Effects of Glucose on Cell Viability and Antioxidant and Anti

Article pubs.acs.org/JPCB

Effects of Glucose on Cell Viability and Antioxidant and Antiinflammatory Properties of Phytochemicals and Phytochemically Modified Membranes Teng Chang,† Chandrasekaran Neelakandan,†,§ Linda DeFine,‡ Thomas Alexander,‡ and Thein Kyu*,† †

Department of Polymer Engineering, University of Akron, Akron, Ohio 44325, United States Department of Pathology and Laboratory Medicine, Summa Health System, Akron, Ohio 44304, United States § Personal Care Division, 3M Company, St. Paul, Minnesota 55144, United States ‡

ABSTRACT: By virtue of antioxidant and anti-inflammable properties, plant-derived phytochemicals such as mangiferin and genistein have attracted considerable attention for functionalization of polymeric hemodialysis (HD) membranes via solution blending. In-vitro dihydrorhodamine (DHR) assay of the genistein-modified membranes revealed drastic reduction in the level of the reactive oxygen species (ROS). In contrast, mangiferin-modified HD membrane manifested the pro-oxidant activity. We suspected that such difference in ROS generation may be attributed to the glucose unit on the xanthone backbone of mangiferin. This hypothesis was confirmed by comparing the ROS levels of genistein versus genistin, and mangiferin versus xanthone and 3,4,5,6-tetrahydroxyxanthone. Phytochemicals without the glucose unit show better antioxidant property related to the glycosides. Anti-inflammatory property was further conducted by measuring the level of TNF-α in blood after contacting with the same selected phytochemicals. Of particular interest is that the glucose unit promotes the generation of TNF-α.

1. INTRODUCTION Hemodialysis (HD) membrane functions like an artificial kidney for removal of extracorporeal waste products from end stage renal disease (ESRD) patients. In HD dialyzer, a bundle of hollow fibers was customarily assembled for blood circulation through the hollow channels, whereas the dialysates such as uremic toxins were filtered out through asymmetric channels in the fiber cross sections. Historically, cellulose was the first polymer used in the HD operation, which have now been switched to synthetic polymers like polyamide (PA), and recently to polysulfone (PSf) and polyether sulfone (PES) fibers.1,2 These synthetic materials are known for their biocompatibility and hydrolytic stability, thus the mechanical strength and membrane integrity can be sustained at high humidities.3−5 HD treatment has been found to raise several complications that affected kidney-failure patients, including dialysis induced oxidative stress,6 undesired inflammation response induced by membrane-blood contact7 and cardiovascular disease.8 Hence, there is a pressing need for multifunctional biomaterials that would be more biocompatible to lower the aforementioned complications. In this regard, a simple and an effective approach has been sought to develop multifunctional biomaterials by utilizing polymer/phytochemical blends,9,10 which also fits perfectly within this scope of development of hemo-compatible biomaterials. Recently, plant-derived phytochemicals have garnered a reputation as alternative medicine by virtue of their natural © 2014 American Chemical Society

origin, abundant and sustained availability, lower toxicity, and perceived health benefits.11 Among various phytochemicals, we have selected two phytochemicals, mangiferin and genistein, to fabricate functional HD membranes. Maginferin is a naturally occurring glucosyl xanthone (2-C-β-D-glucopyranosyl-1,3,6,7tetrahydroxyxanthone) derived from barks and leaves of the mango tree (Mangiferaindica). It is a crystalline compound having a molecular weight of 422 g/mol and a melting temperature of ∼267 °C. Genistein (4′,5,7-trihydroxy isoflavone) is a soybean (Glycine max)-derived polyphenol called isoflavone that contains multiple hydroxyl groups. It has a molecular weight of 270 g/mol and melts at 306 °C. Mangiferin is known to possess antioxidant,12,13 immunomodulatory,14,15 and antidiabetic16 properties. The biological and pharmacological activities of various xanthone derivatives such as mangiferin may be found elsewhere.17 The choice of genistein is due to its antioxidant18,19 and anti-inflammatory properties.20,21 In our previous studies, we have demonstrated the in vitro effects of genistein-modified PA/polyvinyl pyrrolidone (PVP)9 and PES/PVP10 membranes on cytotoxicity, oxidative burst, and inflammatory response of human blood. According to these studies, the incorporation of genistein was found to significantly Received: August 7, 2014 Revised: September 17, 2014 Published: September 23, 2014 11993

dx.doi.org/10.1021/jp5080187 | J. Phys. Chem. B 2014, 118, 11993−12001

The Journal of Physical Chemistry B

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Figure 1. Chemical structures of the phytochemicals: (a) genistein, (b) genistin, (c) mangiferin, (d) xanthone, and (e) 3,4,5,6-tetrahydroxyxanthone.

