Turning now to the central parenchymatous tissues of the aloe leaf, these are tasteless and colourless and of a more or less glutinous nature. These tissues in A. vera yield the aloe gel now so widespread in commercial therapeutic and cosmetic preparations (reviewed elsewhere in this volume). Although not accepted medically because of the difficulty in establishing the dosage, there are very many reports of healing properties, especially for skin complaints. It seems that activity may take place through the immunological system (reviewed Reynolds and Dweck, 1999; also Tizard and Ramamoorthy, chapter 13). Various components have been described from the gel, the principle ones being polysaccharides which give the substance its glutinous nature. Also present are
Figure 3.16 Naphthalene and tetralin derivatives.
Cape aloes compound 2 Isoeleutherol 5-O-glucoside
Figure 3.16 Naphthalene and tetralin derivatives.
glycoproteins for which biological activity has been reported. From time to time mention of the presence of various small molecules is made.
As might be expected much work has been carried out on A. vera although a few other species have been used. In the first modern preparation of polysaccharides from aloe gel mucilage, a water soluble material was produced which was precipitated by ethanol and dialysed to remove the high ash content (13%). It represented 30% of the dry weight of the leaf parenchyma (Robez and Haagen-Smith, 1948). On acid hydrolysis equal quantities of glucose and mannose were formed with a small amount of uronic acid (2%). A similar material was prepared in the course of a patented procedure (Farkas, 1967), which contained about 15% chemically bound calcium and had a molecular weight of around 450,000 daltons, which varied depending on variations in the process. A further short report confirmed the presence of mannose and glucose but in the molar ratio of about 10:1 with added arabinose, galactose and xylose in trace amounts (Segal etal., 1968). A later study reported a mannose glucose ratio of 6:1 for a similar product and fractionated the substance by graded ethanol precipitation into three fractions A1, A2 and B and then A1 into A1a and A1b (Gowda etal., 1979). They were shown to be partially acetylated glucomannans of which B had the highest O-acetyl content and formed the most mucilagenous mixture with water. Further study showed them to be 1 ^ 4 linked and to have molecular weights in excess of 2 X105 daltons.
In contrast analysis of a gel from a different A. vera individual demonstrated a high content of pectic acid (70—85%) separated by ethanol precipitation. The remaining polysaccharides separated by DEAE—cellulose chromatography were shown to be a galactan, a glucomannan and an arabinan (Mandal and Das, 1980a). The D-galactan contained 1 ^ 4 and 1 ^ 6 linkages. The glucomannan contained glucose and mannose in the molar ratio of 1:22 joined by 1 ^4 linkages with some side-chains joined by 1 ^ 6 linkages (Mandal and Das, 1980b). The pectic acid fraction contained mainly galacturonic acid together with galactose and traces of glucose and arabinose. The galactose units were joined by 1 ^ 3 linkages to a linear 1 ^ 4 linked galacturonic acid chain (Mandal etal., 1983). A cruder preparation using just fractional precipitation by ethanol gave a single product characterized as a linear glucogalactomannan with glucose, mannose and galactose in a molar ratio of 2:2:1 (Haq and Hannan, 1981).
An activity-guided purification of gel polysaccharides using both anion-exchange and gel filtration chromatography identified two fractions, B-I and B-II showing inhibition of classical pathway complement activity ('t Hart etal., 1989). They contained mainly mannose together with galactose, glucose and arabinose in ratios 89:4:3:1 for B-I and 22:2:1:1 for B-II, with a molecular weight between 1.5 X105 and 4.8X105 daltons. From the data it appears that there is also considerable activity in non-polysaccharide fractions.
In 1987 an acetylated mannan was reported from A. vera gel as a commercial product, acemannan or carrisyn™ (McDaniel etal, 1987) to which several types of biological activity were attributed (reviewed Reynolds and Dweck, 1999). Subsequent work established the position of the O-acetyl group as equally on either C2 or C3 and on C6 of the mannose moiety (Manna and McAnalley, 1993). A very detailed account of the preparation and purification of this substance and its chemical and physical properties is the subject of two U.S. patents (McAnalley, 1988, 1990), which also mention thera peutic activity. Extensive details of the whole process are given elsewhere in this volume (Chapter 8).
The evidence above indicates that the presence of certain specific polysaccharides is critical for healing. These can vary markedly from product to product, so much so that accurate analysis is necessary to predict activity (Ross etal., 1997).
