used in areas of skin care, cosmetics and wound healing. Acemannan Hydrogelk, a pulp extract, has been used in a hydrogel wound dressing CarrasynR hydrogel for treatment of wounds. The A. vera L. has also been used as a food supplement or a nutraceutical. The carbohydrate composition of the pulp has been described in numerous reports. Various polysaccharides have been detected or isolated from the pulp, including mannan [814], galactan [15], arabinan

[16], arabinorhamnogalactan [17], pectic substance [15,16,18,19] and glucuronic acid-containing polysaccharide [20,21]. However, significant variations on the pulp polysaccharide species were seen in these early studies. For example, several studies identified the mannan as the major polysaccharide of the pulp, whereas other studies, in the absence of mannan, found pectic substance as the primary polysaccharide [15,19]. The reason for such discrepancies was not understood, but was largely attributed to seasonal change and/or different geographic locations [6,15]. The mannan is the most widely studied polysaccharide from A. vera L. It consists of h14-linked mannose residues [9,12,13,15,2225]. The mannan is partially acetylated and thus the term bacemannanQ was coined [23]. Detailed structural studies on A. vera L. were few; there was only one such report to our knowledge [26]. This in turn limited any attempts to identify and isolate individual components and analyze their chemical composition. A clear picture of the structure components in relation to chemical composition is not only important to the basic understanding of the A. vera L. pulp, but also to product characterization and standardization. It is with this goal in mind that we carried out the following studies.

2. Materials and methods 2.1. Materials A. vera L. plants (10 in.) were obtained from H&P Sales (Vista, CA). Bulk-acetylated acemannan (BAM) is an A. vera L. pulp extract manufactured by Carrington Laboratories (Irving, TX) and is referred to as Acemannan Hydrogelk when used in formulations of Carrington wound hydrogels. It is a product-by-process produced in a pharmaceutical facility under cGMP. The process involves homogenization and alcohol precipitation, but no enzyme treatment or charcoal filtration. 2.2. Light microscopy Fresh A. vera L. leaves were sectioned with a surgical blade into 23 mm thick cross or longi-

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tudinal sections. The sections were mounted onto glass slides and observed directly under the light microscope (Olympus BH-2). Leaves from multiple plants were examined. For histological analysis with thin sections, blocks of fresh A. vera L. leaves were fixed in 10% formalin in phosphate-buffered saline (PBS) and sections of 5 Am were prepared and stained with toluidine blue, which was performed by the Histology Laboratory, College of Medicine, Texas A&M University, College Station, TX. 2.3. Electron microscopy Electron microscopy was performed in the Imaging Laboratory, Department of Biology, Texas A&M University, College Station, TX. The protocols for tissue fixing and staining followed that described by Trachtenberg [26]. Briefly, fresh pulp tissue blocks (~1 cm3) were fixed at room temperature in 4% glutaraldehyde in 0.2 M cacodylate HCl buffer (pH 7.2) for 2 h followed by fixing for 2 h in 2% osmium tetroxide in the same buffer. The tissues were dehydrated and sectioned after embedding in resin. The tissue sections were stained with uranyl acetate and examined using a Zeiss 10C electron microscope. 2.4. Isolation of pulp structural components by centrifugation Fresh A. vera L. leaves from several plants were first allowed to drain off the yellow sap from the rind and then cleaned with deionized water. The rind was removed with a sharp blade. The clear pulp was homogenized with a polytron. The homogenized pulp was centrifuged at 3000 rpm (500g, Beckman TJ-6) for 15 min. The pellet was harvested and named pellet I. The supernatant was further centrifuged at 18,000 rpm (25,000g; Beckman JA-20 rotor) for 30 min. The supernatant was removed and saved. The new pellet was collected and named pellet II. Both pellets were washed once with deionized water, i.e., resuspending in deionized water and pelleted down at the respective centrifugation conditions. Pellets and supernatant were then lyophilized (Centrivap, Labconco). The drying process was stopped when

