This article was downloaded by: [University of Glasgow] On: 27 March 2013, At: 04:57 Publisher: Routledge Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Nutrition and Cancer Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/hnuc20
Mammalian lignan production from various foods a
a
a
Lilian U. Thompson , Paul Robb , Maria Serraino & Felicia Cheung
a
a
Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario, M5S 1A8, Canada Version of record first published: 04 Aug 2009.
To cite this article: Lilian U. Thompson , Paul Robb , Maria Serraino & Felicia Cheung (1991): Mammalian lignan production from various foods, Nutrition and Cancer, 16:1, 43-52 To link to this article: http://dx.doi.org/10.1080/01635589109514139
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-andconditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
Mammalian Lignan Production From Various Foods
Downloaded by [University of Glasgow] at 04:57 27 March 2013
Lilian U. Thompson, Paul Robb, Maria Serraino, and Felicia Cheung
Abstract Mammalian lignans such as enterolactone and enterodiol, which are produced in the colon from precursors in foods, have been suggested as playing a role in the cancer-protective effect of vegetarian diets. Despite this, very little is known regarding the amount that is produced from different food products. Therefore, the objective of this study was to determine the production of mammalian lignans from 68 common plant foods by using the technique of in vitro fermentation with human fecal microbiota, which simulates colonic fermentation. Results showed a wide range (21-67,541 µg/100 g sample) in the amount of lignans produced. On the average as a group, the oilseeds produced the highest amounts (20,461 ± 12,685), followed by the dried seaweeds (900 ± 247), whole legumes (562 ± 211), cereal brans (486 ± 90), legume hulls (371 ± 52), whole grain cereals (359 ± 81), vegetables (144 ± 23), and fruits (84 ± 22). The vegetables produced the second highest concentration of lignans (1,546 ± 280) when the data were expressed on a moisture-free basis. Flaxseed flour and its defatted meal were the highest producers of lignans (mean 60,110 ± 7,431). Lignan production with the in vitro method related well to the urinary lignan excretion observed in rats and humans. The data should be useful in the estimation of lignan production from a given diet and in the formulation of high-lignan-producing diet for the purpose of reducing the cancer risk. (Nutr Cancer 16, 43-52, 1991)
Introdbction The incidence of cancer is lower in countries where the diet is either vegetarian or semivegetarian (1-4), but the mechanism whereby vegetarian diets exert their protective effect is still unclear. Although large dietary fiber intake was thought to be partly responsible, numerous studies on the relationship between total dietary fiber intake and reduced colon cancer incidence showed conflicting results (4,5). Presumably, not only the type of fiber but also other substances associated with the fiber-containing foods are determinants of health benefits of vegetarian diets. One of these suggested substances is the lignans and their precursors (6-8). The authors are affiliated with the Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Copyright © 1991, Lawrence Erlbaum Associates, Inc.
