Food Phytochemicals for Cancer Prevention I - American Chemical

Chapter 30

Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 26, 2018 | https://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0546.ch030

Chemopreventive Phytochemicals in Soy and Licorice Diets Affecting Key Rat Enzyme Systems 1

2

3

4

T. E. Webb , P. C. Stromberg , H. Abou-Issa , M . Moeschberger , H. F. Pierson , and R. W. Curley, Jr. 5,7

1

6

2

Departments of Medical Biochemistry, Veterinary Pathobiology, College of Veterinary Medicine, Surgery, and Preventive Medicine, College of Medicine, The Ohio State University, Columbus, OH 43210 National Cancer Institute, Bethesda, MD 20892 Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210 3

4

5

6

As a component of a feeding study of the possible chemopreventive diet additives soybean meal and licorice root extract, simplified ex traction and HPLC methods were developed for the analysis of the soy isoflavones genistein and daidzein and the licorice triterpenoids glycyrrhizic acid and glycyrrhetinic acid. In the diet containing 25% soybean meal, genistein and daidzein were present at about 2-5 μg/g of diet although some variability suggests these isoflavones, espe cially genistein, may not be stable in frozen diet extracts. Markers glycyrrhizic acid and glycyrrhetinic acid showed the 3% licorice extract containing diet to be uniformly mixed and stable with final concentrations of 300 and 20 μg/g of diet each respectively. Of these markers, only glycyrrhetinic acid was reliably detected in the plasma of rats fed the appropriate diet with an observed concentration of 5.83 μg/ml. Recent studies suggest that diet has a marked impact on the incidence of cancer (1) and that this may be due to protective agents in foods, many of which are phyto chemicals (2). Soybeans are known to contain potential chemopreventive isofla vones and protease inhibitors (3) while licorice root contains various flavonoids (4) and triterpenoids (5) of interest. Of particular interest in this study has been the impact of these phytochemical-rich diet additives on the induction of protective enzymes or the suppression of enzymes which may increase cancer risk (6). Such enzymes could serve as intermediate end point markers in chemoprevention studies or provide clues to possible mechanisms of action of food additive phytochemicals. We have evaluated the effects of soybean meal and licorice root extract on twenty 7

Current address: Preventive Nutrition Consultants, 19508 189th Place Northeast, Woodinville, WA 98072 0097-6156/94/0546-0361$06.00/0 © 1994 American Chemical Society Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

362

FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES

potentially important enzyme systems in the male rat as well as the impact of the diets on histopathological and clinical chemistry parameters. The results, reported in detail elsewhere (7), will be summarized below. Of importance for interpreting these results was the need to establish the uniformity of food additive mixing, the stability of the phytochemicals in these modified diets, and the oral absorption of important agents from these diets. Our efforts in this regard are described in more detail herein.


Chemicals Chosen for Study Phytochemicals to be used as analytical markers for diet mixing, stability, and absorption were chosen based on the likelihood of a significant concentration in the blended diet as well as a possibility that the chemicals may be contributors to potential chemopreventive effects of the food additives. In the case of soybean, the major isoflavones genistein ( 1 , Figure 1) and daidzein (2) were selected for monitoring. These isoflavones have been reported to show estrogenic (#), antifungal (9), and antioxidant (10) activities as well as the ability to induce cyto chrome P450 in Streptomyces griseus (11). Thus, these compounds have a broad range of biological activities, many of which may be due to regulation of key enzyme systems of potential impact in chemoprevention. The principal triterpenoid in licorice root is the acidic diglucuronide glycyrrhizic acid (3). This triterpenoid is believed to have estrogenic, antiulcer, and glucocorticoid activities (12) and has been suggested to cause "pseudoaldosteronism" when ingested in large doses (13). The acidic diglucuronide 3 is readily hydrolyzed, especially in vivo (13), and thus 3 and its aglycone glycyrrhetinic acid (4) — which has shown chemopreventive potential (5) — are the two major triterpenoids from licorice root extract which were estimated. A number of novel flavonoids have been isolated from licorice root (Μ Ι 6). From among these, we chose to focus on the isoflavone formononetin (5) and the chalcone licochalcone A (6) because of their presence in reasonable concen tration in the mixed Chinese and Russian licorice root extract used in these studies (Jeffcoat, A.R., Research Triangle Institute, personal communication). The licochalcones have shown radical scavenging/antioxidant activity which may be due to reg ulation of (per)oxidizing enzymes and could be relevant to chemoprevention (16). The principal method we employed for approximate quantitation of the dietary and blood levels of these six agents was high performance liquid chromato graphy (HPLC) separation and comparison of chromatographic peak areas with standard curves prepared using an appropriate range of known analyte concen trations. Materials and Methods Standard glycyrrhizic acid and glycyrrhretinic acid were from Aldrich Chemical (Milwaukee, WI), genistein from I C N Biochemicals (Cleveland, OH), daidzein from Spectrum Chemical (Gardena, CA), formononetin from Indofine Chemical (Somerville, NJ), and licochalcone A from Arthur D. Little (Cambridge, M A ) . A l l solvents and buffers were HPLC grade from Fisher Scientific (Pittsburgh, PA). Nylon, 0.45 μηι syringe filters were from Fisher Scientific. Ultraviolet spectra were performed on a Beckman Instruments (San Ramon, CA) DU-40 spectrophotometer. H P L C analyses were performed on a Beckman Instruments Model 332 gradient liquid chromatograph system equipped with a Beckman Model 164 variablewavelength U V detector and Kipp and Zonen (Delft, Holland) B D 41 dual channel Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