Aloe arborescens is another species very widespread in cultivation and used medicinally, especially in the Far East. A partially acetylated mannan was isolated from the gel of this plant as a single substance precipitated by acetone and having a molecular weight of 15,000 daltons (Yagi etal., 1977). Separation of the polysaccarides by gel filtration yielded three fractions A, B and C (Yagi etal, 1986). Fraction A was a 1 ^ 6 linked glucan, with a molecular weight of 15,000daltons. Fraction B contained arab-inose and glucose in the molar ratio 3:2 with 2 ^ 6 linkages, with a molecular weight of 30,000daltons. Fraction C was a 1 ^4 linked acetylated mannan containing 10% acetyl groups, with a molecular weight of40,000 daltons. Acetyl groups were on the C2 or C3 and C6 portions, recalling the structure of acemannan. Meanwhile elsewhere, an acidic polysaccharide isolated by fractional precipitation of copper complexes was shown to consist of 1 ^ 3 and 1 ^4 linked glucose and glucuronic acid in the ratio 9:1 and to have a molecular weight of around 3.6 X103 daltons (Hranisavlyevic-Jakovlyevic and Miljkovic-Stojunovic, 1981). It was accompanied by an unspecified polyglucan.
In another study, ion exchange chromatography separated two fractions named arboran A and arboran B, said to diminish plasma glucose level in mice (Hikino etal, 1986). Both fractions contained protein, 2.5% and 10.4% respectively, although it is not certain if this was joined to the polysaccharide. Arboran A contained mainly galactose, glucose and rhamnose in the molar ratios 10:3:3 and traces of fucose, arabinose, xylose and mannose with 16.7% O-acetyl groups and a molecular weight of 1. 2 X104 daltons. Arboran B contained only glucose and mannose in the molar ratio 10:3 with 5.3% O-acetyl groups and a molecular weight of 5.7 X 104daltons. Another preparation using separation on an ion exchange gel resulted in three polysaccharide fractions. The acidic fraction with a molecular weight of 5 X 104 daltons contained arabinose and galactose in a molar ratio of 1:1 with traces of rhamnose and glucose and 6% glucuronic acid. The two neutral fractions had molecular weights of 1. 2 X 104 and 1 X106 daltons and a mannose — glucose molar ratio of 95:5 with 1 ^ 4 linking. Both contained O-acetyl groups located at C6 and C2 3 (Wozniewski etal., 1990).
Aloe saponaria is another species with reputed therapeutic properties and its gel yielded three polysaccharides separated by fractional precipitation. The main component (77%) was an acetylated mannan. One of the minor components was a polyglucan and the other contained mannose, glucose and galactose in the ratio 5:4:1 (Gowda, 1980). Elsewhere a polysaccharide was isolated as the main component (c70%) of the gel. Here, the material harvested earlier yielded a 1 ^ 4 linked mannan with acetyl groups (18%) on C6 and a molecular weight of 1.5 X 104daltons, while material harvested later was a 1 ^ 4 linked acetylated mannan with 5 % glucose units and a molecular weight of 6.6X 104daltons (Yagi etal, 1984). Interestingly, immuno-adjuvant properties had been reported for the gel of a Madagascan endemic, A. vaombe, Decorse et Poiss. (incorrectly cited as A. vahombe in text) and a polysaccharide isolated by ethanol precipitation. Further purification by gel filtration yielded a fraction with a glucose to mannose ratio of 1:3 and one acetyl unit on each glucose unit (Radjabi etal., 1983; Radjabi-Nassab etal, 1984). Furthermore, critical separation by these methods separated this entity, with a molecular weight of 1 X105 daltons, from three other acetylated glucomannans with weights of 2.5 X103, 2 X104 and above 105 daltons. This latter large molecule also contained protein (Vilkas and Radjobi-Nassab, 1986). A very ornamental aloe with strap-shaped leaves, A. plicatilis Mill., contained an acetylated glucomannan with a molecular weight of 1.2 X 104daltons, and a glucose to mannose ratio of 1:2.8 (Paulsen etal., 1978). On the other hand a pure acetylated mannan was the major component of A. vanbalenii gel (Gowda, 1980). The minor polysaccharides were a glucan and a galactoglucomannan. A different array of compounds was reported from the gel of A. ferox Mill. where 14 distinct polysaccharide entities were distinguished, most of which were arabinogalactans or rhamnogalacturonans (Mabsuela etal, 1990).
