Selective Adsorption of Vegetable Tannins onto Collagen Fibers

Vegetable tannins are widely distributed in the plant kingdom. In many cases, it is necessary to remove tannins from natural beverages and medicinal p...
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Ind. Eng. Chem. Res. 2003, 42, 3397-3402

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Selective Adsorption of Vegetable Tannins onto Collagen Fibers Xue pin Liao, Zhong bing Lu, and Bi Shi* The Key Laboratory of Leather Chemistry and Engineering of Ministry of Education, Sichuan University, Chengdu 610065, P. R. China

Vegetable tannins are widely distributed in the plant kingdom. In many cases, it is necessary to remove tannins from natural beverages and medicinal plant extracts due to their physiological toxicity. In the present work, an adsorbent for this purpose was prepared on the basis of hide collagen fibers. Its adsorption selectivity for tannins was studied, using tannic acid as a target component to be removed and tea polyphenols, isoflavones, and baicalin as probe molecules. The experiment results showed that hide collagen fiber is able to remove polyphenols containing the galloyl group from aqueous solution effectively, and the extent of adsorption for tannic acid is almost 100%, while slightly adsorbing probe molecules. As a comparison, the adsorption property of macroreticular resin which was usually suggested to be used for removal of tannins was also investigated. It showed no significant adsorption selectivity in the present experiments. The mechanism of adsorption selectivity of collagen fibers for tannins was also discussed. Introduction Vegetable tannins (plant polyphenols) constitute one of the most numerous and widely distributed categories in the plant kingdom, with more than 8000 phenolic structures currently known.1 Tannins are found in approximately 80% of woody and 15% of herbaceous dicotyledonous species and can occur at high levels in some forages, feeds, and foods.2 Tannins are polyphenolic compounds of plant origin, which are of two distinct types,3 hydrolyzable tannins (polyesters of gallic acid and polysaccharides) and condensed tannins (polymerized products of flavan-3-ols and flavan-3,4-diols, or a mixture of the two), although other tannins occur which are combinations of these two basic structures. Generally, only the plant polyphenols with molecular weight between 500 and 3000 Da are called tannins.4 Numerous studies have demonstrated the harmful effect of tannins on animals and humans.5 In many experiments, it has been shown clearly that the ability of tannins to form strong complexes with proteins causes negative effects on appetite and nutrient utilization, particularly of proteins, in herbivores.6-9 The injection site of the patient would be red and swollen if tannins had not been removed from the injection made from plants such as red-rooted salvia.10 Tannic acid (hydrolyzable tannin) had shown hepatic necrosis in humans and grazing animals.11 Ingestion of high level of tannins can cause gastroenteritis and congestion of the intestinal wall in rats.12 Tannins can affect the utilization of vitamins and minerals,13 and inhibition of digestive enzymes.14 There is more and more proof indicating that the hydrolyzable tannins are more toxic than condensed tannins. Hydrolyzable tannins, such as tannic acid, are easily degraded in the biological system by nonspecific esterases, and the hydrolyzed products could lead to organ (particularly liver and kidney) toxicity once the level in blood is beyond the detoxification capability of these organs.15-17 The condensed tannins with larger molecules are not hydrolyzed in the biological sys* To whom correspondense should be addressed. E-mail: [email protected].

