330 JOURNAL OF COSMETIC SCIENCE Table I Analysis Results on Three Samples of Collagen Suitable for Cosmetic Applications Test AteloHelogen ® CLR Collagen® Collasol® Protein content (%w/v) 1.05% 0.28% 4.00% pH 4.7 3.8 4.2 pl 8.9 8.9 8.9 Arsenic 1 ppm 1 ppm 1 ppm Heavy metals 5 ppm 5 ppm 5 ppm Dry weight 1.15% 5.07% 5.25% Conductivity 0.45 ms 19.0 ms 42.0 ms Ash content 0.1% 1.2% 1.1% Hydration regain 21% 3% 7% In the present study, the levels of these components were below the detection limit for all three samples. An important feature of a collagen product is its collagen protein concentration. For fully soluble and essentially pure collagen preparations with concentrations above about 2-3 mg/ml (or solutions after clarification by centrifugation), a Biuret assay (16) against appropriate collagen standards is simple and rapid. However, non-collagenous impuri­ ties could interfere with this determination, leading to higher values. Total nitrogen content can also be readily determined by Kjeldahl analysis. This value allows the collagen concentration of the sample to be estimated, using a conversion factor based on the known structure of collagens (17). This factor is dependent on the collagen type and the species of origin. Again, this method can give erroneous values for collagen content if any non-collagenous proteins are present. Hydroxyproline is found as an amino acid that is characteristic of collagenous proteins, and therefore provides a specific method for determination of collagen content, separate from any other proteins that may be present. Hydroxyproline content and the total amino acid composition can be determined by amino acid analysis (see below). Hydroxyproline content can also be determined by a specific colorimetric assay (18). The use of dry weight is not suitable to determine collagen content, as some preparations contain a significant salt content, as can be seen from the ash content of samples (Table I). The conductivity of a sample can confirm the presence of salts (Table I). For example, AteloHelogen® shows a negligible ash content, and has a very low conductivity, whereas both CLR Collagen® and Collasol® both have a significant ash content and significant conductivities, although these measures are not necessarily linked, as they depend on the nature of the salts present (Table I). The pH values for the preparations examined in this study varied (Table I). These values may be important in developing formulations. The presence of salts may affect the pH, as they may be buffering the collagen solution (Table I). Samples of collagen that contain oligomers are more soluble under low pH conditions-at higher pH the collagen oligo­ mers become less soluble and may precipitate (10). On the other hand, at a higher pH, such as neutral pH, a minimum salt content of around 0.15 M NaCl is needed to ensure collagen solubility (10). Thus, the higher pH of the AteloHelogen® collagen with a very low salt content (Table I) indicates a highly monomeric collagen preparation. WATER REGAIN The intrinsic water-binding property of collagen is critical to its excellent performance
COLLAGEN EVALUATION 331 as a humectant in cosmetic preparations. An estimate of this water-binding capacity may be obtained from the water regain by dry samples held under constant humidity (32%) (Table I). It can be seen that all collagen samples in the present study showed water regain after drying, but the extent or rate varied between the samples for the present method at 32% relative humidity. When samples were held at 87% relative humidity (saturated Na2CO3 ), all absorbed sufficient water to form solutions or wet slurries of collagen (data not shown). The variation between samples could reflect the presence of salts. The conductivity and ash content data (Table I) show that AteloHelogen® has a very low salt content, whereas the other two samples both contain salts. The nature of the salts (which was not determined) could appear to increase the collagen water regain if they are particularly hygroscopic, but this is not apparent in the present study. Alternatively, they could reduce the water regain, by slowing down the rate at which equilibrium is attained, for example. An alternative approach to compare samples would be to examine them after extensive dialysis to remove any salts present. SPECTROSCOPY Commercial collagen preparations are usually colorless solutions or they may be white if there is any insoluble material present. The principal use, therefore, of UV/visible spectroscopy is not to detect colored impurities, but rather to assess the level of turbidity that may be present in a soluble collagen preparation. For clear samples, it may also be used to detect and potentially quantitate any UV-absorbing preservatives that may be present. Material may be assessed as supplied or after dilution, for example, either in water so as to minimize changes in pH or in dilute acetic acid so as to ensure the solubility of non-cross-linked components, as in certain preparations it is possible that soluble collagen has formed into fibrils at neutral pH. As an illustration, comparison of the three test collagens shows that AteloHelogen® was particularly transparent as supplied, whereas both CLR Collagen® and Collasol® were turbid (Figure lA). After dilution in water to equivalent concentrations of 1 mg/ml, the clarity of Collasol® was still poor, while that for CLR Collagen® was significantly improved, although still much less than AteloHelogen® (Figure lB). The solubility of Collasol® was improved in acetic acid (data not shown). IR spectroscopy (Figure 2) provides a method for showing protein identity in the sample. It may also show the presence of organic buffers or preservatives. IR spectroscopy also has potential for assessing the extent of collagen denaturation in a collagen preparation, as any gelatine present changes the relative intensity of the bands at 1660 cm- 1 and 1633 cm- 1 (19). However, this requires high resolution. In the present study, the resolution of the IR spectra was not sufficient, and also seemed to vary between preparations (Figure 2). Thus IR spectroscopy may not be a convenient method for estimating the content of denatured collagen (gelatine) present in a sample. The collagen/gelatine content of fully clear solutions may otherwise be examined by ORD or CD spectroscopy, where standard values have been reported (20,21), but this requires access to specialized equipment. Sample clarity is an issue: insoluble collagen interferes and can be removed by centrifu­ gation, but the resulting analysis is not representative of the total sample. Loss of gelatine components after brief proteolysis, where collagen is stable, followed by colla­ gen precipitation, also provides a convenient method for estimating gelatine content.
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