Aloe Vera Broker

Note a N & O are brokers. P was supplied by a consumer product manufacturer. Other footnotes are as per Table 8.12A.

Note a N & O are brokers. P was supplied by a consumer product manufacturer. Other footnotes are as per Table 8.12A.

expected for pure aloe (c.1,000 |S) at the 2C limit. Measuring the ionic strength by conductivity gave one of the clearest signals for maltodextrin/aloe fraud and misrepresentation of spray-dried material as freeze-dried.

Liquid samples can be among the most difficult to analyze because of the wide variety of adulterants encountered (Pelley etal., 1998). Among the aloe liquids (1—7) examined in Table 8.10, only one (5) would have been identified by conductivity as suspicious. Among the questionable liquids (8—13) four would have been flagged by conductivity as questionable (8, 9, 12, 13) and the remaining two (10, 11) would be on the borderline of normal.

In conclusion, all of the three basic analytical parameters, total solids, divalent metal cations and ionic strength, have their different places in the laboratory analysis of A. barbadensis materials. Solids measured by evaporation and weighing should be measured in every Quality Control laboratory. Because of the cost of the equipment, quantitation of metal cations, including the monovalent cations, will remain a test sent to an outside laboratory as part of preparation for IASC certification. Conductivity meters are cheap (under US$300) and easy to use. The measurement of conductivity should be one of the first things a laboratory does and a conductivity meter should be out on the production floor next to the pH meter and the refractometer.

Malic acid and other organic acids

We have examined the relationship between the composition of matter of Aloe barbadensis gel, the microbial physiology of aloe-associated micro-organisms and the HPLC analysis of aloe. This relationship was first suggested in 1993 when we established that an organic acid, at that time termed 'E peak' and now known to be malic acid, was a process-stable, freshness-sensitive analyte (Pelley etal., 1993). Malic acid is universally present in ARF materials (Table 8.4) and the Texas A&M population (Wang and Strong, 1995). However, the content of malic acid varies greatly and therefore the range of acceptable malic acid values for IASC Certification is quite wide (Table 8.3). Malic acid makes an acceptable certification parameter because its content does not vary during standard processing (Pelley etal, 1993). The determination of malic acid is relatively straightforward. For purposes of certification, the IASC has specified an HPLC method (IASC, 1991). This utilizes a 4X250mm bonded amino column eluted isocratically at 1 ml per minute with a buffer consisting of 70% acetonitrile in 0.05 M phosphate buffer, pH 5.6. The eluate is monitored by UV absorbance at 205 nm. Samples of aloe liquids are adjusted to IASC '1:1' concentration (assuming that the label claim is correct). Currently, dry samples are suspended to 0.83 g% concentration. In all cases samples are filtered through 0.2 |m filter prior to use and 20 |l of sample is injected. Currently, the system is calibrated using chemically pure malic acid. Previously, ARF standard sample was employed for this purpose (Pelley etal., 1998). Figure 8.13 below illustrates typical chromatograms. It should be noted that the AOCA has recently approved a somewhat more complex method for the determination of malic acid in fruit juices (AOAC, 2000b).

Malic acid is a desirable analyte because of its ability to act as a marker for 'freshness'. As Figure 8.13 illustrates, it is the major organic acid in fresh native gel. Bacteria such as bacilli that do not grow well in aloe do not alter the malic acid content. Some of the Gram —ve rods (Panel B) that grow in aloe, assimilate malic acid and produce other organic acid such as lactate. Aloe-associated micrococci (Panel C) are very efficient at

Figure 8.13 Effect of bacterial growth upon the organic acid content of A. barbadensis gel. ARF Standard Sample gel incubated with A) Bacillus sp., B) Cedecea sp., C) Micrococcus sp. and D) gel stored without processing or preservation. This figure uses data originally described in Pelley et al.

(1993) which should be consulted for experimental conditions.

Figure 8.13 Effect of bacterial growth upon the organic acid content of A. barbadensis gel. ARF Standard Sample gel incubated with A) Bacillus sp., B) Cedecea sp., C) Micrococcus sp. and D) gel stored without processing or preservation. This figure uses data originally described in Pelley et al.

(1993) which should be consulted for experimental conditions.

assimilating malic acid and in many cases produce lactate. Aloe gel that has 'rotted' has a pattern quite similar to that of Micrococcus-innoculated aloe and although all micrococci produce lactate, all 'spoiled' aloe materials do not contain lactate.