30. WEBB ET AL.

Soy and Licorice Diets and Key Rat Enzyme Systems 363

1 Genistein R = H, R = OH, R = H 2 Daidzein R = R = R = H 5 Formononetin = H, R = H, R = C H 1

1

2

2

3

3

2

3

3

Ο

6 Licochalcone A Figure 1. Structure of assayed phytochemicals.

Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

364



recorder. Separations were performed on Zorbax-ODS 250 χ 4.6 mm columns (Mac-Mod Analytical, Chadds Ford, PA) equipped with a matching pre-column. Mobile phase flow rates were 1 ml/min and all sample handling and chromato graphy was performed under yellow light at ambient temperature. Animals and Diets. Male Fischer 344 rats (Harlan Labs, Indianapolis, IN) were entered into the studies at approximately 50 days old and a weight of 175-180 g. Powdered diets were replenished weekly and water provided ad libitum. Powdered AIN-76A diet was from Dyets (Bethlehem, PA). Toasted, defatted soybean meal was from Central Soya (Fort Wayne, IN). Licorice root extract was supplied through A . D. Little by McAndrew's and Forbes (Camden, NJ). Essentially isocaloric diets were prepared from control diet to contain (w/w) 25%, 12.5%, or 3.13% soybean meal (SBM), 3%, 1.5%, or 0.38% licorice root extract (LIC), and all nine combinations thereof. Diets were prepared by mixing in a Reynolds com mercial food mixer for 15 min, removed and mixed manually, then blended an additional 10 min in the mixer. Diets were stored for a maximum of two weeks. Diets were fed for 1 and 3 months to individual groups of rats. Diet Extraction Procedures. Briefly, 1 g of chosen diet was shaken for 1 hr at 25°C with 5 ml hexane, centrifuged, and the hexane (containing no analyte by U V / H P L C analysis) discarded. Diet residue was resuspended in methanol and extracted as above, centrifuged, and the methanol layer removed. The methanol extract was syringe filtered, rotary evaporated, and stored at -20°C for reconsti tution in 1 ml methanol prior to HPLC analysis of a suitable aliquot. Blood Extraction Procedures. Plasma aliquots (500 μΐ) from two rats in each experimental group were combined, shaken 30 sec with 1 ml hexane, centrifuged, the analyte free hexane layer discarded, and the process repeated. Methanol (10 ml) was added to the aqueous phase, mixed for 30 min, centrifuged, and the liquid phase removed. The extract was rotary evaporated and stored at -20°C for reconsti tution in 1 ml methanol prior to HPLC analysis. H P L C Analysis Conditions. The phytochemicals were separated and quantitated by reverse phase HPLC using 10 m M ammonium acetate-containing mobile phases of (a) methanol/water 60:40 for 1, 2, and 3, (b) methanol/water 85:15 for 4, (c) methanol/water 65:35 for 5, and (d) methanol/water 80:20 for 6. Analyses were performed under the direction of a Good Laboratory Practices consultant (Ms. Kathleen M . Zajd) who required that samples be run in duplicate with two injections (runs) per sample. If intra- and inter-sample variability was less than 5%, then the average of averages was also recorded. If intersample variability was greater than 5%, a third duplicate injection was done. If analysis of this sample agreed with one of the previous two, it replaced the other and the average of averages was recorded, otherwise all three results were reported. Analyte concen trations were estimated by the standard curve method. That is, an appropriate range of known analyte amounts were chromatographed and plotted versus corresponding peak area and the curve determined by linear regression. Chromatographic peak areas were approximated manually according to the relationship that peak area equals peak height at maximum times peak width at half height: A = h χ w . This method has been estimated to be 94% accurate and show 2.6% precision (77). Standard curves were verified after every twelve HPLC samples. m a x