vacuum level reached below 50 Am Hg. The weight of dried materials was determined immediately after the drying process. The supernatant was also subjected to alcohol precipitation by mixing with 3 volumes of 100% ethanol. The resulting precipitates were collected by centrifugation and re-dissolved in water before being lyophilized as described above. Acemannan Hydrogelk was similarly fractioned by centrifugation. It was first dissolved in water at 2 mg/ml. The first centrifugation was performed at 1000 rpm (180g) for 15 min. The pellets were washed with deionized water as above. 2.5. Anthrone assay Anthrone assay was carried out according to the method described by Dische [27] with modifications. The 0.2% anthrone in concentrated H2SO4 was freshly prepared for each assay. d-Mannose at various concentrations (0, 0.031, 0.063, 0.125, 0.25, 0.5 and 1 mg/ml) was used as the standard. Fifty microliters of samples or d-mannose standard was added to the wells of a 96-well plate in triplicate in a column-wise manner. A multiple channel pipette was used to add 100 Al anthrone reagent to each well and mix the contents in a row-wise manner so that standard and samples in each row were handled in a consistent manner. The mixtures were kept at room temperature for 15 min and color development was measured by a microplate reader (MR600, Dynatech) at 630 nm. 2.6. Uronic acid assay The m-hydroxyldiphenyl uronic acid assay was carried out as described by Blumenkratz and AsboeHansen [28]. Briefly, samples or standards in 200 Al deionized water were mixed with 1.2 ml concentrated H2SO4 containing 0.0125 M sodium tetraborate and then immediately put on ice. The samples were then placed in boiling water for 5 min followed by cooling in an ice-water bath. A total of 20 Al of 0.15% (w/v) m-hydroxyldiphenyl in 0.5% NaOH was then added to each reaction. After mixing, the samples were kept at room temperature for 30 min. The absorbance at OD520 nm was then determined. Galacturonic acid (Gal A) was used to

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generate a standard curve (0, 1, 2, 4, 6, 8 and 10 Ag). Mannose was used as a neutral sugar control. All samples were tested at 20 Ag (the high end of the linear range) or less by weight. 2.7. Sugar composition analysis 2.7.1. FACE Fluorophore-assisted carbohydrate electrophoresis (FACE) is a fast and simple technique for sugar composition analysis. It has not been adapted for analyzing complex plant polysaccharides that contain many special non-mammalian sugars, but is very useful for initial composition profiling. It was carried out accordingtotheprocedureprovidedwiththeFACEsugar composition analysis kit (Glyco). Briefly, polysaccharides were hydrolyzed with 2 N trifluoroacetic acid at 100 8C for 5 h and then labeled with the fluorescent dye (AMAC, 2-aminoacridone) and separated by electrophoresis. Monosaccharide bands were visualized under UV light (Fotodyne 3-3000). The gel images were captured using GS-5000 digital imaging system (Alpha Innotech)andtheopticaldensitiesofthemonosaccharide bands were measured using the FluorChem software (Alpha Innotech). Besides the neutral sugar standards provided in the kit, Gal A was also used. Because rhamnose and arabinose migrate closely with mannose in the FACE system (Glyco), the band corresponding to the mannose standard may not be only composed of mannose. 2.7.2. Cellulose analysis Isolated cell wall fibers prepared as above were analyzed for neutral sugar composition under the conditions allowing detection of glucose and other sugars in cellulose by Econotech Services (BC, Canada). The cell wall sample was hydrolyzed with sulfuric acid and alditol acetates generated were analyzed by gas chromatography. Inositol was used as an internal standard.

Fig. 1. Gross view of A. vera L. leaf sections. (a) Cross section and (b) longitudinal section. Closed arrows indicate the rind; open arrow indicate the pulp; open arrow heads indicate the vascular bundle.

leaf, outer green rind and inner clear pulp, were clearly visible. Vascular bundles were tubular structures located in the pulp, but adjacent to the green rind. The number of these bundles varied, depending on the size of leaves. The majority of the pulp consisted of water; the dry matter only accounted for 0.9 % (w/w, S.D.=0.33) based on three separate drying experiments. 3.2. Light microscopy Examination of sections (23 mm) of fresh A. vera L. leaves showed that the pulp consisted of large clear mesophyll cells with a hexagonal or elongated hexagonal shape (Fig. 2a, b and c). The size of these cells was very large and some were more than 1000 Am (or 1 mm) long. The walls of these cells were transparent (Fig. 2c). No apparent difference in appearance was noted between cross and longitudinal sections. Under higher magnification, the cell walls could be clearly seen (Fig. 2c). When thin sections (~5 Am) of leaf blocks fixed with formalin were examined, only cell walls were seen in the pulp (Fig. 3). Cellular organelle-like structures were only observed in the green rind and vascular bundles (Fig. 3). 3.3. Electron microscopy

3. Results 3.1. Gross leaf structure A cross and longitudinal sections of an A. vera L. leaf are shown in Fig. 1. The two distinct parts of the Electron microscopy revealed, in addition to cell walls, only the cell membranes along with a very limited number of cellular organelles in the pulp