Tables 8.10, 8.11 and 8.12 illustrate some of the patterns of malic acid distribution in commercial aloe materials. Among liquids studied, malic acid was present in all seven of the materials determined by multiparameter analysis to be authentic A. barbadensis (Table 8.10: 1-7). It should be noted that in three of these (Table 8.10: 5-7) the levels of malate were low. This is consistent with the suspicion that these materials are WLE. As discussed earlier, WLE inherently has a high bacterial content and control of bacterial proliferation in these preparations is difficult. Liquid materials suspected of severe adulteration or fraud, possessed no detectable malate. Analysis of powdered material yielded similar findings. Of eleven materials consistent with authentic aloe (Table 8.11: 2, 3, 5, 6; Table 8.12A: 1-11) seven (Table 8.11: 2, 3, 6; Table 8.12A: 1-3, 6) had normal levels of malic acid, three (Table 8.11: 5; Table 8.12A: 4, 7) had low but detectable amounts and only one (Table 8.12A: 5) was devoid of malate. Here, there was also an association between commercial materials with low malate and a WLE origin (Table 8.11: 5; Table 8.12A: 7). There were two commercial materials with low to absent malate (Table 8.12A: 4, 5) that were likely to have originated as gel. Commercial materials that were consistent with maltodextrin (Table 8.11: 8-10; Table 8.12B: 8-16) were devoid of malate with the exception of sample 10 in Table 8.11. The possibility that a small amount of aloe is present in this commercial material is strengthened by the conductivity and the presence of metallic cations. Lastly there are three commercial materials that were alleged to be 100% pure aloe but which on examination were more consistent with spray-dried aloe (Table 8.12C). HPLC analysis for malic acid is negative, suggesting that these materials were manufactured with very low quality aloe that had already undergone bacterial spoilage.

In conclusion, after over a decade in use as an IASC certification standard, malic acid is well established as a quality control analyte. No feedstock should vend without the malic acid content on the certificate of analysis, nor should any consumer product manufacturer buy aloe without knowing this. Now that 'E Peak' is known to be malic acid, it is possible that disreputable manufacturers will aduterate with malic acid just as they adulterated with maltodextrin. Until that day arrives, malate will remain at the center of our analytical armamentarium.

Anthraquinones

Anthraquinones have been discussed elsewhere (Chapters 3 and 7) and it is surprising that the aloe gel industry uses essentially no anthraquinone quality control. The IASC does not employ anthraquinones in its certification process because the content of these molecules radically changes during the charcoal absorption of routine processing. Furthermore, the cathartic action of anthraquinones by and large does not enter into the claims promulgated by either the cosmetic or drink industries that utilize Aloe barbadensis gel and WLE extracts. Therefore, in the U.S. anthraquinones remain of research interest only.

Although TLC analysis is not strictly quantitative, it is capable of analyzing dozens of samples an hour. We use Merck G-60 silica gel plates, developed with toluene — ethyl acetate — methanol — water — formic acid = 10:40:12:6:3. The only post-development technique used is to heat the plates and expose them to ammonia vapor in order to develop THA zones. For the determination of aloin, aloe emodin and the chromone glycosides and their esters, several HPLC techniques were used, all with C18 reversed phase columns. The simplest separation of aloin and the chromones uses isocratic 70% aqueous methanol. Tests for anthraquinone aglycones uses isocratic 90% aqueous methanol. In order to measure both the aglycones and the glycosides a gradient of 30% to 80% methanol in water is useful. In all cases elution is monitored by UV-absorption. For estimation of single compounds, the X maximum of that compound is used. When multiple compounds are being analysed, 256 or 280nm is used as a universal wavelength. Unfortunately, the only reference compounds available in useful purity are aloe emodin or barbaloin (Sigma-Aldrich Co. Ltd).

In the future, measuring the presence and amount of anthraquinones in aloe gel and WLE preparations applied to the skin or orally ingested may become more important. On the beneficial side, we demonstrate in Figure 8.11, that activated charcoal treatment removes a number of biological activities perhaps due to either anthraquinones or chromones. On the regulatory side, Europeans have traditionally closely monitored anthraquinone content in products because of the concern over the potential carcino-genicity of these compounds. This began when Mori etal. (1985) found chryazin caused gastrointestinal cancer in rats. These concerns have been extended to aloe emodin (Westendorf etal, 1990; Wolfe etal, 1990; Heidemann etal, 1996). This controversy has passed relatively unnoticed in America. Recently (Strickland etal., 2000) it was found that topically applied aloe emodin in ethanol solution interacts with ultraviolet radiation to cause the development of pigmented skin tumors. Scientifically these observations are extremely important because hitherto it has been very difficult to develop murine models for melanoma (Bardeesy etal, 2000; Berkelhammer etal, 1982; Kelsall and Mintz, 1998; Kusewitt and Ley, 1996; Romerdahl etal, 1989; Takizawa etal, 1985). It is likely that in the future anthraquinones will come under increasing scrutiny.

Polysaccharides

Besides the anthraquinones, polysaccharides are the most studied aloe components. Strickland etal. (Chapter 12) critically review the various studies of the structure of aloe

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Aloe and Your Health

Aloe and Your Health

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