Purification and Antioxidant Activities of Soybean Isoflavones

. The formation of the four COPs, 5,6α-EP, 5,6β-EP, 7-keto, and 7β-OOH occurred quickly and steadily increased within the 48 hr of reaction. However, the formation rates were different, with about 21percent of the cholesterol being oxidized to 7β-OOH from 24hr to 48hr of reaction, while only 7.03, 2.29, and 2.44 percent of cholesterol was oxidized to 5,6α-EP, 5,6β-EP and 7-keto respectively from 24hr to 48hr of reaction. (Degradation) (Epoxidation) Degradation products Cholesterol 5,6-EP (Free radical chain reactions) 7-OOH (Reduction) 7-OH 7-keto (Dehydration) (Dehydrogenation) Figure 4. Major pathways of cholesterol oxidation. 33 Figure 5. Chromatogram of cholesterol oxidation products after 24 hr of oxidation with 5 mM AAPH and 10 mM cholesterol: (peak 1) 5,6α-EP; (peak 2) 5,6β-EP; (peak 3) 7- keto; (peak 4) 7β-OOH. Figure 6. Percentage of cholesterol oxidized to 5,6α-EP, 5,6β-EP, 7-keto, and 7β-OOH accelerated by AAPH at different oxidation times: (!) 7β-OOH; (!) 5,6α-EP; (") 7- keto; (#) 5,6β-EP. AU 0.00 0.01 0.02 0.03 0.04 Minutes 5.00 10.00 15.00 1 2 3 4 0 5 10 15 20 25 30 35 40 0 12 24 36 48 Time (hr) Pe rc en ta ge 34 4.2.2 Antioxidant Activities of Genistein, Daidzein, and Glycosides Mixture from Defatted Soybean Flour The structure-activity relationships for antioxidant activities of flavonoids have been studied by different groups, with the same conclusion that hydroxyl substitutions on the B-ring and the A-ring are the determinants of antioxidant capacity. Hydroxyl substitutions on the B-ring especially affect the antioxidant potencies of flavonoids. In the case where the B-ring could not contribute to the antioxidant activities of flavonoids, hydroxyl substitutions on the A-ring would become a larger determinant of flavonoid antioxidant activity (Arora et al., 1998; Silva et al., 2002). The two pharmacophores, the 4’ hydroxyl group on the B-ring and the hydroxyl groups on the AC-ring (Figure 1), which could be hydrogen/electron donators to scavenge free radicals, account for soybean isoflavones’ antioxidant activity (Heijnen et al., 2002). Although genistein and daidzein have the same B-ring structure, because genistein has one more hydroxyl group than daidzein on the AC-ring, genistein might have higher free radical scavenging capacity than daidzein. In a cell culture model with HL-60 cells, genistein was revealed to exert higher antioxidant activities than daidzein to inhibit TPA-induced H2O2 formation and xanthine/xanthine oxidase-caused O2- generation (Wei et al., 1995). Genistein also was shown to prevent glucose mediated LDL oxidation more effectively than daidzein (Exner et al., 2001). However, this is not always true. Neither genistein nor daidzein had influence on LDL oxidation resistance, while both exerted extremely high antioxidant activities against LDL oxidation after being esterified (Meng et al., 1999). The authors postulated that esterification of soybean isoflavones provided them lipophilicity needed for incorporation of soybean isoflavones into LDL. So the similarities between soybean isoflavones and chemicals that need protection is of significant importance relative to 35 soybean isoflavones’ antioxidant activities because the analogous characteristics could enable soybean isoflavones to be closer to their target chemicals thus exerting antioxidant effects more efficiently. In addition, it has been suggested that an antioxidant mechanism other than free radical scavenging reaction may account for soybean isoflavones’ antioxidant effects since they exerted different reactivity to the oxidative stress caused by copper or peroxy radicals (Kerry and Abbey, 1998; Hwang, et al., 2000). For example, it was found that both free radical scavenging and iron chelating describe flavonoids’ antioxidant activities, although free radical scavenging might play the major role (Acker et al., 1996). Figure 7 shows the percentage of cholesterol oxidized to 5,6α-EP, 5,6β-EP, 7- keto, and 7β-OOH in the genistein, daidzein, or control group after 24 hr or 48 hr of reaction. Both genistein and daidzein exhibited very high antioxidant activity because each group had significantly less COPs than control group after 24 hr or 48 hr of reaction (p<0.05). The percentage of