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Intact cellular organelles such as nuclei, chloroplasts and mitochondria were only observed in the green rind and vascular bundles (data not shown). Thus, these mesophyll cells in the pulp appeared to be non-living cells, probably playing a role in water storage. 3.4. Isolation and identification of the pulp structural components The homogenized fresh pulp was centrifuged in a sequential manner to yield three fractions (see Section 2). They were pellet I obtained by low-speed centrifugation, pellet II obtained by high-speed centrifugation and the supernatant or the viscous liquid gel after the high-speed centrifugation, which accounted for 16.2% (F3.8), 0.70% (F0) and 83.1% on a dry weight basis, respectively (Table 1). The experiments for measuring pellets I and II were repeated multiple times and the average values from two experiments using larger fresh pulp sample sizes (N100 g) are shown in Table 1. The pellets were considered the insoluble materials, whereas liquid gel was considered the soluble material. The liquid gel was subjected to alcohol precipitation and a white precipitate was obtained along with the alcohol solution containing the alcohol soluble materials. Under a microscope, the pellet I materials exhibited a fiber-like appearance at low magnification (4), but appeared to be clear transparent

Fig. 2. Microscopic examination of fresh pulp. Sections (23 mm) of fresh A. vera L. leaves were directly observed under microscope. (a) Pulp (4 magnification), (b) pulp along with green rind (4 magnification), (c) pulp under a higher magnification (10 magnification). The filled arrow indicates the green rind, the empty arrow indicates the clear pulp and open arrow heads indicate the cell walls of pulp mesophyll cells.

(Fig. 4a and b). The cellular organelles observed in the pulp all appeared to be degenerated. In some areas, the remains from the broken or degenerated cellular organelles could clearly be seen (Fig. 4b).

Fig. 3. Histological examination of A. vera L. leaves. Blocks of fresh A. vera L. leaves were fixed in 10% formalin in PBS, sectioned and stained with Alcian blue. Magnification, 10. The filled arrow indicates the clear pulp, the empty arrow indicates the green rind and the open arrow head indicates the vascular bundle.

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Y. Ni et al. / International Immunopharmacology 4 (2004) 17451755 Table 1 Proportions and carbohydrate contents of three primary components in A. vera L. pulp % of dry Carbohydrate Uronic acid pulpa content (Gal A) (w/w) content Cell wall fibers (pellet I) Microparticles (pellet II) Liquid Alcohol-insoluble gel (precipitates) Alcohol-soluble
a

16.2% (F3.8) 0.70% (F0) 7.5% 75.6%

93% (w/w) b50% (w/w) 0.22% (w/v)

34% (w/w) 8% (w/w) b0.015% (w/v)

The dried pulp accounts for 0.9% (w/w) of the wet pulp.

sheet materials that still maintain some original cell structures could be observed. These clear sheets were therefore identified as the broken pulp mesophyll cell wall fragments or cell wall fibers.

Fig. 4. Electron microscopic examination of A. vera L. leaf pulp. The fresh A. vera L. leaf pulp were fixed in 5% paraformadehyde, embedded, sectioned and stained with uranyl acetate. Open arrow indicates the cell membrane, the closed one indicates the cell wall and open arrowhead indicates the degenerated cellular organelles.

sheets at higher magnifications (10 and 40) (Fig. 5a and b). They were usually very large being several hundred Am across, but could be much smaller if homogenized for a longer period of time. They exhibited an identical appearance to the cell walls seen in the pulp sections (Fig. 2c), i.e., being thin, clear and transparent. When pulp was homogenized to a lesser extent, larger pieces of such

Fig. 5. Isolated A. vera L. pulp mesophyll cell walls. They were examined and photographed using an Olympus BH-2 microscope. 40 magnification (a and b).