tems18,19 and, therefore, are not absorbed into the blood stream.20 It was reported that tannic acid is of a higher toxicity to carp compared to that of quebracho tannin, a typical condensed tannin.21 Most physiologically active compounds coming from medicinal plant extracts are phenolic substances, and the extracts often contain tannins.22 Generally, tannins in medicinal plant extracts are considered as useless or harmful components, especially the hydrolyzable tannins, and should be removed before further processing and practical use. Removal or isolation of tannins is also an important event when using bioassays involving tannins in medical23,24 or ecological studies.25,26 Precipitating with gelatin or heavy metal ions and adsorbing by polymer resins can remove tannins from plant extracts.11 Electrochemical removal of tannins from aqueous solution,27 removal of tannic acid by adsorption of zirconium pillared clay,28 and removal of phlorotannins using insoluble polyvinylpolypyrrolidone29 were also reported. However, precipitating by gelatin impairs the quality of the product and decreases the stability of storage, precipitating with heavy metal ions introduces heavy metal ions into the product, and adsorbing by resins has limited adsorption selectivity. The electrochemical method destroys organic molecules of plant extracts, including functional components. Tannins, having a molecular weight between 500 and 3000 Da, are soluble in polar solution and are distinguished from other polyphenolic compounds by their ability to precipitate proteins, and tannins are traditionally used as a tanning agent in leather manufacture. Therefore, the ability of tannins to form a strong association with proteins might promise a method to remove tannins from plant extracts. The hide collagen fibers are insoluble in water and much more stable than gelatin. Its primary sequence is basically a tripeptide repeat, (Gly-X-Y)100-400, where X is often proline and Y sometimes hydroxyproline.30,31 Hide collagen fiber contains functional groups, like -COOH, -NH2, -CONH2, and -CONH-, which any synthetic material cannot be compared with. The interaction of tannins and hide collagen fibers is due to hydrogen bond and hydrophobic bond associations. The multipoint hydrogen bond com-

10.1021/ie0209475 CCC: $25.00 © 2003 American Chemical Society Published on Web 06/10/2003

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Figure 1. HPLC chromatogram of the mixed solution of tea polyphenols and tannic acid before and after adsorption by collagen fiber. Initial content of tea polyphenols and tannic acid in the solution was 1000 mg/L, respectively. Mobile phase: A ) aqueous solution of 0.2% CH3CN, B ) CH3OH. Column: Hypersil ODS C18, 4.0 × 150, 5µm. Flow rate: 1.2 mL/min. Detector: DAD, 280 nm. Injection volume: 20 µL. Gradient: 0-20 min with 0-50% B, 20-25 min with 50-100% B.

bination would ensure highly selective adsorption of tannins onto collagen fibers in comparison with other components. However, native hide collagen fiber is easily attacked by chemicals and bacteria and has limited hydrothermal stability. Therefore, a proper modification of hide collagen fibers should be carried out. In the present work, the adsorbent was prepared by cross-linking hide collagen fibers using formaldehyde. Tea polyphenols, isoflavones of soybean, and baicalin, which are typical physiologically functional components with phenolic structure, were selected as probe molecules to investigate the adsorption selectivity of collagen fibers. Experimental Section Materials. White hide powder, produced according to standard procedures32 and commercially available,

Figure 2. HPLC chromatogram of tea polyphenols before and after adsorption by collagen fibers. Initial concentration of tea polyphenols was 1000 mg/L. Chromatographic conditions were as same as those for Figure 1.

was used as collagen fibers. Tannic acid was the analytical reagent. The reagent described as tea polyphenols was a mixture extracted from Chinese green tea, containing (+)-catechin (C), (-)-epicatechin (EC), (-)epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-gallocatechin gallate (GCG), (-)-epigallocatechin gallate (EGCG), caffeine, and other unknown components. The total content of polyphenols in the extract is over 95%. Isoflavones was obtained from soybean mainly including daidzin (7,4′-dihydroxy-isoflavone-7-O-glucose) and genistein (5,7,4′-trihydroxl-isoflavone-7-Oglucose). Baicalin (5,6-dihydroxy-flavone-7-O-glucose) was obtained from the roots of scutellaria baicalensis georgi. All standard samples were purchased from Sigma Co. Macroreticular resin (polyvinylpolypyrrolidone, PVPP) is a commercial product (Divergan RS, BASF) for adsorption purpose. Cross-Linking of Hide Collagen Fibers. The hide collagen fiber was cross-linked by formaldehyde with the following procedures. A 3 g portion of hide powder

Table 1. HPLC Parameters of Tea Polyphenols and the Extent of Adsorption of the Components on Collagen Fibers components EGC, C caffeine EC EGCG GCG ECG

before adsorption retention time (min) peak area 9.176 10.672 11.935 11.343 12.814 14.038