cholesterol oxidized to 5,6α-EP, 5,6β-EP, 7-keto and 7β- OOH accelerated by AAPH with either 1 mM genistein or 1 mM daidzein treatment after 24 hr or 48 hr of reaction is listed and compared in Table 2. Genistein had higher antioxidant activities against the formation of 5,6β-EP and 7-keto than daidzein (p=0.0007 and p=0.0615 for 5,6β-EP and 7-keto at 24 hr of reaction; p=0.0010 and p=0.0019 for 5,6β-EP and 7-keto at 48 hr of reaction). However, the difference between genistein and daidzein to inhibit the formation of 5,6α-EP and 7β-OOH was not significant (p>0.05). Soybean isoflavones have different affinity to their substrate and their antioxidant activities are substrate dependent: the higher the affinity that isoflavones have to their substrate, the more efficiently they would exert their antioxidant activities. 36 Figure 7. Percentage of cholesterol oxidized to 5,6α-EP, 5,6β-EP, 7-keto and 7β-OOH accelerated by AAPH with or without soybean isoflavone treatment after 24 hr or 48 hr of reaction: (bars represent from left to right in each group) control; genistein; daidzein. Significant differences in percentage (p< 0.05) are expressed by a different letter at each oxidation time. Table 2. Comparison of percentages of cholesterol oxidized to 5,6α-EP, 5,6β-EP, 7-keto and 7β-OOH accelerated by AAPH with either 1 mM genistein or 1 mM daidzein treatment after 24 hr or 48 hr of reaction (date were represented as mean ± SD) Genistein (%) Daidzein (%) p 24 hr 5,6α-EP 0.09±0.02 0.13±0.04 0.6297 5,6β-EP 0.21±0.03 0.44±0.01 0.0007 7-keto 0.35±0.02 0.54±0.03 0.0615 7β-OOH 0.24±0.08 0.55±0.12 0.1654 48hr 5,6α-EP 0.18±0.01 0.39±0.06 0.4811 5,6β-EP 0.33±0.03 0.75±0.06 0.0010 7-keto 0.62±0.13 1.05±0.04 0.0019 7β-OOH 0.54±0.03 1.62±0.10 0.2136 0 2 4 6 8 10 12 14 24hr 48hr pe rc en ta ge 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 24hr 48hr pe rc en ta ge 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 24hr 48hr pe rc en ta ge 0 5 10 15 20 25 30 35 40 24hr 48hr pe rc en ta ge (1) 5,6α-EP (2) 5,6β-EP (3) 7-keto (4) 7β-OOH a b b a b b a b b a b b a c b a c b a c b a c b 37 For example, genistein was found to have higher affinity to liposomal membrane than daidzein and aglycones had higher affinity to liposomal membrane than glycosides (Murota et al., 2002). According to Sigma production information, daidzein has higher lipophility than genistein (daidzein has one less hydroxyl group than genistein), so daidzein might have greater ability to associate with cholesterol in the small droplets of the emulsion. Thus daidzein could exert its antioxidant activity more efficiently than genistein, which would compensate for the fact that daidzein has lower radical quenching capacity and explain the similar antioxidant activity between genistein and daidzein against cholesterol oxidation to 5,6α-EP and 7β-OOH. As with the formation of different COPs under different conditions, the formation of COPs might have different susceptibility to certain antioxidants. For example, the formation of 7-keto was retarded by BHA, BHT, and a-tocopherol markedly, but the formation of 7α-OH and 7β-OH was reduced to a lesser degree (Csallany et al., 2002). The percentages of cholesterol oxidized to 5,6α-EP, 5,6β-EP, 7-keto, or 7β-OOH accelerated by AAPH with or without soybean isoflavone treatment at different reaction times are shown in Figure 8. The rates for the formation of 7β-OOH, 7-keto, 5,6α-EP, and 5,6β-EP were calculated as the slopes of the lines. They all decreased dramatically with either genistein or daidzein treatment compared to control (Table 3). Both genistein and daidzein had significantly stronger inhibition to the formation of 7β-OOH than to 7- keto. 7β-OOH, which could be dehydrated to 7-keto, is the precursor of 7-keto in the cholesterol oxidation reaction initiated by free radicals (Figure 3), so soybean isoflavones might have stronger inhibition to peroxidation of cholesterol, the former step of chain reaction, than to dehydration, the later step of the chain reaction. The formation of 5,6α- 38 Figure 8. Percentage of cholesterol oxidized to 5,6α-EP, 5,6β-EP, 7-keto, and 7β-OOH accelerated by AAPH with or without soybean isoflavone treatment at different reaction times: (!) control; (") daidzein; (#) genistein. Table 3. Percentage of formation rates for 7β-OOH, 