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The pellet II materials mostly appeared to be microparticles under a microscope with a size of ~1.0 Am as measured under the electron microscope by negative staining. Their sizes were similar to those of degenerated cellular organelles found in the pulp mesophyll cells. Thus, these microparticles are most likely those degenerated cellular organelles possibly along with some cell wall fragments of similar sizes that could be generated during homogenization process. The liquid gel was inevitably from the content of these mesophyll cells. It was a clear solution following high-speed centrifugation. It could be further split into two portions by alcohol precipitation, i.e., alcohol-insoluble (precipitates) and alcohol-soluble materials. The alcohol precipitates recovered by low speed centrifugation, which accounted for 7.5% of the pulp on a dry weight basis, could be dissolved in water after being dried, producing a very viscous solution at 2 mg/ml or higher. The distribution of these three components based on dry weight is shown along with the alcohol insoluble and soluble portions of the liquid gel in Table 1. It is clear that the liquid gel accounted for most of the pulp by dry weight and only a small part of it was precipitated by alcohol. The same three components were also found in a pulp-based product, Acemannan Hydrogelk. On the dry weight basis, the liquid (soluble materials) and cell wall component were dominant, accounting for 7080% and 2030%, respectively, along with a small amount of microparticles (12%). The cell wall component had an identical appearance as those isolated from fresh pulp. More than 50% of the materials in the liquid portion were carbohydrates, which were primarily the mannan (see below). 3.5. Carbohydrate analysis of the pulp structural components 3.5.1. Total carbohydrate content The total carbohydrate content as measured by anthrone assay showed that cell wall fibers were the richest in carbohydrate (93%, w/w) (Table 1). The microparticles had a carbohydrate content of b50% (w/w). The supernatant or liquid gel contained only 0.22% (w/v) of carbohydrate (Table 1). However, the alcohol precipitates derived from the gel was highly

rich in carbohydrates (N50%, w/w), indicating that the precipitates were mostly polysaccharides. 3.5.2. Uronic acid content The uronic acid assays showed that cell wall fibers, microparticles and liquid gel were very different in the uronic acid (UA) content, having a content of 34% (w/ w), 8% (w/w) and b0.015% (w/v), respectively (Table 1). The polysaccharides precipitated from the gel by alcohol did not have an increased UA content (b3%, w/w) as compared to the original liquid gel. The uronic acid detected was fount to be galacturonic acid (Gal A) by the sugar composition analysis (see below). These data further supports the identification of pellet 1, pellet 2 and soluble materials as distinctly separate components as cell wall fibers, microparticles and the liquid gel, respectively. 3.5.3. Sugar composition The sugar composition profile of cell wall fibers, microparticles and the polysaccharides precipitated from the supernatant or liquid gel were distinct as shown by the FACE analysis (Fig. 6 and Table 2). It was found that Gal A, galactose and mannose were the predominant sugars in cell wall fibers (pellet I), microparticles (pellet II) and the polysaccharide preparation (precipitates by ethanol) from the liquid gel, respectively (Tables 1 and 2). These results further substantiate the establishment of these three distinct components. The mannan, the most widely studied pulp polysaccharide, was clearly located in the liquid gel. In the mannan preparation, glucose was also detected. A small amount of Gal A was also detected in the microparticles (Fig. 6). The same carbohydrate composition were also found in the cell wall fibers, microparticles and soluble portion of the Acemannan Hydrogelk. 3.5.4. Analysis of the cellulose component in the cell wall fibers The condition for sugar composition analysis described above does not allow detection of the glucose in the insoluble cellulose, a primary component of plant cell walls. To confirm the identity of the cell walls fibers, they were subjected to neutral sugar analysis under conditions allowing break-down of cellulose and

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Fig. 6. Sugar composition analysis of pulp components by FACE. Cell walls, microparticles and polysaccharides precipitated from liquid gel were hydrolyzed with 2 N TFA and resulting monosaccharides were labeled with the labeling reagent and separated by electrophoresis. Galacturonic acid (Gal A) was treated similarly as a Gal A standard.

detection of released glucose residues. The results showed that glucose was the most predominant of total 5 neutral sugars (arabinose, xylose, mannose, galactose and glucose) analyzed, accounting for 69.2% of the total with other sugars accounting for 2.711.8%. On the dry weight basis, these five sugars made up 43.9% of the cell wall materials with glucose making up 30.4% (w/w). A small amount of acid-insoluble materials (lignin, 2.6%) was also detected. Thus, these results together indicated that the clear transparent sheets or fibers are indeed cell walls.