433.06 3000.92 866.48 847.56 582.83 391.97

peak height 63.62 375.59 105.23 71.56 52.51 32.09

after adsorption retention time (min) peak area 9.197 10.687 11.917 11.382 12.881 14.048

351.15 2791.47 664.08 58.17 49.40 23.43

peak height

extent of adsorption, %

48.57 360.34 83.32 3.71 2.60 2.23

18.91 6.98 23.36 93.14 91.52 94.02

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Figure 3. HPLC chromatogram of isoflavones before and after adsorption by collagen fiber. Initial concentration of isoflavones was 1000 mg/L. Mobile phase: A ) H2O, B ) CH3OH. Column: Hypersil ODS C18, 4.0 × 250, 5 µm. Flow rate: 0.6 mL/min. Detector: DAD, 254 nm. Injection volume: 20 µL. Gradient: 0-10 min 15% B to 40% B, 10-45 min 40% B to 55% B.

was wet back in 500 mL of distilled water for 24 h at room temperature. After filtrating, the hide powder was put into 200 mL of 30 g/L formaldehyde solution and shaken on constant temperature oscillator at 25 °C for 2 h. Then, the temperature was elevated to 35 °C, and the pH was adjusted to 8.0 with sodium bicarbonate solution. After shaking for another 2 h and stilling for 12 h, the collagen fiber was fully washed, filtrated, and dried. Adsorption Procedures. The adsorbents of collagen fibers and macroreticular resin were enclosed in bags made by filter gauze, respectively, (0.5 g for each in dry basis) and soaked in distilled water for 24 h. After washing with distilled water, the bags were put into 100 mL of sample solutions and shaken at 25 °C. For collagen fiber, adsorption time was 100 min, but it was 180 min for macroreticular resin. Analysis Methods. The contents of compounds in sample solutions before and after adsorption were determined by HPLC (Agilent1100, HP Co.) with column Hypersil ODS C18 at 25 °C. The mobile phases and other chromatographic conditions are recorded in each analysis. Results and Discussion Adsorption of Collagen Fibers to Polyphenols. Figure 1a is the HPLC chromatogram of the mixed

Figure 4. HPLC chromatogram of the mixed solution of baicalin and tannic acid before and after adsorption by collagen fiber. Initial content of baicalin and tannic acid in the solution was 580 and 500 mg/L, respectively. Mobile phase: A ) aqueous solution of 0.4% H3PO4, B ) CH3OH, B/A ) 45:55. Column: Hypersil ODS C18, 4.0 × 250, 5 µm. Flow rate: 1.0 mL/min. Detector: DAD, 280 nm. Injection volume: 20 µL. No gradient.

aqueous solution of tea polyphenols and tannic acid. Figure 1b is the HPLC chromatogram of the residual solution after adsorption by collagen fibers. Control experiments had indicated that the elutes with retention time e15 min are the components of tea polyphenols, and the ones of retention time g15 min are the components of tannic acid. It is clear that tannic acid, the typical hydrolyzable tannin with average molecular weight 1700, was nearly completely removed by adsorption of collagen fiber. However, the adsorption of collagen fiber to tea polyphenols is limited. Further experiments showed that the adsorption of collagen fiber to tea polyphenols in aqueous solution is highly selective, as illustrated in Figure 2 and Table 1. It can be seen that EGCG, ECG, and GCG, which have the galloyl group, were remarkably adsorbed, while the extent of adsorption of EGC, C, and EC, which are polyphenols without the galloyl group, was small. Also, the caffeine, which is an alkaloid, has a very small extent of adsorption. This implies that the extent of adsorption of polyphenols onto collagen fiber is mainly dependent on the galloyl group, the predominant functional group of hydrolyzable tannins. Tannic acid includes 10 galloyl groups on average, so it can be strongly absorbed by collagen fibers and removed from extract completely. The adsorption selectivity of collagen fibers to tannins can be further confirmed when the adsorptions of