7-keto, 5,6α-EP, and 5,6β-EP decreased by either genistein or daidzein treatment compared to control (unit for formation rate: percentages per hour) 7β-OOH 7-keto Treatment Formation rate Decreased percentage Rate Decreased percentage Genistein 0.0254 97.24% 0.0273 64.91% Daidzein 0.0498 91.06% 0.0397 48.97% Control 0.5570 0.0778 5,6α-EP 5,6β-EP Treatment Formation rate Decreased percentage Rate Decreased percentage Genistein 0.0065 96.02% 0.0154 82.32% Daidzein 0.0093 94.30% 0.0324 62.80% Control 0.1632 0.0871 0 2 4 6 8 10 12 14 0 12 24 36 48 Time(hour) Pe rc en ta ge 0 1 2 3 4 5 0 12 24 36 48 Time(hour) Pe rc en ta ge 0 1 2 3 4 5 0 12 24 36 48 Time(hour) Pe rc en ta ge 0 5 10 15 20 25 30 35 40 0 12 24 36 48 Time(hour) Pe rc en ta ge (1) 5,6α-EP (2) 5,6β-EP (3) 7-keto (4) 7β-OOH 39 EP and 5,6β-EP are usually investigated together because they have nearly the same formation pathway. The formation of 5,6α-EP and 5,6β-EP were studied separately in this study and the inhibition to the formation of 5,6α-EP by either genistein or daidzein treatment was much stronger than to 5,6β-EP, which could arise from differences in stability between 5,6α-EP and 5,6β-EP, or favorable chiral structure of genistein and daidzein to 5,6α-EP. 7β-OOH had a much higher formation rate than 7-keto and the formation rate of 5,6α-EP was also higher than that of 5,6β-EP, so the inhibition by soybean isoflavone might be related to the formation rate of COPs: the higher the formation rate, the stronger the inhibition from soybean isoflavones. These results suggest that different biomarkers should be used in the study of cholesterol oxidation under different conditions and more than one biomarker should be used to study the antioxidant activity of compounds of interest when using a cholesterol oxidation model. The percentages of cholesterol oxidized to 5,6α-EP, 5,6β-EP, 7-keto, and 7β- OOH accelerated by AAPH with or without glycosides mixture treatment at different reaction times are shown in Figure 9. The formation rate calculated as the slope of the lines for each COPs is shown in Table 4. In a manner similar to geinistein and daidzein, the glycosides mixture had much stronger inhibition to the formation of 7β-OOH than to 7-keto and had stronger inhibition to the formation of 5,6α-EP than to 5,6β-EP. In order to compare the antioxidant activities between aglycones and the glycosides mixture, it was assumed that antioxidant activity would be linear within the molar concentration range used in this study. Although genistein, daidzein, and the glycosides mixture had different molar concentrations in the reaction system, according to this assumption, their antioxidant activities could be compared relative to molar concentration and the results 40 Figure 9. Percentage of cholesterol oxidized to 5,6α-EP, 5,6β-EP, 7-keto, and 7β-OOH accelerated by AAPH with or without glycosides mixture treatment at different reaction times: (!) control; (!) glycosides mixture. Table 4. Percentage of formation rates for 7β-OOH, 7-keto, 5,6α-EP, and 5,6β-EP as affected by glycosides mixture compared to control (unit for formation rate: percentages per hour) 7β-OOH 7-keto Treatment Formation rate Decreased percentage Rate Decreased percentage Mixture 0.1872 66.39% 0.0657 15.55% Control 0.5570 0.0778 5,6α-EP 5,6β-EP Treatment Formation rate Decreased percentage Rate Decreased percentage Mixture 0.0330 79.78% 0.0615 29.39% Control 0.1632 0.0871 0 2 4 6 8 10 12 14 0 12 24 36 48 Time(hour) Pe rc en ta ge 0 1 2 3 4 5 0 12 24 36 48 Time(hour) Pe rc en ta ge 0 1 2 3 4 5 0 12 24 36 48 Time(hour) Pe rc en ta ge 0 5 10 15 20 25 30 35 40 0 12 24 36 48 Time(hour) Pe rc en ta ge (1) 5,6α-EP (2) 5,6β-EP (3) 7-keto (4) 7β-OOH 41 are shown in Table 5. The glycosides mixture had lower antioxidant activities than the aglycones, genistein and daidzein (p<0.05), which might arise from the fact that glycosides have one less hydroxyl group because of the binding of glucose. As the aglycones have higher affinity to liposomal membrane than glycosides (Murota et al., 2002), aglycones might also have higher affinity to cholesterol droplets than glycosides because of their higher lipophilicity, so alycones would exert antioxidant activities against cholesterol oxidation more efficiently than glycosides. Also, the glycosides