The picture emerges from the six Aloe species investigated so far of a number of polysaccharides with mannose as the predominant monomer and molecular weights ranging of several orders of magnitude from 103 to 106 daltons. The patented compound with the greatest number of reported therapeutic activities is acemannan which is an acetylated mannan about 8X 104daltons in size (see Chapter 4).
Nitrogen analysis of leaf extracts and of crude or partially purified aloe gel preparations has always yielded positive results. Thus Rowe and Parks (1941) reported 1.39% dry weight of nitrogen in the outer leaf tissues (rind) of A.vera but gave no figure for the gel. Another early report of A. vera gel reported 2.9% protein of the leaf parenchyma dry weight (Roboz and Haagen-Smit, 1948). A later assay gave a protein content of 0.01% for aloe 'juice' (sic) (Gjerstad, 1971) and recognized hydroxyproline, histidine and cystine as free acids. Another analysis recognized 17 common amino acids in the free state, of which arginine was the most abundant (Waller etal., 1978). Similar results were obtained elsewhere (Khan, 1983), with arginine again being especially of note, together with glutamic acid. In A. ferox, on the other hand, asparagine was reported as the most abundant, followed by glutamine, alanine and histidine (Ishikawa etal, 1987). Alanine, proline, lysine and glutamic acid were prominent in A. arborescens leaves, accompanied by other free protein amino acids (Yagi etal., 1987). Elsewhere, aspartic acid, glutamic acid and serine were noted in A. vera gel (Baudo etal, 1992).
Lectin activity had been reported in aloe preparations (e.g. Fujita etal., 1978) and is the subject of Chapter 5, so the presence of glycoproteins could be presumed. Two such substances were separated by Sephadex chromatography from the whole leaves of A. arborescens and given codes P-2 and S-1. Both were confirmed as glycoproteins, with 18% and 50% neutral carbohydrate and molecular weights of 1. 8 X 104 and 2.4 X104daltons (Suzuki etal, 1979). They were designated as Aloctin A and Aloctin B. A quite separate entity was isolated about the same time and named ATF 1011, later shown to activate T cells (Yoshimoto etal., 1987). Another preparation was subsequently made and examined by polyacrylamide gel electrophoresis which confirmed the molecular weights and extended the range of biological activities (Saito, 1993). Separation of a protein fraction from A. arborescens 'fresh leaf juice' (sic) by DEAE cellulose and Sepharose 6B chromatography was also described (Yagi etal., 1986). In addition to the expected amino acids the hydrolysed protein fraction contained glucose, mannose, galactose, glucosamine, galactosamine and N-acetylglucosamine in the ratio 2:2:1:1:4:1. Two glycoproteins were separated by ammonium sulphate precipitation, both of which contained mannose, arabinose, glucose, galactose and glucosamine (Kodym, 1991). The outer green tissues of the leaves yielded several proteins separated on DEAE cellulose, of which one had lectin activity and a molecular weight of 3.5 X 104daltons (Koike etal, 1995).
Lectins were also reported from A. vera gel (Winters, 1993). Polyacrylamide gel electrophoresis revealed at least 23 polypeptides from the mature leaf, of which 13 occurred in the gel (Winters and Bouthet, 1995). One of these which had a molecular weight of 1.8 X 104 daltons resembled the Aloctin A, mentioned above (Bouthet etal., 1996), while six others appeared common to this species and to A. arborescens which itself had a total of nine. In the same investigation 11 polypeptides were noted in A. saponaria reflecting those observed in A. vera. Elsewhere a glycoprotein fraction promoting cell proliferation in vitro was shown to contain a single entity of molecular weight 2.9X 104daltons and to contain 11% carbohydrate (Yagi etal, 1997).
A different type of protein has been isolated from A. arborescens leaves. Ultrafiltration of extracts yielded a fraction with a molecular weight in excess of 1.0 X105 which had bradykininase activity (Fujita etal., 1976) and it was characterized as a carboxypepti-dase (Fujita etal., 1979), specifically a serine carboxypeptidase (Ito etal., 1993). In a separate study, two glycoprotein fractions were separated by Sepharose chromatogra-phy, one of which was shown to have bradykininase activity (Yagi etal, 1987). The molecular weight was 4.0X 104daltons and the other properties appeared to be identical with an entity previously described as glycoprotein A (Yagi etal., 1986). These authors had previously obtained a bradykininase from A. saponaria containing mainly mannose as the carbohydrate moiety (Yagi etal, 1982).
A number of enzymes were extracted and separated by starch gel electrophoresis for use as genetic markers to identify hybrids between A. arborescens and A. ferox (van der Bank and van Wyk, 1996).
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