Downloaded by [University of Glasgow] at 04:57 27 March 2013
Lignans are a group of substances that differ in structure but generally contain a dibenzylbutane skeleton (7-9). While previously thought to be constituents of higher plants only, lignans have recently been detected as well in biologic fluids of animals and humans (8,10-13). Although the evidence that lignans can reduce the risk for cancer is still unclear, the biologic properties of lignans and epidemiological data suggest their potential to do so. Many lignans have antitumor, antimitotic, antioxidant, weak estrogenic, and antiestrogenic activities (6,8,9,12-15), and some have been shown to prevent the growth of many tumors studied in the chemotherapy program of the US National Cancer Institute (14). Furthermore, urinary excretion of mammalian lignans has been found to be lower in nonvegetarians and in postmenopausal women with breast cancer compared with healthy controls (7,8,16,17). Despite the potential significance of the lignans in reducing cancer risk, very little is known regarding which foods contain them or their precursors (7,8,18). Mammalian lignans, primarily enterolactone [/rc«^-2,3-Ws(3-hydroxybenzyl)-7-butyrolactone] and enterodiol [2,3-fc(3-hydroxybenzyl)butan-l,4-diol] (10,12,13), are believed to be derived predominantly from plant lignans or other precursors in the diet (7,8). Earlier studies in germ-free rats (19) and humans administered antibiotics (20) had established that mammalian lignan production depends on the presence of bacteria in the intestinal tract. In vitro studies have also demonstrated the efficient production of mammalian lignans from the dietary precursors by human fecal flora (21). These suggest that the primary site of their production is the cecum and colon. Recently, we developed a method of in vitro fermentation using human fecal microbiota to simulate colonic fermentation (22-30). Because mammalian lignan appears to be produced during fermentative events in the cecum and colon, this in vitro method may be suitable for screening foods for the presence of plant lignans or other precursors of mammalian lignans. The objective of this study was to determine the production of mammalian lignans from various food products by using such a technique. Materials and Methods
The food samples studied for mammalian lignan production are listed in Table 1. All samples were obtained from the local supermarket and ground to pass through a 60-mesh sieve. Fruits and vegetables were freeze-dried prior to grinding. Moisture content of all fresh and freeze-dried samples was determined by an oven-drying method (31) to allow the estimation of the lignan production on both the wet (as-is) and dry basis. Samples were subjected to in vitro simulations of colonic fermentation with fresh fecal microbiota collected from normal healthy volunteers as described in our recent papers (22-30). Briefly, samples (0.5-1.0 g) were weighed into 100-ml serum bottles. Fermentation medium (40 ml) (22) was added to the samples approximately 12-24 hours prior to the start of the incubation to ensure complete hydration of the samples. The contents of the serum bottles were reduced by the addition of 2 ml reducing solution and flushing with carbon dioxide until the solution turned colorless (32). The reducing solution was prepared by mixing (1:1, vol/vol) freshly prepared solutions of Na2S-9H2O (1.25 g/100 ml) and cysteine-HCl with KOH (1.25 g and 5.0 g/100 ml, respectively). The bottles were sealed with a butyl rubber stopper and crimped metal seal (33) and stored in the refrigerator overnight to limit the possibility of microbial growth. At one to two hours prior to incubation, the bottles were placed in a 37°C water bath. Fresh human feces were collected from healthy individuals who had been eating unspecified Western diet and had not taken antibiotics for six months or more. Each fecal sample was collected into a tared blender containing 400 ml collection media, which had been warmed to 37° C and was oxygen free. The collection media consisted of distilled water,