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Figure 5. HPLC chromatogram of the mixed solution of tea polyphenols and tannic acid before and after adsorption by macroreticular resin. Initial content of tea polyphenols and tannic acid in the solution was 500 mg/L, respectively. Mobile phase: A ) aqueous solution containing 11%CH3CN and 10 mmol ClCH2COOH, B ) CH3OH. Column: Hypersil ODS C18, 4.0 × 250, 5 µm. Flow rate: 1.0 mL/min. Detector: DAD, 280 nm. Injection volume: 20 µL. Gradient: 0-15 min 0% B to 50% B. Table 2. HPLC Parameters of Isoflavone and Mixed Solution of Baicalin and Tannic Acid and the Extent of Adsorption of the Components on Collagen Fiber before adsorption retention component time (min) daidzin genistin baicalin tannin acid

14.324 17.174 7.043 2.186

after adsorption

peak area

retention time (min)

peak area

extent of adsorption, %

11188.5 24993.7 4899.22 20691.4

14.517 17.246 7.054 2.180

10942.4 22967.0 4527.28 1891.68

2.20 8.11 7.59 90.86

Table 3. HPLC Parameters of the Mixed Solution of Tea Polyphenols and Tannic Acid and the Extent of Adsorption of the Components on Macroreticular Resin before adsorption retention components time (min) EGC C EC caffeine EGCG GCG ECG tannic acid

3.586 4.807 5.998 6.338 7.751 7.977 8.712 9.0-17.5

after adsorption

peak area

retention time (min)

peak area

extent of adsorption, %

220.97 311.05 558.72 1672.79 698.33 518.83 538.62 22126.1

3.550 4.731 5.897 6.300 7.667 7.848 8.593 9.0-17.5

215.05 278.15 491.98 587.09 414.47 388.02 297.56 6222.33

2.68 10.58 11.95 64.90 40.63 25.21 44.76 71.88

isoflavones and baicalin were investigated, as shown in Figures 3 and 4 and Table 2. For these experiments, the mixed solvent of H2O and CH3OH (50:55, v/v) was used to obtain well-dissolved sample solutions. The

Figure 6. HPLC chromatogram of tea polyphenols before and after adsorption by macroreticular resin. Initial concentration of tea polyphenols was 500 mg/L. Mobile phase: aqueous solution containing 11% CH3CN and 10 mmol of ClCH2COOH. Column: Hypersil ODS C18, 4.0 × 250, 5 µm. Flow rate: 1.0 mL/min. Detector: DAD, 280 nm. Injection volume: 20 µL. No gradient. Table 4. HPLC Parameters of Tea Polyphenols and the Extent of Adsorption of the Components on Macroreticular Resin before adsorption retention components time (min) EGC C caffeine EC EGCG GCG ECG

5.275 6.995 7.362 12.084 13.088 17.615 41.518

after adsorption

peak area

retention time (min)

peak area

225.77 201.78 1594.38 423.25 652.90 415.67 186.00

5.312 7.045 7.454 12.173 13.176 17.735

183.49 155.39 736.02 245.96 220.87 109.90

extent of adsorption, % 18.73 22.99 53.84 41.89 66.17 73.56 100

isoflavones and baicalin are biologically active polyphenols containing no galloyl group, and thus, their extent of adsorption on collagen fiber is small. These results strongly imply that collagen fiber can be used as a highly selective adsorption material for tannins existing in plant extracts, such as herbal medicines, particularly for the removal of hydrolyzable tannins that are rich in galloyl groups. It is well-known that the reaction of polyphenol and collagen is synergistic effect of hydrophobic association and hydrogen bonding, and it had been proved that the galloyl group of polyphenols plays the most important role in the interaction, due to its having both the functions undertaking hydrophobic and hydrogen bond associations with collagen fibers.33 Hence, the adsorption selectivity of collagen fibers to polyphenols observed in this research is in correspondence with the interaction mechanism of polyphenols (tannins) and collagen.