h

30. WEBB ET AL.

Soy and Licorice Diets and Key Rat Enzyme Systems

Summary of Biological Results


While described in detail elsewhere (7), a summary of the impact of the diets on the rats and on key enzymes in the liver and intestinal mucosa is appropriate here. Food Consumption and Body Weights. Food consumption for the rats fed the individual diets was similar to controls except that it was 8% lower for those fed the 3% LIC diet and 23% higher for animals consuming the 25% S B M . In contrast, all animals in these groups showed weight gain of 5-15% above those of controls. For the combined diets, consumption was higher than the control group for all groups with the highest being 20% above control for the 25% S B M + 0.38% LIC fed rats. Weight gain ranged from 6% less than control for the 25% S B M + 0.38% L I C to a 13% increase with the 12.5% S B M + 0.38% LIC. Most groups were essentially identical to the controls. None of the differences were statistically significant. Histopathology and Blood Chemistry. The consumption of the diets for either one or three months caused no anatomical lesions, observable by histopathological analysis, which were attributable to the S B M or LIC diet additives. The same was true for the hematological profile of these animals. The SBM-containing diets, however, were found to cause a concentration-dependent decrease in serum cholesterol and increases in serum alkaline phosphatase, blood urea nitrogen, and phosphorous concentrations. Enzymology. A limited number of enzyme systems assessed showed changes in activity due to addition of S B M or LIC to the diet. Thus, both S B M and L I C addition caused concentration-related inductions (up to 50%) in the activities of hepatic glutathione transferase, catalase, and protein kinase C. Likewise, most S B M and LIC containing diet caused reductions (up to 50%) in liver ornithine decarboxylase activity. None of the diet additives caused other than marginal effects on enzyme activity in the intestinal mucosa nor were any additive, synergistic, or antagonistic effects observed upon combining the diet additives. Analysis of Compounds of Interest Soy Diet Extract Analysis. Because of their similar chromatographic behavior, extracts of SBM-containing diet could be prepared and analyzed simultaneously for the contained quantity of 1 and 2. Results of the HPLC analysis for the quantity of 2 in the extracts are tabulated in Table I. Samples were subjected to a variety of storage conditions and were drawn from different layers in the food mixer to assess the uniformity of diet blending. While there is substantial intersample variability for this analyte, there does not appear to be deterioration of 2 or nonuniform diet mixing. Analysis of these same extracts for the presence of 1 is presented in Table II. As opposed to the previous compounds analyzed, 1 showed a surprising variation in apparent concentration in different replicate samples. For example, this is the only compound which seemed to show any variation with regard to region of the diet mixer from which feed sample was drawn. That is, there appeared to be a significantly higher concentration of 1 at the mixer bottom. While this might appear unlikely in view of the relatively uniform distribution of the soy flavonoid 2 (Table


365

366


I), the third samples for this species showed consistently much higher concen trations of 1 than the first two samples. These third samples were prepared and analyzed sooner after extraction than the earlier extracts (which were prepared ca. 2 months before analysis). Since these earlier samples were stored frozen before anal ysis, it is tempting to speculate that the resorcinol-like structure of 1 might make it significantly more susceptible to oxidative degradation than the simple phenolic 2.