44
Nutrition and Cancer 1991
o
Table 1. Mammalian Lignan Production From Different Foods" As-Is (Wet) Basis
Downloaded by [University of Glasgow] at 04:57 27 March 2013
Foods Oilseeds Flaxseed meal Flaxseed flour Rapeseed Soybean Sunflower Peanuts Mean ± SEM Cereal brans Oat bran Corn bran Wheat bran Barley bran Rice bran Mean ± SEM Whole cereals Triticale Wheat Purple rice Oats Brown rice Sorghum Corn Rye
Barley Mean ± SEM
Dry Basis
Enterolactone
Enterodiol
Total
Enterolactone
Enterodiol
Total
8,517 11,818
59,024 40,861 155 170 195 56
9,841 12,980 1,111
68,204 44,877
975 693 201 105
67,541 52,679 1,130
78,045 57,857 1,288
863 396 161
767 216 110
3,718 ± 2,088 265 168 269 243 134
216 ± 27 519 411 340 251 169 199 199 69 41
244 ± 52
16,743 ± 10,757 386 480 298 140 47
270 ± 79 405 79 80 89 128 56 31 91 74
115 ± 37
20,461 ± 12,685 651 648 567 383 181
486 ± 90 924 490 420 340 297 255 230 160 115
359 =fc 81
4,171 ± 2,330 303 182 296 276 151
242 ± 31 591 476 385 279 190 222 227 78 46
277 ± 59
177 188 209 58
18,952 ± 12,262 440 522 327 158 53
300 ± 87 461 91 92 99 143 62 36 103 83
130 ± 43
955 425 168
23,123 ± 14,414
743 704 623 434 204
542 ± 99 1,052 567 477 378 333 284 263 181 129
407 ± 93 (continued)
Table 1. (Continued) Dry Basis
Downloaded by [University of Glasgow] at 04:57 27 March 2013
As-Is (Wet) Basis
n
8
Foods Vegetables Garlic Squash Asparagus Carrot Sweet potato Broccoli Leek Green pepper Turnip Cauliflower Beet Snow pea Iceberg lettuce Onion String bean Potato Brussels sprout Boston lettuce Cabbage Bok choy Mushroom Watercress Radish Celery Cucumber Tomato Fiddle head Mean ± SEM
Enterolactone 81 271 136 284 240 161 24 162 78 68 109 60 58 11 40 33 57 27 30 44 43 20 25 17 18 11 14
78 ± 15
Enterodiol 326 110 238 62 55 65 174 33 78 77 26 62 63 101 56 50 18 47 34 14 13 28 10 14 11 10 7
66 ± 14
Total 407 381 374 346 295 226 198 195 156 145 135 122 121 112 96 83 75 74 64 58 56 48 35 31 30 21 21
144 ± 23
Enterolactone
Enterodiol
208
842
4,509 1,641 2,410
1,835 2,862
1,478
522 186 594
166
1,193
2,458 921 760 859
499 921 856 201
1,026 1,254
1,053 1,357
103 499 161 388 538 404 958 451 301 453 288 369 75 135
924 705 248 123
816
875 ± 186
1,112 473 293 132 420 185 230 216 156 65
674 ± 120
Total 1,050 6,344 4,503 2,932 1,002 2,072 1,359 2,957 1,840 1,616 1,060 2,079 2,611 1,027 1,204 409 511
1,650 877
1,251 583 721 638 518 585 331 200
1,553 ± 265
•z Downloaded by [University of Glasgow] at 04:57 27 March 2013
o
Dried whole legumes Lentil Kidney bean Alfalfa seed Navy bean Faba bean Yellow pea Pinto bean Mean ± SEM Legume hulls Navy bean Lentil Kidney bean Field pea bean Faba bean Mean ± SEM Fruits Pear Plum Strawberry Banana Orange Canteloupe Apple Mean ± SEM Dried seaweeds Mekuba Hijiki Mean ± SEM
789 329 328 352 129 169 154
321 ± 85 370 278 299 262 143
270 ± 37
112 47 41 55 27 21 34
48 ± 12 167 266
217 ± 50
998 232 170 108 88 44 47
241 ± 129 165 119 91 49 79
101 ± 20
69 98 38 14 12 16 1
35 ± 13 980 387
684 ± 297
1,787 561 498 460 217 213 201
562 ± 211 535 397 390 311 222
371 ± 52 181 145 79 69 39 37 35
84 ± 22 1,147 653
900 ± 247
864 377 341 399 145 185 173
355 ± 93 412 309 334 291 160
301 ± 41
670 247 400 228 192 241 223
314 ± 64 184 297
241 ± 56
1,092
1,956
266 180 144 99 48 53
643 521 543 244 233 226
269 ± 140 184 133 102 54 88
112 ± 22
410 519 376 56 84 185 6
624 ± 231
596 442 436 345 245
413 ± 58 1,080 766 776 284 276 426 229
234 ± 76
548 ± 124
1,083
1,267
432
758 ± 326
a: Values are ^g/100 g sample. They are also means of 2 fermentation experiments, each with samples fermented and analyzed in duplicate.