viscous and accounts for most of the pulp by weight and volume, i.e., the primary contributor to the succulent nature of the pulp. These three components are distinct to each other both morphologically as well as with respect to sugar composition. The liquid gel is rich in mannan, the cell wall in Gal A and the microparticles in galactose. Such correlation has not been described previously and is summarized in Fig. 7. The mannan is clearly a soluble polysaccharide present in the liquid gel and also the basis for the viscoelastic property of the liquid gel. The richness of cell walls in Gal A strongly suggests the presence of a high level of pectic substance or pectin. The microparticles contain the galactose-rich polysaccharide. The microparticles may possibly also include some very small fragments of cell walls generated during homogenization, besides the degenerated cellular organelles. The discrepancy on pulp polysaccharide species (mannan, pectic substance and others) in early studies has been largely attributed to the seasonal change or difference in the geographic location [6]. However, from our work, it now appears to be the result of detecting or isolating only one polysaccharide, but not others, and then identifying it (in the absence of others) as the primary polysaccharide by different investigators. Based on our findings, this earlier inconsistency is in large part due to lack of a clear understanding of the pulp structure and its components. The pulp had been largely treated as a single homogenous entity. The situation was further complicated by use of the terms such as mucilaginous jelly or

Table 2 Sugar composition of A. vera L. pulp components, the ethanol precipitates of liquid gel, microparticle and cell wall, by FACE analysis (% total sugars detected) Liquid gel (ethanol precipitate) Mannose Fucose Glucose Galactose Gal A Othersa Total 62.9 0 13.1 1.5 0.6 21.9 100 Microparticle 32.2 0 20.6 40.2 2.8 4.2 100 Cell wall 20.4 0 12.3 6.4 38.4 22.50 100

4. Discussion The present studies identified and isolated three structural components from A. vera L. pulp, i.e., cell wall, microparticles and liquid gel. The liquid gel is considered as a structural component because it is

a Sugar bands whose identity was not identified based on the standard used.

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Fig. 7. Schematic representation of A. vera L. leaf pulp structure and its components.

gel which implies a homogenous entity. Thus, the pulp contains all these polysaccharides. What was not realized was the effect of the difference in pulp processing and extraction methods that may lead to enrichment or elimination of certain pulp components and hence the polysaccharides associated with them. Femenia et al. [29] made an important step forward in trying to isolate individual components. The issue of lack of understanding of cell wall was brought forward. However, a clear separation of the liquid gel from cell wall was not achieved. Preparation of AIR (alcohol insoluble residue), a common practice when studying polysaccharides, was applied without clear separation of the homogenized pulp into the soluble and insoluble. Alcohol causes the mannan to precipitate or become insoluble and cell walls are insoluble with or without alcohol treatment. Thus, the resulting AIR is still a mixture. The pulp cell wall is a component that has not been characterized before. It is a significant component of the pulp cells, accounting for 16.2% on a dry weight basis. Uniquely, it possesses an unusually high level of Gal A (34%, w/w). Such a high level of Gal A is seldom found in plant cell wall materials. The high Gal A content suggests a high content of pectic substance or pectin which usually contains ~10% of neutral sugars. Thus, the pectic substance in pulp cell wall materials could be up to 4050% (w/w). Such a high pectic substance content suggests that the pulp cell walls could have significant cholesterol-lowering and detoxifying effects [3032]. This component is well preserved in Acemannan Hydrogelk along with the mannan. Similar findings were also made with another pulp-based product, MannapolR, which has a higher content of cell wall component (data not shown).

We have extracted an unique pectin from the cell wall fibers prepared from pulp as well as rinds that is chemically and functionally distinct from all known pectins or other polymers. The details of this pectin will be presented elsewhere. Plant mucilaginous polysaccharides are characterized with a relatively high content of uronic acid [33]. The mucilaginous polysaccharide (mannan) of A. vera L., however, is different in that it does not contain any uronic acid; no uronic acid has been detected in the mannan purified by chromatography, although uronic acid or Gal A has been detected in many crude mannan preparations from A. vera L. pulp. This mannan component has been detected in all Aloe species analyzed so far although differences in mannoseglucose ratio, molecular weight and extent of acetylation have been noted [21,34,35]. This polysaccharide is the basis for the pseudoplastic flow behavior of the liquid gel from the pulp [35,36]. But once the mannan is degraded by heat or enzyme, the gel becomes less viscous and exhibits a Newtonian flow property [37]. The light and electron microscopy found no intact cellular organelles, strongly suggesting that mesophyll cells are non-living cells and thus likely act as the water storage cells or sacs. This observation is consistent with that by Trachtenberg [26], which showed that in mature mesophyll cells only degenerated plastids were found. These observations are further supported by the findings that CAM does not occur in the pulp [3]. It is important to note that the alcohol soluble materials in the liquid gel constitute the largest portion by weight. They are likely small molecules. It is known that the pulp contains various organic and