Table I. Amount of Daidzein (2) Detected in Diet Extracts Amount ([ig/g diet) Sample 1 Sample 2 Sample 3 Average

Diet 25% S B M diet (-20°C storage) 25% S B M diet (top of mixer, -20°C) 25% S B M diet (middle of mixer, -20°C) 25% S B M diet (bottom of mixer, -20°C) 25% S B M diet (4°C 2 wks, then -20°C) 25% S B M diet (RT 1 wk, then -20°C) S B M (4°C 4 wks, then -20°C) S B M (-20°C)

1.66 5.79 2.77 3.81 1.66 2.51 10.12 10.69

1.66 3.42 3.42 31.00 1.63 2.61 12.19 33.50

1.66 8.99 8.05 3.48 1.65 2.56 21.17 —

Table II. Amount of Genistein (1) Detected in Diet Extracts Amount (^g/g diet) Sample 1 Sample 2 Sample 3 Average

Diet 25% S B M diet (-20°C storage) 25% S B M diet (top of mixer, -20°C) 25% S B M diet (middle of mixer, -20°C) 25% S B M diet (bottom of mixer, -20°C) 25% S B M diet (4°C 2 wks, then -20°C) 25% S B M diet (RT 1 wk, then -20°C) S B M (4°C 4 wks, then -20°C) S B M (-20°C)

1.66 0.96 0.54 5.79 1.53 5.54 7.22 7.75

1.70 0.61 0.90 5.57 1.62 5.33 7.12 10.42

1.68 5.29 5.98 5.68 4.34 5.44 7.17 —

As a test of the above hypothesis, some of the third samples, now frozen for ca. 6 weeks, were reanalyzed for content of 1. The samples from the top, middle, and bottom of mixer showed 4.27, 4.65, and 4.21 μg/g of 1 respectively. In addition, a fresh set of fourth samples analogous to those above were prepared and showed 4.87, 4.95, and 4.29 μg/g of 1 respectively. Lastly, the sample above containing 4.21 μg/g of 1 was bubbled with compressed air for one hour and upon reanalysis showed 3.54 μg/g of genistein. The above offered rationale for variability of the concentration of 1 is plausible based on observations about sample age and the apparent impact of com pressed air on the sample. These experiments are by no means conclusive,



30. WEBB ET AL.

Soy and Licorice Diets and Key Rat Enzyme Systems 367

however, and one might suggest possible alternative explanations. For example, the earlier samples analyzed for content of 1 spent a considerably longer time as dried extracts at -20°C in glass vials. Perhaps this flavonoid binds avidly to glass in this type of situation, effectively reducing its concentration. Alternatively, the proteinbinding ability of these soy flavonoids could conceivably result in sample concentration variability, although we have no direct experimental confirmation of either supposed binding phenomena. In summary, we do not believe there is any significant variation in the concentration of 1 in the diet mix nor any decomposition of this flavonoid in the diet mix, but there may be some decomposition of this component over time when stored as a frozen diet extract. An overlay of chromatograms representing extracts of control diet, diet spiked with 1 and 2, and diet containing 25% S B M is shown in Figure 2. Licorice Extract Diet Analysis. Extracts of 3% LIC-containing diet were prepared and analyzed for the presence of 3. Again, samples were subjected to several storage conditions and drawn from different layers of the food mixer to assess the uniformity of diet blending. The results in Table III suggest that while there was some intersample variability, there was no significant degradation of 3 under any of the conditions and 3 appeared to be uniformly mixed in the diet. Note that the LIC samples were diluted 30-fold for analysis because of the high concentration of 3 and to approximate the 3% LIC diet concentrations. Similarly, the diet extracts were analyzed for the presence of the triterpenoid aglycone 4. As for 3, the results shown in Table IV suggested some intersample variability but no evidence of non-uniform mixing of diet nor significant degradation of 4 under any of the conditions.

Table III. Amount of Glycyrrhizic Acid (3) Detected in Diet Extracts Amount (^g/g diet) Sample 1 Sample 2 Sample 3 Average

Diet 3% LIC diet (-20°C storage) 3% LIC diet (top of mixer, -20°C) 3% LIC diet (middle of mixer, -20°C) 3% LIC diet (bottom of mixer, -20°C) 3% LIC diet (4°C 2 wks, then -20°C) 3% LIC diet (RT 1 wk, then -20°C) LIC (4°C 4 wks, then -20°C) LIC (-20°C)

296.59 378.06 241.30 393.71 303.94 404.53 400.27 549.53

291.88 307.02 383.12 280.41 304.09 368.65 314.48 —

294.23 210.77 206.51 242.41 304.01 251.60 259.39 339.19

It was not decided until late in this study to employ 5 and 6 as flavonoid markers of the LIC diet and its consumption. Nonetheless, we did find that as with all the other phytochemical markers investigated, when these two compounds were added to control diet our extraction procedures readily removed the analyte from the spiked diet.