729
998 ± 269
Downloaded by [University of Glasgow] at 04:57 27 March 2013
fermentation medium, and reducing solution as described previously (15:5:2, vol/vol). The feces were diluted with the collection media (66.6 g wet feces/1), blended for 30 seconds to detach cellulolytic microbiota from the fecal fiber matrix, and then squeezed through a 41-/tm nylon membrane and filtered through glass wool to remove fiber particulates. This inoculum (10 ml) was injected into each serum bottle. All inoculations were finished within 10-15 min after fecal collection. Fecal contents, solutions, and containers were kept under constant flow of carbon dioxide during fecal collection and inoculum preparation to maintain anaerobiosis. Serum bottles were swirled at regular intervals. At the end of 48 hours, serum bottles were opened, 1 ml copper sulfate solution (10 g/1) was added to inactivate the microorganisms, and the final solutions were analyzed for mammalian lignans (enterolactone and enterodiol). The lignans were determined in duplicate with a slightly modified capillary gas chromatographic procedure (34,35). They were extracted from 20 ml of in vitro fermentation digesta on a reverse-phase octadecysilane bonded silica cartridge (Speed C-18; Applied Separations, Bethlehem, PA), which was then washed with 5 ml distilled water. The lignans that were absorbed to the silica particles were eluted with 4 ml methanol and taken to dryness, and the glucuronide conjugates were redissolved in 5 ml of sodium acetate buffer (0.5 M, pH 4.5) and hydrolyzed enzymatically overnight at 37°C with 50 jtl /3-glucuronidase preparation (Sigma Chemical, St. Louis, MO). The unconjugated lignans were then extracted from the hydrolysate with another C-18 cartridge and further purified and isolated on a DEAE Sephadex ion-exchange column that had been prepared in the OH-form in methanol. Lignans were recovered from the gel by eluting with a solution of methanol saturated with carbon dioxide gas, derivatized with a solution of hexamethylidisilazane-trimethylchlorosilane-pyridine (3:2:1 by vol; Sigma Chemical) with heating at 60°C for 15 min, and then analyzed by using a gas chromatograph with flame ionization detector (HewlettPackard, Avondale, PA) and hydrogen as carrier gas. Internal standards used were 5a-androstron-3/3,17/3-diol and stigmasterol (Steraloids, Wilton, NH). The results represent the mean of two fermentation runs, each with different fecal donors and each sample done in duplicate. Because fermentation was done for 48 hours, the variability between fermentation runs is low (
Nutrition and Cancer Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/hnuc20
Mammalian lignan production from various foods a
a
a
Lilian U. Thompson , Paul Robb , Maria Serraino & Felicia Cheung
a
a
Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario, M5S 1A8, Canada Version of record first published: 04 Aug 2009.
To cite this article: Lilian U. Thompson , Paul Robb , Maria Serraino & Felicia Cheung (1991): Mammalian lignan production from various foods, Nutrition and Cancer, 16:1, 43-52 To link to this article: http://dx.doi.org/10.1080/01635589109514139
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-andconditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
Mammalian Lignan Production From Various Foods
Downloaded by [University of Glasgow] at 04:57 27 March 2013
Lilian U. Thompson, Paul Robb, Maria Serraino, and Felicia Cheung
Abstract Mammalian lignans such as enterolactone and enterodiol, which are produced in the colon from precursors in foods, have been suggested as playing a role in the cancer-protective effect of vegetarian diets. Despite this, very little is known regarding the amount that is produced from different food products. Therefore, the objective of this study was to determine the production of mammalian lignans from 68 common plant foods by using the technique of in vitro fermentation with human fecal microbiota, which simulates colonic fermentation. Results showed a wide range (21-67,541 µg/100 g sample) in the amount of lignans produced. On the average as a group, the oilseeds produced the highest amounts (20,461 ± 12,685), followed by the dried seaweeds (900 ± 247), whole legumes (562 ± 211), cereal brans (486 ± 90), legume hulls (371 ± 52), whole grain cereals (359 ± 81), vegetables (144 ± 23), and fruits (84 ± 22). The vegetables produced the second highest concentration of lignans (1,546 ± 280) when the data were expressed on a moisture-free basis. Flaxseed flour and its defatted meal were the highest producers of lignans (mean 60,110 ± 7,431). Lignan production with the in vitro method related well to the urinary lignan excretion observed in rats and humans. The data should be useful in the estimation of lignan production from a given diet and in the formulation of high-lignan-producing diet for the purpose of reducing the cancer risk. (Nutr Cancer 16, 43-52, 1991)
Introdbction The incidence of cancer is lower in countries where the diet is either vegetarian or semivegetarian (1-4), but the mechanism whereby vegetarian diets exert their protective effect is still unclear. Although large dietary fiber intake was thought to be partly responsible, numerous studies on the relationship between total dietary fiber intake and reduced colon cancer incidence showed conflicting results (4,5). Presumably, not only the type of fiber but also other substances associated with the fiber-containing foods are determinants of health benefits of vegetarian diets. One of these suggested substances is the lignans and their precursors (6-8). The authors are affiliated with the Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Copyright © 1991, Lawrence Erlbaum Associates, Inc.