mixture is composed of genistin and daidzin, together with glycitin that might have lower antioxidant activity because of its methoxyl substitution instead of hydroxyl substitution on the B-ring (Figure 1) (Arora et al., 1998). The study of the different antioxidant activities between glycosides and aglycones would be mute for in vivo study because most of the glycosides would be converted to aglycones by intestinal microorganisms during absorption (Adlercreutz, 1995). However, this could have significance in vitro. For example, it may be beneficial to treat the soy products in a way that would convert glucosides to aglycone forms in order to increase antioxidant capacity. Table 5. Comparison of percentages of cholesterol oxidized to 5,6α-EP, 5,6β-EP, 7-keto and 7β-OOH accelerated by AAPH with 1 mM different soybean isoflavone treatment after 24 hr or 48 hr of reaction (date were represented as mean ± SD) Genistein (%) Daidzein (%) Glycosides mixture (%) p 24 hr 5,6α-EP 0.09±0.02 0.13±0.04 0.34±0.06 0.0009 5,6β-EP 0.21±0.03 0.44±0.01 0.60±0.06 <0.0001 7-keto 0.35±0.02 0.54±0.03 0.64±0.14 0.0120 7β-OOH 0.24±0.08 0.55±0.12 1.58±0.27 0.0002 48hr 5,6α-EP 0.18±0.01 0.39±0.06 1.69±0.36 0.0002 5,6β-EP 0.33±0.03 1.75±0.06 1.09±0.10 <0.0001 7-keto 0.62±0.13 1.05±0.04 1.25±0.06 0.0003 7β-OOH 0.54±0.03 1.62±0.10 7.09±1.19 <0.0001 42 4.3 Antioxidant Activities of Soybean Isoflavones on tert-Butyl Hydroperoxide- Induced Oxidative Stress in SVEC4-10 Cells 4.3.1 Growth Curve of SVEC4-10 Cells SVEC4-10 is an endothelial cell line derived by SV40 transformation of endothelial cells from mouse auxiliary lymph node vessels. They have epithelial morphology and form branching tube-like networks when adhering and growing on the surface of culture plates. The growth curve of SVEC4-10 cells was a sigmoidal line (Figure 10). Cells grew slowly when the density was below 2×105 cell/cm2 and then divided and grew very fast from the density of 2×105 cells/cm2 to 5×105 cells/cm2. After that, the growth of SVEC-4-10 reached a plateau because the growth area had been occupied completely. In order to decrease variance, the plated density of 2×105 cells/cm2 was applied in each experiment so the density on the third day would be within the lag phase of growth. Figure 10. The growth curve of SVEC-4-10 cells with the plated cell density of 8.7×104 cell/cm2 0 200000 400000 600000 0 1 2 3 4 5 6 Day Ce lls /c m 2 43 4.3.2 Cytotoxicity of Genistein and Daidzein According to pharmaceutical studies, plasma concentration of soybean isoflavones could range from 0.55 mM to 0.86 mM after two weeks administration of soy beverage (Barnes et al., 1996). However, in this study, the concentration that did not alter the viability of SVEC4-10 cells over a 24 hr incubation compared to control cells only ranged from 0 µM to 1 µM. Both genistein and daidzein caused cell viability to decrease in a dose dependent manner when the concentration reached above 1 µM (Figure 11). Figure 11. Effect of soybean isoflavones on the viability of SVEC4-10 cells over a concentration ranging from 0 µM to 50 µM for 24 hrs (cell viability was expressed by percentage of total cellular activity): (#) daidzein; (!) genistein. Genistein caused a more severe decrease in cell viability than daidzein at the same concentration. Cell viabilities after 24 hr of genistein or daidzein treatment are listed and compared in Table 6. Only at the concentrations of 2.5 µM and 5 µM was there no significant difference between the genistein and daidzein’s cytotoxicity (p=0.2105 for 2.5 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 Concentration (uM) Pe rc en ta ge 44 µM and 0.0782 for 5 µM). Significant differences were found when the concentrations were above 5 µM. For example, at 50 µM, daidzein treatment maintained 88.73 ± 12.11% of cell viability, while genistein treatment maintained only half of the cell viability compared to control. At 100 µM, daidzein treatment maintained 83.10 ± 7.62% of cell viability, while genistein treatment caused the cell viability to decrease to 9.73 ± 8.91%. Obviously, genistein had a higher cytotoxicity than daidzein to SVEC4-10 cells. The anti-proliferation action of soybean isoflavones has been studied with a number of cancer cell lines and many mechanisms have been postulated, such as modulation of