368


Figure 2. HPLC overlay of control diet extract ( 1 and 2 (····), and extract of 25% S B M diet (

), extract of diet spiked with ).


30.

WEBB ET AL.


While we did not have sufficient time to quantitate either of these two materials in the experimental diets as was done for 3 and 4 (Table ΙΠ and IV), using the appropriate H P L C method described above, both 5 and 6 were found to be present in the LIC-containing diet.


Table IV. Amount of Glycyrrhetinic Acid (4) Detected in Diet Extracts Amount (^g/g diet) Sample 1 Sample 2 Sample 3 Average

Diet 3% LIC diet (-20°C storage) 3% LIC diet (top of mixer, -20°C) 3% LIC diet (middle of mixer, -20°C) 3% LIC diet (bottom of mixer, -20°C) 3% LIC diet (4°C 2 wks, then -20°C) 3% LIC diet (RT 1 wk, then -20°C) LIC (4°C 4 wks, then -20°C) LIC (-20°C)

15.46 20.12 20.03 21.06 18.90 18.19 109.36 136.66

25.77 25.42 17.81 17.86 25.69 20.25 107.66

18.03 23.09 21.78 24.44 27.52 30.11 142.52

108.51 139.59

Combined Diet Extract Analysis. Due to time constraints and because 4 was the only substance we could reliably detect in rat blood extracts (see below), a brief survey of the combined L I C - S B M diet was conducted to determine whether there was any unusual interactions that occurred with this diet combination leading to any enhanced/reduced recovery of 4. This approach was chosen to maximize the opportunity to acquire quality data of importance to the study. With this in mind, two samples of the diet prepared as a 25% S B M + 3% LIC combination were extracted and found to contain 26.46 and 27.20 μg/g of 4 respectively. Comparing these results with the data in Table IV, it would appear there is little influence on the observed concentration of this substance in the diet after combining the S B M and LIC in a single diet. Blood Extract Analyses. As with the diet extracts, preliminary experiments were conducted in which it was found that all six of our chosen analytes when added to control rat plasma were extractable from control rat plasma using our methods. Further experiments were conducted on the plasma samples from rats receiving the low concentrations of feed additive in the preliminary study. As opposed to the diet extracts, the presence of interfering substances extracted from the plasma posed a more significant problem using our simple techniques in the face of apparently very low concentrations of the compounds under investigation. In these early surveys, it appeared that perhaps only 4 could be reliably identified under our established protocols and, with perhaps some difficulty, 2 might also be quantified. Thus, we turned to study of plasma extracts from the rats fed the diets con taining the high dietary additive conditions for one month. In these samples, while it was somewhat easier to observe 4 and even 2 in the appropriate samples, the remaining compounds remained difficult to unequivocally identify and quantitate in the absence of feeding radiolabeled test compound. In the case of 2, we have observed the compound in 2 of 6 of the plasma samples investigated with estimated


369


370


concentrations of 2.67 and 2.68 μg/ml respectively (detection limit estimated to be 1.25 μg/ml). The aglycone 4 has clearly been observed in all 4 samples evaluated with an apparent concentration of 5.84 ± 0.43 μg/ml. Since 4 was the only marker we could reliably detect in the plasma of LICfed rats, as with the diet combination, we explored the possibility that feeding the combined 25% S B M + 3% LIC diet to rats might have some impact on the ob served concentration of 4. Thus, two of these rat blood samples were extracted and found to contain 7.63 and 8.04 μg/ml of 4 respectively. While insufficient samples were analyzed to make any firm statistical comparison, these values do not appear to differ dramatically from those found above for the LIC diet fed rats alone. Summary and Conclusions Long-term feeding to Fisher 344 rats of S B M - or LIC-containing diets had little remarkable deleterious effect on the animals. At least four hepatic enzyme systems potentially important with regard to chemoprevention, however, experienced changes in their level of activity because of the diet additives (7). Increasing doses of S B M - and LIC-containing diets caused concentration-dependent inductions (up to 50%) of liver glutathione transferase, catalase and protein kinase C activities and reductions (up to 50%) of hepatic ornithine decarboxylase activity. These enzymes are generally thought to be protective or indicative of lowered risk for cancer. Using the triterpenoids 3 and 4 as markers and as representative agents likely to influence the chemopreventive activity of licorice root extract, H P L C analysis suggested the experimental LIC-containing diets were evenly mixed and the phytochemical components were stable in the diet. The final concentrations of 3 and 4 in these diets were about 300 and 20 μg/g of 3% LIC-containing diet respec tively. The potentially important licorice root flavonoids 5 and 6 were also found to be present in the LIC diet but were not quantitated. The important isoflavones 1 and 2 were used as markers for phytochemical stability in the soybean meal containing diets. For both of these components, there was greater intersample variability in the apparent concentration of the compounds. Thus, while both isoflavones showed concentrations of about 2-5 μg/g in 25% SBM-diet, some evidence gathered suggested that long-term storage of frozen SBM-diet extracts resulted in apparent decreases in isoflavone content. While not clear, this observation may be due to oxidative degradation, binding to the glass vial walls or avid protein-binding of these isoflavones. The mixing of these two dietary additives in a single feed appeared to have little additional effect on the animals nor the concentration or stability of the assayed phytochemicals. Finally, after long-term feeding of the diets containing the high S B M or LIC concentrations, using our methods few of the target phytochemicals could be reliably detected in the plasma of the treated rats. Only triterpenoid 4 was readily detected as a marker for LIC phytochemical absorption with a concentration of 5.84 ± 0.43 μg/ml. The soy isoflavone 2 was detected less consistently and showed a concentration of about 2.68 μg/ml in about one-third of the plasma samples investigated. It would appear that given our protocols and methods, detection of absorbed dietary compounds 1-6 is difficult, with the exception of 4, in the absence of more sensitive techniques such as the feeding of radiolabeled phytochemicals.