Downloaded by [University of Glasgow] at 04:57 27 March 2013
Lignans are a group of substances that differ in structure but generally contain a dibenzylbutane skeleton (7-9). While previously thought to be constituents of higher plants only, lignans have recently been detected as well in biologic fluids of animals and humans (8,10-13). Although the evidence that lignans can reduce the risk for cancer is still unclear, the biologic properties of lignans and epidemiological data suggest their potential to do so. Many lignans have antitumor, antimitotic, antioxidant, weak estrogenic, and antiestrogenic activities (6,8,9,12-15), and some have been shown to prevent the growth of many tumors studied in the chemotherapy program of the US National Cancer Institute (14). Furthermore, urinary excretion of mammalian lignans has been found to be lower in nonvegetarians and in postmenopausal women with breast cancer compared with healthy controls (7,8,16,17). Despite the potential significance of the lignans in reducing cancer risk, very little is known regarding which foods contain them or their precursors (7,8,18). Mammalian lignans, primarily enterolactone [/rc«^-2,3-Ws(3-hydroxybenzyl)-7-butyrolactone] and enterodiol [2,3-fc(3-hydroxybenzyl)butan-l,4-diol] (10,12,13), are believed to be derived predominantly from plant lignans or other precursors in the diet (7,8). Earlier studies in germ-free rats (19) and humans administered antibiotics (20) had established that mammalian lignan production depends on the presence of bacteria in the intestinal tract. In vitro studies have also demonstrated the efficient production of mammalian lignans from the dietary precursors by human fecal flora (21). These suggest that the primary site of their production is the cecum and colon. Recently, we developed a method of in vitro fermentation using human fecal microbiota to simulate colonic fermentation (22-30). Because mammalian lignan appears to be produced during fermentative events in the cecum and colon, this in vitro method may be suitable for screening foods for the presence of plant lignans or other precursors of mammalian lignans. The objective of this study was to determine the production of mammalian lignans from various food products by using such a technique. Materials and Methods
The food samples studied for mammalian lignan production are listed in Table 1. All samples were obtained from the local supermarket and ground to pass through a 60-mesh sieve. Fruits and vegetables were freeze-dried prior to grinding. Moisture content of all fresh and freeze-dried samples was determined by an oven-drying method (31) to allow the estimation of the lignan production on both the wet (as-is) and dry basis. Samples were subjected to in vitro simulations of colonic fermentation with fresh fecal microbiota collected from normal healthy volunteers as described in our recent papers (22-30). Briefly, samples (0.5-1.0 g) were weighed into 100-ml serum bottles. Fermentation medium (40 ml) (22) was added to the samples approximately 12-24 hours prior to the start of the incubation to ensure complete hydration of the samples. The contents of the serum bottles were reduced by the addition of 2 ml reducing solution and flushing with carbon dioxide until the solution turned colorless (32). The reducing solution was prepared by mixing (1:1, vol/vol) freshly prepared solutions of Na2S-9H2O (1.25 