Ind. Eng. Chem. Res., Vol. 42, No. 14, 2003 3401 Table 5. HPLC Parameters of Tea Polyphenols and the Extent of Adsorption of the Components on Collagen Fibers with and without Cross-Linkinga before adsorption

after adsorption by collagen fibers with cross-linking

after adsorption by collagen fibers without cross-linking

component

retention time (min)

peak area

retention time (min)

peak area

extent of adsorption, %

retention time (min)

peak area

extent of adsorption, %

EGC C caffeine EC EGCG GCG ECG

5.182 6.892 7.092 11.792 12.596 16.696 39.868

471.57 343.65 3545.56 896.30 1233.48 764.39 384.71

5.164 6.855 7.149 11.723 12.562 16.668 -

389.58 314.35 3225.31 740.21 272.22 154.99 -

17.39 8.53 9.03 17.42 77.93 79.72 100

5.127 6.808 7.071 11.626 12.418 16.414 -

402.81 323.59 3289.42 784.77 223.65 147.48 -

14.58 5.84 7.22 12.44 81.87 80.71 100

a Initial concentration of tea polyphenols was 1000 mg/L. Mobile phase: aqueous solution containing 11% CH CN and 10 mmol of 3 ClCH2COOH. Column: Hypersil ODS C18, 4.0 × 250, 5 µm. Flow rate: 1.0 mL/min. Detector: DAD, 280 nm. Injection volume: 20 µL. No gradient.

Adsorption of Macroreticular Resin to Polyphenols. Figure 5 illustrates the HPLC chromatograms of aqueous solutions of tea polyphenols and tannic acid before and after adsorption by macroreticular resin. Control experiments had indicated that the elutes with retention time e10.5 min are the components of tea polyphenols, and the ones of retention time g10.5 min are the components of tannic acid. The extents of adsorption to EGCG, GCG, and ECG were only 40.61%, 25.21%, and 44.76%, respectively, but were over 90% adsorbed by collagen fiber adsorbent (Table 1). It is worth noting that the caffeine, little adsorbed by collagen fibers, was significantly adsorbed by macroreticular resin as shown in Table 3. The adsorption selectivity of macroreticular resin to tea polyphenols containing the galloyl group is obviously lower. Figure 6 is the HPLC chromatogram of tea polyphenols in aqueous solution before and after adsorption by macroreticular resin. The extent of adsorption of the components is listed in Table 4. It can be found that macroreticular resin still has a relatively stronger adsorption ability to the components containing the galloyl group (EGCG, GCG, and ECG), but this selectivity of adsorption is not as remarkable as collagen fibers, as shown in Table 1. This should be the reason leading to lower adsorption selectivity of macroreticular resin to tannic acid, compared to collagen fibers. Influence of Formaldehyde Cross-Linking on Selective Adsorption of Collagen Fibers to Polyphenols. For enhancing chemical and hydrothermal stabilities, the collagen fibers used in the experiments had been cross-linked by formaldehyde. The influence of the cross-linking reaction on adsorption ability of collagen fibers had been investigated in the research. As an example, the absorption of tea polyphenols on collagen fibers with and without cross-linking is compared in Table 5. Indeed, all the control experiments show that the cross-linking reaction has little influence on absorption capacity and adsorption selectivity of collagen fibers to polyphenols. It is well-known that the cross-linking reaction of formaldehyde takes place at the -NH2 and -NH- groups of the side chains in collagen fibers, which would reduce hydrogen bond association of polyphenols on collagen fibers. However, it had been proven that the hydrogen bond association of polyphenols (tannins) on collagen fibers is predominantly dependent on peptide chains existing regularly and numerously in collagen fibers.34,35 So, the cross-linking reaction could somewhat reduce hydrogen bond association of polyphenols on collagen fibers, but the influence is very limited. Consequently, the change of collagen