estrogen receptor signal and inhibition of tyrosine kinase. However, in a test with yeast estrogen screening, only genistein was found to induce an estrogen signal, while no signal was detected for either daidzein or glycitein (De-Boever and Verstraete, 2000). SVEC4-10 cells have characteristics similar to cancer cells because they could grow indefinitely without special addictives. After a latency period of 14 weeks, they would induce spindle tumors with some of the histo-pathologic characteristics of human Kaposi Sarcoma (O’Connell et al., 1991). Table 6. Comparison of cell viabilities after different concentrations of genistein or daidzein treatment (data were represented as mean ± SE; probability of p<0.05 was considered statistically significant) Concentration (µM) 2.5 5 10 25 Genistein (viability %) 94.69±5.09 80.34±11.00 66.64±13.81 56.55±9.62 Daidzein (viability %) 99.11±0.79 96.42±4.37 96.19±3.57 92.59±7.26 P 0.2105 0.0782 0.0230 0.0066 Concentration (µM) 50 100 Genistein (viability %) 46.52±10.07 9.73±8.91 Daidzein (viability %) 88.73±12.11 83.10±7.62 P 0.0097 0.0004 45 In order to avoid any possible cytotoxic effects, the concentration of 1 µM, where daidzein treatment maintained 100± 0% of cell viability and genistein treatment maintained 99.2 ± 1.39%, was chosen to test their antioxidant activity in cells. 4.3.3 Cytotoxicity of tBOOH Oxidative stress induced by tBOOH could cause DNA damage and membrane integrity loss, ultimately leading to cell death. Figure 12 shows the effect of tBOOH ranging from 100 µM to 500 µM on cell viability of SVEC4-10 cells. A dose-dependent effect was evident. At 100 µM, tBOOH caused the cell viability to decrease to 73.01 ± 15.38% of the control after 5 hr of incubation. A similar decrease was obtained after 3 hr of incubation with 250 µM tBOOH and after 2 hr with 500 µM tBOOH. On the basis of this result, to investigate the antioxidant activity of soybean isoflavones, a concentration of 500 µM was chosen in order to induce a substantial oxidative stress and cause the cell viability to decrease substantially compared to control. Figure 12. Decreased viability of SVEC4-10 cells in response to increasing concentration of tBOOH (cell viability was expressed by percentage of total cellular activity): (!) 0 µM; (#) 100 µM; (") 250 µM; (!) 500 µM. 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 Time (hr) Pe rc en ta ge 46 4.3.4 Antioxidant Activities of Genistein and Daidzein After being absorbed, soybean isoflavones would either circulate in the blood steam or be incorporated into tissues. Circulating soybean isoflavones might offer protective activity as much as incorporated isoflavones, however, almost all of the previous research that has been done in this area used incorporated isoflavones to study soybean isoflaovnes’ antioxidant activities in vivo or in vitro. In order to elucidate how soybean isoflavones exert their antioxidant activity in vivo, two approaches were used here to investigate the protective activity of genistein and daidzein against tBOOH induced oxidative stress in SVEC4-10 endothelial cells. In the first approach (method A), soybean isoflavones were added into the cell culture media together with tBOOH at the same time, which simulated the circulating isoflavones because soybean isoflavones had not been incorporated into SVEC4-10 cells before tBOOH treatment. The result is shown in Figure 13. Different from what had been expected, no significant difference was found between the cell viability of control group and that of experimental groups within 5 hr of tBOOH treatment. In the second approach (method B), which was designed to simulate the incorporated soybean isoflavones, cells were incubated with soybean isoflavone solution for 24 hrs and the non-attached genistein and daidzein was washed away before the cell was treated with tBOOH. Thus, only incorporated soybean isoflavones would be in this experimental model. The results are shown in Figure 14. As with the results from method A, no significant difference was found between the cell viability of control group and that of experimental groups. The results demonstrated that soybean isoflavones, whether incorporated or non- incorporated, did not exert enough antioxidant activities against tBOOH induced 47 Figure 13. Cellular viability after soybean isoflavone treatment against oxidative stress caused by tBOOH on SVEC4-10 cells (method A) (cell viability was expressed by percentage of total cellular activity): (!) genistein; (#) daidzein; (") control. Figure 14. Cellular viability after soybean isoflavone treatment against oxidative stress caused by tBOOH on SVEC4-10 cells (method B) (cell viability was expressed by percentage of total cellular activity): (!) genistein; (#) daidzein; (") control. 