g/100 ml) and cysteine-HCl with KOH (1.25 g and 5.0 g/100 ml, respectively). The bottles were sealed with a butyl rubber stopper and crimped metal seal (33) and stored in the refrigerator overnight to limit the possibility of microbial growth. At one to two hours prior to incubation, the bottles were placed in a 37°C water bath. Fresh human feces were collected from healthy individuals who had been eating unspecified Western diet and had not taken antibiotics for six months or more. Each fecal sample was collected into a tared blender containing 400 ml collection media, which had been warmed to 37° C and was oxygen free. The collection media consisted of distilled water,
44
Nutrition and Cancer 1991
o
Table 1. Mammalian Lignan Production From Different Foods" As-Is (Wet) Basis
Downloaded by [University of Glasgow] at 04:57 27 March 2013
Foods Oilseeds Flaxseed meal Flaxseed flour Rapeseed Soybean Sunflower Peanuts Mean ± SEM Cereal brans Oat bran Corn bran Wheat bran Barley bran Rice bran Mean ± SEM Whole cereals Triticale Wheat Purple rice Oats Brown rice Sorghum Corn Rye
Barley Mean ± SEM
Dry Basis
Enterolactone
Enterodiol
Total
Enterolactone
Enterodiol
Total
8,517 11,818
59,024 40,861 155 170 195 56
9,841 12,980 1,111
68,204 44,877
975 693 201 105
67,541 52,679 1,130
78,045 57,857 1,288
863 396 161
767 216 110
3,718 ± 2,088 265 168 269 243 134
216 ± 27 519 411 340 251 169 199 199 69 41
244 ± 52
16,743 ± 10,757 386 480 298 140 47
270 ± 79 405 79 80 89 128 56 31 91 74
115 ± 37
20,461 ± 12,685 651 648 567 383 181
486 ± 90 924 490 420 340 297 255 230 160 115
359 =fc 81
4,171 ± 2,330 303 182 296 276 151
242 ± 31 591 476 385 279 190 222 227 78 46
277 ± 59
177 188 209 58
18,952 ± 12,262 440 522 327 158 53
300 ± 87 461 91 92 99 143 62 36 103 83
130 ± 43
955 425 168
23,123 ± 14,414
743 704 623 434 204
542 ± 99 1,052 567 477 378 333 284 263 181 129
407 ± 93 (continued)
Table 1. (Continued) Dry Basis
Downloaded by [University of Glasgow] at 04:57 27 March 2013
As-Is (Wet) Basis
n
8
Foods Vegetables Garlic Squash Asparagus Carrot Sweet potato Broccoli Leek Green pepper Turnip Cauliflower Beet Snow pea Iceberg lettuce Onion String bean Potato Brussels sprout Boston lettuce Cabbage Bok choy Mushroom Watercress Radish Celery Cucumber Tomato Fiddle head Mean ± SEM
Enterolactone 81 271 136 284 240 161 24 162 78 68 109 60 58 11 40 33 57 27 30 44 43 20 25 17 18 11 14
78 ± 15
Enterodiol 326 110 238 62 55 65 174 33 78 77 26 62 63 101 56 50 18 47 34 14 13 28 10 14 11 10 7
66 ± 14
Total 407 381 374 346 295 226 198 195 156 145 135 122 121 112 96 83 75 74 64 58 56 48 35 31 30 21 21
144 ± 23
Enterolactone
Enterodiol
208
842
4,509 1,641 2,410
1,835 2,862
1,478
522 186 594
166
1,193
2,458 921 760 859
499 921 856 201
1,026 1,254
1,053 1,357
103 499 161 388 538 404 958 451 301 453 288 369 75 135
924 705 248 123
816
875 ± 186
1,112 473 293 132 420 185 230 216 156 65
674 ± 120
Total 1,050 6,344 4,503 2,932 1,002 2,072 1,359 2,957 1,840 1,616 1,060 2,079 2,611 1,027 1,204 409 511
1,650 877
1,251 583 721 638 518 585 331 200
1,553 ± 265
•z Downloaded by [University of Glasgow] at 04:57 27 March 2013
o
Dried whole legumes Lentil Kidney bean Alfalfa seed Navy bean Faba bean Yellow pea Pinto bean Mean ± SEM Legume hulls Navy bean Lentil Kidney bean Field pea bean Faba bean Mean ± SEM Fruits Pear Plum Strawberry Banana Orange Canteloupe Apple Mean ± SEM Dried seaweeds Mekuba Hijiki Mean ± SEM