30. WEBB ET AL.


Acknowledgment This study was supported by the Division of Cancer Prevention and Control, National Cancer Institute under Contract NO1-CN-05261-01.


Literature Cited 1. Doll, R.; Peto, R. J. Natl. Cancer Inst. 1981, 66, 1191-1308. 2. Ames, B. Science 1983, 221, 1256-1258. 3. Messina, M.; Barnes, S. J. Natl. Cancer Inst. 1991, 83, 541-546. 4. Mitscher, L . Α.; Drake, S.; Gollapudi, S. R.; Harris, J. Α.; Shankel, D. M. In Antimutagenesis and Anticarcinogenesis Mechanisms; Shankel, D. M., Ed.; Plenum: New York, 1986; pp 153-165. 5. Wang, Ζ. Y . ; Agarwal, R.; Zhou, Z. C.; Bickers, D. R.; Mukhtar, H . Carcino genesis 1991, 12, 187-192. 6. Sparins, V. C.; Chuan, J.; Wattenberg, L. W. Cancer Res. 1982, 42, 205-209. 7. Webb, T. E.; Stromberg, P. C.; Abou-Issa, H . ; Curley, R. W., Jr.; Moeschberger, M. Nutr. Cancer, 1992, 18, 215-230. 8. Drane, H . M.; Patterson, D. S. P.; Roberts, Β. Α.; Saba, N . Food Cosmet. Toxicol. 1980, 18, 425-427. 9. Kramer, R. P.; Hindorf, H.; Jha, H . C.; Kallage, J.; Zilliken, F. Phytochemistry 1984, 23, 2203-2205. 10. Pratt, D. E.; Birac, P. M . J. Food Sci. 1979, 44, 1720-1722. 11. Saviaslani, F. S.; Kunz, D. A . Biochem. Biophys. Res. Commun. 1986, 141, 405-410. 12. Duke, J. A . Handbook of Medicinal Herbs; CRC Press: Boca Raton, FL, 1985; pp 215-216. 13. Sakiya, Y.; Akada, Y.; Kawano, S.; Miyauchi, Y . Chem. Pharm. Bull. 1979, 27, 1125-1129. 14. Litivenko, V. I. Dokl. Akad. Nauk SSR 1964, 155, 600-606. 15. Mitscher, L . Α.; Park, Y . H . ; Clark, D.; Beal, J. L . J. Nat. Prods. 1980, 43, 259-269. 16. Hatano, T.; Kagawa, H.; Yasuhara, T.; Okuda, T. Chem. Pharm. Bull. 1988, 36, 2090-2097. 17. Yost, R. W.; Ettre, L . S.; Conlon, R. D. Practical Liquid Chromatography: An Introduction; Perkin-Elmer: Norwalk, CT, 1980; pp 216-241. RECEIVED July 27,

1993


371

Food Phytochemicals for Cancer Prevention I - American Chemical

Recommend Documents