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 Time (hr) Pe rc en ta ge 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 Time (hr) Pe rc en ta ge 48 oxidative stress in SVEC4-10 cells. The reason might be that the concentration of 1 µM was too low for soybean isoflavones to exhibit any discernable antioxidant activities in method A. The incorporation of soybean isoflavones into LDL has been studied and the incorporation ratio was very low (approximately 3-4%), so copper-mediated oxidation of control LDL and LDL isolated from plasma pre-incubated with genistein was not significantly different (Kerry and Abbey, 1998). Tikkanen et al., (1998) examined the different oxidation resistances between control LDL and LDL pre-incubated with soybean isoflavones but suggested that it was not soybean isoflavones’ antioxidant activities directly but modified LDL that was produced by circulating isoflavones that promoted the oxidation resistance of LDL. In this research, the incorporation ratio of soybean isoflavones into cells might be very low due to the low concentration of 1 µM and the limited incubation time of 24 hr may have limited the potential antioxidant activity of soybean isoflavones in method B. Soybean isoflavones have also been reported to promote endogenous antioxidant enzymes, by which they exhibit their special “antioxidant activity”, but the limited incubation time in our research decreased the possible influence that soybean isoflavones might exhibit on the antioxidant system of cells. Moreover, the toxic effects that originated from tBOOH may include S-thiolation of some key proteins thus inhibiting protein synthesis (Latour, et al., 1999), the effects of which could not be recovered by any antioxidant activity alone. These reasons help to explain why treatments had the same cell viabilities as controls in this research. Therefore, some modifications are needed in order to improve this cell culture model, such as increasing the treatment concentration, increasing the treatment duration, or changing the oxidative stress initiator. 49 Perhaps of even greater importance could be that this cell culture model is not suitable for the study of antioxidant activities, but it might be applied to the study of anti-caner activities of soybean isoflavones that is becoming a more and more popular research area. The cell viabilities of the 500 µM group (C group) in tBOOH cytotoxicity study and the two control groups in method A (A group) and method B (B group) are listed in Table 5. All three of these groups used 500 µM tBOOH treatment, but both A and B groups had higher cell viability than C at the same treatment time. The only difference among these groups was that there was 0.7% of dimethyl sulfoxide (DMSO) in A and B groups but not in the C group. The cell viability difference might come from DMSO that exerted some antioxidant activity against tBOOH induced oxidative stress, and thus increased the cell viabilities in both A and B groups compared to C group. Table 7. Comparison of cell viabilities of the 500 µM group (C group) in cytotoxicity study of tBOOH and the control groups in method A (A group) and method B (B group) Group/Time (hr) 1 2 3 4 5 A (%) 90.58±2.40 84.84±5.84 79.88±6.60 73.84±8.80 64.86±7.94 B (%) 93.3±8.15 91.53±8.61 89.12±8.79 84.81±6.47 79.35±9.46 C (%) 84.66±8.09 71.08±13.03 64.09±11.79 58.75±14.47 52.14±18.27 50 Chapter 5 Conclusion Soybean isoflavones, including genistein, daidzein, and glycitein and their derivatives, exist mainly in the glycoside form in the soybean. In order to prevent any possible enzymatic hydrolysis that may break down the glycoside groups, methanol was used as solvent to extract soybean isoflavones from defatted soy flour. A reversed phase HPLC method was developed and used to purify soybean isoflavone extract and the purity of