789 329 328 352 129 169 154
321 ± 85 370 278 299 262 143
270 ± 37
112 47 41 55 27 21 34
48 ± 12 167 266
217 ± 50
998 232 170 108 88 44 47
241 ± 129 165 119 91 49 79
101 ± 20
69 98 38 14 12 16 1
35 ± 13 980 387
684 ± 297
1,787 561 498 460 217 213 201
562 ± 211 535 397 390 311 222
371 ± 52 181 145 79 69 39 37 35
84 ± 22 1,147 653
900 ± 247
864 377 341 399 145 185 173
355 ± 93 412 309 334 291 160
301 ± 41
670 247 400 228 192 241 223
314 ± 64 184 297
241 ± 56
1,092
1,956
266 180 144 99 48 53
643 521 543 244 233 226
269 ± 140 184 133 102 54 88
112 ± 22
410 519 376 56 84 185 6
624 ± 231
596 442 436 345 245
413 ± 58 1,080 766 776 284 276 426 229
234 ± 76
548 ± 124
1,083
1,267
432
758 ± 326
a: Values are ^g/100 g sample. They are also means of 2 fermentation experiments, each with samples fermented and analyzed in duplicate.
729
998 ± 269
Downloaded by [University of Glasgow] at 04:57 27 March 2013
fermentation medium, and reducing solution as described previously (15:5:2, vol/vol). The feces were diluted with the collection media (66.6 g wet feces/1), blended for 30 seconds to detach cellulolytic microbiota from the fecal fiber matrix, and then squeezed through a 41-/tm nylon membrane and filtered through glass wool to remove fiber particulates. This inoculum (10 ml) was injected into each serum bottle. All inoculations were finished within 10-15 min after fecal collection. Fecal contents, solutions, and containers were kept under constant flow of carbon dioxide during fecal collection and inoculum preparation to maintain anaerobiosis. Serum bottles were swirled at regular intervals. At the end of 48 hours, serum bottles were opened, 1 ml copper sulfate solution (10 g/1) was added to inactivate the microorganisms, and the final solutions were analyzed for mammalian lignans (enterolactone and enterodiol). The lignans were determined in duplicate with a slightly modified capillary gas chromatographic procedure (34,35). They were extracted from 20 ml of in vitro fermentation digesta on a reverse-phase octadecysilane bonded silica cartridge (Speed C-18; Applied Separations, Bethlehem, PA), which was then washed with 5 ml distilled water. The lignans that were absorbed to the silica particles were eluted with 4 ml methanol and taken to dryness, and the glucuronide conjugates were redissolved in 5 ml of sodium acetate buffer (0.5 M, pH 4.5) and hydrolyzed enzymatically overnight at 37°C with 50 jtl /3-glucuronidase preparation (Sigma Chemical, St. Louis, MO). The unconjugated lignans were then extracted from the hydrolysate with another C-18 cartridge and further purified and isolated on a DEAE Sephadex ion-exchange column that had been prepared in the OH-form in methanol. Lignans were recovered from the gel by eluting with a solution of methanol saturated with carbon dioxide gas, derivatized with a solution of hexamethylidisilazane-trimethylchlorosilane-pyridine (3:2:1 by vol; Sigma Chemical) with heating at 60°C for 15 min, and then analyzed by using a gas chromatograph with flame ionization detector (HewlettPackard, Avondale, PA) and hydrogen as carrier gas. Internal standards used were 5a-androstron-3/3,17/3-diol and stigmasterol (Steraloids, Wilton, NH). The results represent the mean of two fermentation runs, each with different fecal donors and each sample done in duplicate. Because fermentation was done for 48 hours, the variability between fermentation runs is low (