fibers in adsorption characteristics is almost inconsequential with cross-linking modification. Conclusions Collagen fiber is a highly selective adsorption material for polyphenols containing galloyl groups, particularly, hydrolyzable tannins that are rich in galloyl groups. It could be employed as an adsorbent for the removal of physiologically toxic polyphenols existing in plant extracts, such as herbal medicines. Cross-linking with aldehyde is able to increase chemical and hydrothermal stabilities of the collagen fiber while its adsorption capacity and adsorption selectivity are not remarkably influenced. Acknowledgment This research was financially supported by The Foundation of Ph.D. Education Disciplines in Chinese University. Literature Cited (1) Bravo, L.; Manas, E.; Saura-Galixto, F. Dietary nonextractable condensed tannins as indigestible compounds: effects on faecal weight, and protein and fat excretion. J. Sci. Food Agric. 1993, 63, 63. (2) Bryant, J. P.; Reichardt, B. P.; Clausen, T. P. Chemically mediated interactions between woody plants and browsing mammals. J. Range Manage. 1992, 45, 18. (3) Haslam, E. Plant polyohenols-vegetable tannins revisited; Cambridge University Press: Cambridge, 1989. (4) Shun, D. W. Chemistry of vegetable tannins; China Forestry Press: Beijing, 1992. (5) Lu, Y.; Bennick, A. Interaction of tannin with human salivary proline-rich proteins. Arch.f Oral Biol. 1998, 43, 717. (6) Silanikove, N.; Perevolotsky, A.; Provenza, F. D. Use of tannin-binding chemicals to assay for tannins and their negative postingestive effects in ruminants. Anim. Feed Sci. Technol. 2001, 91, 69. (7) Robbins, C. T.; Hanely, T. A.; Hagerman, A. E.; Hjeljord, O.; Baker, D. L.; Schwartz, C. C.; Mautz, W. W. Role of tannins in defending plants against ruminants: reduction in protein availability. Ecology 1987, 68, 98. (8) Silanikove, N.; Gilboa, A.; Nir, I.; Perevolotsky, A.; Nitsan, Z. Effect of a daily supplementation of polyethylene glycol on intake and digestion of tannin-containing leaves (quercus calliprins, Pistacia lentiscus and Ceratonia siliqua) by goats. J. Agric. Food Chem. 1996, 44, 199. (9) Silanikove, N.; Gilboa, N.; Nitsan, Z. Interactions among tannins, supplementation, and polyethylene glycol in goats fed oak leaves. Anim. Sci. 1997a, 64, 479. (10) Xu, M. C.; Wang, C. R.; Xu, M. C.; Shi, Z. Q.; Zhang, S.; Li, H. T.; Shi, R. F.; Fan, Y. G.; He, B. L. Adsorption of tannin

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from aqueous solution onto macroporous cross-linked poly(N-VinylAcetamide) via hydrogen bonding. Chin. J. React. Polym. 2000, 9 (1), 23. (11) Chung, K. T.; Wei, C. I.; Johnson, M. G. Are tannins a double-edged sword in biology and health? Trends Food Sci. Technol. 1998, 9, 168. (12) Chung, K. T.; Wong, T. Y.; Wei, C. I.; Huang, Y. W.; Lin, Y. Tannins and human health: A review. Crit. Rev. Food Sci. Nutr. 1998, 38 (6), 421. (13) Mitjavila, S.; Lacombe, C.; Carrera, G.; Derache, R. Tannic acid and oxidized tannic acid on the functional state of rat intestinal epithelium. J. Nutr. 1977, 107, 2113. (14) Ahmed, A. E.; Smithard, R.; Ellis, M. Activities of the enzymes of the pancreas, and the lumen and mucosa of the small intestine in growing broiler cockerels fed on tannin-containing diet. Br. J. Nutr. 1991, 65, 189 (15) Garg, S. K.; Makkar, H. P. S.; Nagal, K. B.; Sharma, S. K.; Wadhwa, D. R.; Singh, B. Toxicological investigations into oak (Quercus incana) leaf poisoning in cattle. Vet. Hum. Toxicol. 1992, 34, 161. (16) Mueller-Harvey, I.; McAllan, A. B. Tannins: Their biochemistry and nutritional properties. Adv. Plant Cell Biochem. Biotechnol. 1992, 1, 151. (17) Makkar, H. P. S. Antinutritional factors in foods for livestock. In Animal Production in Developing Countries; Gill, M., Owen, E., Pollott, G. E., Lawrence, T. L. J., Eds.; BSAP Occas. Publ. 16; British Society of Animal Production: Penicuik, U.K., 1993; pp 69-85. (18) McLeod, M. N. Plant tanninsstheir role in forage quality. Nutr. Abstr. Rev. 1974, 44, 803. (19) Makkar H. P. S.; Blu¨mmel, M.; Becker, K. Formation of complexes between polyvinyl pyrrolidone and polyethylene glycol with tannins and their implications in gas production and true digestibility in vitro techniques. Br. J. Nutr. 1995a, 73, 897. (20) Terrill, T. H.; Waghorn, G. C.; Woolley, D. J.; McNabb, W. C.; Barry, T. N. Assay and digestion of 14C-labeled condensed tannins in the gastrointestinal tract of sheep. Br. J. Nutr. 1994, 72, 467. (21) Becker, K.; Makkar, H. P. S. Effects of dietary tannic acid and quebracho tannin on growth performance and metabolic rates of common carp (Cyprinus carpio L.). Aquaculture 1999, 175, 327. (22) Kolesnikov, M. P.; Gins, V. K. Phenolic substances in medicinal plants. Appl. Biochem. Microbiol. 2001, 37 (4), 392.