the final product reached 94% based on an analytical HPLC analysis. Genistein might have higher antioxidant activities than daidzein because it has the same B-ring structure as daidzein yet has one more hydroxyl group on the AC-ring. No significant difference was found between the antioxidant activities of daidzein and genistein against cholesterol oxidation to 5,6α-EP and 7β-OOH, although genistein exerted higher inhibition to the formation of 5,6β-EP and 7-keto than daidzein. The substrate dependent characteristic of soybean isoflavones helps to explain the above result. Because daidzein has higher lipophilicity than genistein (daidzein has one less hydroxyl group than genistein), daidzein might have greater ability to associate with cholesterol in the small droplets of the emulsion, thus exerting antioxidant activity more efficiently than genistein, which could compensate for daidzein’s lower radical quenching capacity. The cholesterol oxidation model results also indicated that isoflavone aglycones might have higher antioxidant activities than their glycosides. This would not be important in vivo since most of the glycosides would be cleaved to form 51 aglycones by intestinal microorganisms during their absorption in the GI system but could have significance in cholesterol containing foods. Cholesterol could be oxidized to form different COPs under different conditions and the formation of COPs might have different susceptibility to certain antioxidants. In this cholesterol oxidation model, all three treatments, genistein, daidzein, and the glycosides mixture, had higher inhibition to the formation of 7β-OOH than to 7-keto and had higher inhibition to the formation of 5,6α-EP than to 5,6β-EP. This suggests that different biomarkers should be used in the study of cholesterol oxidation under different conditions and more than one biomarker should be used when using a cholesterol oxidation model to study antioxidant activity. Genistein had much higher cytotoxicity than daidzein to SVEC4-10 cells. In order to compare the antioxidant activities of genistein and daidzein at the same level, the concentration of 1 µM was selected. However, contrary to what had been expected, no significant difference was found between the cell viability of control group and that of experimental groups treated with either genistein or daidzein in both methods. This implies that neither genistein nor daidzein exerted discernable antioxidant activities against oxidative stress induced by tBOOH in SVEC4-10 cells. The reasons may be the low concentration of 1 µM and the limited incubation time of 24 hr, or could be due to the inhibition of protein synthesis caused by tBOOH that could not be overcome by antioxidant. Therefore, some modifications are needed in order to improve the cell culture model, such as increasing the treatment concentration, increasing the treatment duration, or changing the oxidative stress initiator. Also, perhaps the cell culture model is 52 not suitable for antioxidant activities study, but might be utilized in the study of anti- caner activity of soybean isoflavones, which is becoming a popular research area. 53 References Acker S. A.; Berg D. J.; Tromp M. N.; Griffioen D. H.; Bennekom W. P.; Vijgh W. J.; Bast A. Structural aspects of antioxidant activity of flavonoids. Free Radic. Biol. Med. 1996, 20, 331-342. 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D.; Neuroprotective and neurotrophic efficacy of phytoestrogens in cultured hippocampal neurons. Exp. Biol. Med. 2002, 227, 509-519. 61 Vita The author was born in Zhejiang, China, on December 24, 1977. She earned the degree of Bachelor of Science from the Department of Pharmacology at Zhejiang University in 1999, and then she went to the Graduate School of Zhejiang University majoring in medicinal chemistry. In 2000, she suspended her graduate study in China and set up a private company as a general manager and technical manager. One year later, she came to the United States to continue her graduate study at Louisiana State University. In August 2003, she will receive the degree of Master of Science from the Department of Food Science at Louisiana State University. After that, she is going to Duke University to attend a doctoral program in the Department of Pharmacology and Cancer Biology.