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Y. Ni et al. / International Immunopharmacology 4 (2004) 17451755 [8] Segal A, Taylor JA, Eoff JC. A re-investigation of the polysaccharide material from aloe vera mucilage. Lloydia 1968;31:423. [9] Yagi A, Makino K, Nishioka I, Kuchino Y. Aloe mannan, polysaccharide, from Aloe arborescens var. natalensis. Planta Med 1977;31:17 20. [10] Yagi A, Nishimura H, Shida T, Nishioka I. Structure determination of polysaccharides in Aloe arborescens var. natalensis. Planta Med 1985:213 8. [11] Waller GR, Mangiafico S, Ritchey CR. A chemical investigation of Aloe barbadensis Miller. Proc Okla Acad Sci 1978; 58:69 76. [12] Gowda DC, Neelisidaiah B, Anjaneyalu YV. Structural studies of polysaccharides from aloe vera. Carbohydr Res 1979;72: 201 5. [13] Radjabi-Nassab F, Ramiliarison C, Monneret C, Vilkas E. Further studies of the glucomannan from Aloe vahombe (liliaceae): II. Partial hydrolyses and NMR 13C studies. Biochimie 1984;66:563 7. [14] tHart LA, van den Berg AJJ, Kuis L, van Dijk H, Labadie RP. An anti-complementary polysaccharide with immunological adjuvant activity from the leaf parenchyma gel of aloe vera. Planta Med 1989;55:509 12. [15] Mandal G, Das A. Structure of the D-galactan isolated from Aloe barbadensis Miller. Carbohydr Res 1980;86: 247 257. [16] Mandal G, Das A. Structure of the glucomannan isolated from the leaves of Aloe barbadensis Miller. Carbohydr Res 1980;87:249 56. [17] Mabusela WT, Stephen AM, Botha MC. Carbohydrate polymers from Aloe ferox leaves. Phytochemistry 1990;29:3555 8. [18] Rowe TD, Parks LM. Phytochemical study of aloe vera leaf. J Am Pharm Assoc 1941;30:262 6. [19] Ovodova RG, Lapchik VF, Ovodova YS. Polysaccharides in Aloe arborescens. Him Prir Soedin 1975;11:3 5. [20] Hranisavljevic-Jakovljevic M, Miljkovic-Stojanovic J. Structural study of an acidic polysaccharide isolated from Aloe arborescens Mill: I. Periodate oxidation and partial acid hydrolysis. Glas Hem Drus Beogr 1981;46:269 73. [21] Wozniewski T, Blaschek W, Franz G. Isolation and structure analysis of a glucomannan from the leaves of Aloe arborescens var. miller. Carbohydr Res 1990;198:387 91. [22] Paulsen BS, Fagerheim E, Overbye E. Structural studies of the polysaccharide from Aloe plicatilis Miller. Carbohydr Res 1978;60:345 51. [23] Manna S, McAnalley BH. Determination of the position of the O-acetyl group in a h-(14)-mannan (acemannan) from Aloe barbardensis Miller. Carbohydr Res 1993;241:317 9. [24] Vilkas E, Radjabi-Nassab F. The glucomannan system from aloe vahombe (liliaceae): III. Comparative studies on the glucomannan components isolated from the leaves. Biochimie 1986;68:1123 7. [25] Yagi A, Hamada K, Mihashi K, Harada N, Nishioka I. Structure determination of polysaccharides in Aloe saponaria (Hill) haw. (liliaceae). J Pharm Sci 1984;73:62 5. [26] Trachtenberg S. Cytochemical and morphological evidence for the involvement of the plasma membrane and plastids in

inorganic compounds including malic acid. Also they likely include lipid components from cell membranes that are solubilized by alcohol treatment. A. vera L. plant has enjoyed a long history of being a medicinal plant. Today, components of this plant are widely used in medical devices, cosmetics, and nutraceuticals. However, the methods by which the plant is processed vary greatly from product to product. This likely results in final preparations that have a very different chemical composition. For example, the use of cellulase and charcoal filtration, the classical method for preparation of Aloe products, leads to elimination of the cell wall components; if the cellulase is not pure or contains mannanase, the mannan component in the pulp may also be broken down or eliminated. The mannan, however, can be well preserved by using ethanol precipitation. Different components may have different functions. In addition, functions of a polysaccharide and monosaccharides constituting it are totally different. Thus, understanding of the process in relation to the chemical composition of the final products is essential. It is hoped that the present findings may help lay a foundation for further analysis and development of better defined products, and thus a better understanding of why various products derived from A. vera L. exhibit broadly different biological activities.

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