(23) Wall, M. E.; Taylor, H.; Ambrosio, L.; Davis, K. Plant antitumor agents III: A convenient separation of tannins from other plant constituents. J. Pharm. Sci. 1969, 58, 839. (24) Be´ress, A. A.; Wassermann, O.; Bruhn, T., Be´ress, L. A new procedure for the isolation of ant-HIV compounds (polysaccharides and polyphenols) from the marine alga Fucus vesiculosus. J. Nat. Prod. 1993, 56, 478. (25) Pavia, H.; Toth, G. B. Induced chemical resistance to herbivory in the brown seaweed ascophyllum nodosum. Ecology 2000, 81, 3212. (26) Toth, G. B.; Pavia, H. Lack of phlorotannin induction in the brown seaweed Ascophyllum nodosum in response to increased copper concentration. Mar. Ecol.: Prog. Ser. 2000, 192, 119. (27) Buso, A.; Balbo, L.; Giomo, M. Electrochemical Removal of tannins from aqueous solution. Ind. Eng. Chem. Res. 2000, 39, 494. (28) Vinod, V. P.; Anirudhan, T. S. Sorption of tannic acid on zirconium pillared clay. J. Chem. Technol. Biotechnol. 2002, 77 (1), 92. (29) Toth, G. B.; Pavia, H. Removal of dissolved brown algal phlorotannins using insoluble polyvinylpolypyrrolidone (PVPP). J. Chem. Ecol. 2001, 27 (9), 1899. (30) Bailey, A. J.; Paul, R. G. Collagen: A not so simple protein. J. Soc. Leather Technol. Chem. 1998, 82, 104. (31) Haslam, E. Vegetable tannage: where do the tannins go? J. Soc. Leather Technol. Chem. 1997, 82, 45. (32) Lu¨, X. R. A brief discussion on manufacture of hide powder for tannin analysis. Linchan Huaxue Yu Gongye 2000, 20(1), 71 (33) Shi, B.; He, X.; Haslam, E. Gelatin-polyphenol interaction. J. Am. Leather Chem. Assoc. 1994, 89, 98. (34) Thampuran, K. R. V.; Doraikannu, A.; Ghosh, D. Certain fundamentals of vegetable tannin chemistry. Leather Sci. 1978, 25, 309. (35) Shuttleworth, S. G.; Russell, A. E.; Williams-Wynn, D. A. Further studies on the mechanism of vegetable tannage: Part VI General conclusions. J. Soc. Leather Trades’ Chem. 1968, 52, 486.

Received for review November 25, 2002 Revised manuscript received April 2, 2003 Accepted May 2, 2003 IE0209475