bath, i.e., reverse osmosis water, at 25 °C. The coagulated membranes were peeled-off from the glass plate, rinsed with excess water, and then vacuum-dried at room temperature. The blood was collected from volunteers for the needed biomedical tests. Ten milliliters of venous blood was collected in lithium heparin Vacutainer tubes (Becton Dickinson, Rutherford, NJ) and used immediately after the collection. 2.3. Cell Viability Assay for the Determination of Cell Toxicity. The cell viability assay has been demonstrated in our previous papers.9,10 The effect of phytochemicals, unmodified membranes, and modified membranes on cell viability was determined using whole blood. A nucleic acid dye, 7-aminoactinomycin D (7-AAD) (BD-Via-Probe from BD Bioscience), was used as a viability probe based on uptake of 7-AAD. Fresh blood mixed with the dye was used as negative control, whereas blood incubated with certain sample at 37 °C served as the experimental samples. Briefly, 100 μL of fresh blood was pipetted into separate polypropylene tubes and mixed with 2 mL of ammonium chloride lysis buffer solution, which contained 8.26 g of ammonium chloride, 1 g of potassium bicarbonate, and 0.037 g of ethylenediaminetetraacetic acid dissolved in 1 L of deionized water. The tubes were allowed to stand at room temperature for 10 min during which most of the red blood cells were lysed. The tubes were centrifuged at room temperature for 5 min at 1500 rpm. The supernatant was discarded, and the cells were washed twice and centrifuged with 2 mL of phosphate buffer saline (PBS, Baxter, pH = 7.4) solution. The supernatant was discarded again. The samples were added to 20 μL of 7-AAD dye and then stored under dark conditions for 10 min to permit cell uptake. The cells were resuspended with 500 μL of PBS, and the cell suspension was analyzed using a flow cytometer (EPICS XL-MCL, Beckman Coulter) supported by System II software. 7-AAD is a nucleic acid dye, which is designed in such a way that it cannot enter live cells. The number of cells in each sample was adjusted between 10k and 20k cells/mL using PBS. The fluorescence signal from the dead cells was measured at FL3 red channel (650 nm). 2.4. Dihydrorhodamine 123 Assay for the Determination of ROS Levels. The DHR assay for determining antioxidant property was demonstrated by our previous literature.9,10 To determine the effect of the phytochemical or membrane oxygen radical generation, the standard DHR assay was modified as follows. The assay consisted of one positive control tube, several sample tubes, and one negative control tube. The positive control tube contained 100 μL of blood, 25 μL of DHR, and 25 μL of PMA; the total contents were made up to a total volume of 1 mL with PBS. The effect of oxygen radical generation is tested in what follows using the sample

suppress the reactive oxygen species (ROS) and proinflammatory cytokines levels. In our preliminary exploration of the antioxidant response of mangiferin-modified membranes, we found surprisingly that mangiferin showed an unexpected trend of the ROS elevation upon contact with the blood. We hypothesized that the glucose unit on the hydroxyxanthone backbone of mangiferin may be responsible for increasing the pro-oxidant property. In this article, we mixed glucose with genistein to elucidate the influence of glucose on the ROS generation. The possible role of glucose units as the pro-oxidant was investigated by comparing the ROS activities of genistein versus genistein, and mangiferin versus its derivatives such as xanthone and 3,4,5,6-tetrahydroxyxanthone (hereafter called tetrahydroxyxanthone). Furthermore, we also determined the anti-inflammatory response by the presence of the glycone unit in the molecular structures of the phytochemicals.

2. EXPERIMENTAL METHODS 2.1. Materials. Aromatic poly(amide) (PA), TROGAMID T5000, was kindly provided by Degussa Corporation. Poly(ether sulfone) (PES), UltrasonE 6020P, and polysulfone (PSf), Ultrason S 6010, were both amorphous polymers that were kindly provided by BASF Corp. (Wyandotte, MI). A reagent grade DMSO (dimethyl sulfoxide), was purchased from Sigma-Aldrich (St. Louis, MO). Genistein was bought from Riotto Botannical Company (Xi’an, China). Mangiferin (>98% purity) was obtained from MDidea Exporting Division (Yin Chuan, China). Xanthone, 3,4,5,6-tetrahydroxyxanthone, and phorbolmyristate acetate (PMA) were purchased from SigmaAldrich (St. Louis, MO). Genistin was supplied by Xi’an Tonking Biotech Company (Xi’an, China). Dihydrorhodamine 123 (DHR) was obtained from Invitrogen (Carlsbad, CA). The chemical structures of the phytochemicals were depicted in Figure 1. Blood was donated by the first author and healthy volunteers. The amount of blood collected met the criteria for exemption from Institutional Review Board oversight. 2.2. Formations of the Unmodified and Phytochemically Modified Membranes. PA, PES, and PSf pellets were vacuum-dried at 80 °C for 24 h to remove moisture and then dissolved in DMSO. Mangiferin and genistein were dried under vacuum at 80 °C for 24 h and then dissolved into solution using DMSO. Subsequently, various amounts of mangiferin or genistein solution were added to the polymer solution. The solutions were homogenized for 48 h by stirring mechanically and the entrapped air, if any, was removed under vacuum at room temperature. The homogeneous solutions were then cast in the form of a film of predetermined thickness on a precleaned glass slide, followed by immersion into a nonsolvent 11994



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tube containing 100 μL of blood, 25 μL of DHR, 25 μL of PMA and the appropriate sample (for example, unmodified membranes or modifying agents or modified membranes). Both unmodified as well as modified membranes were cast and dried under sterile conditions inside a biosafety laminar flow hood. For the assay, two small circular samples (4 mm in diameter) were punched out from the cast films and placed inside the test tubes. After the membrane samples were added, the entire contents were incubated at 37 °C for 3 h (to simulate hemodialysis conditions) subjected to continuous shaking. After the incubation, the membrane samples were removed from the blood and agitated with 1 mL of PBS to dislodge the cells that were attached to the membrane. For the case of pure phytochemicals, the samples were prepared by dissolving the chemicals in DMSO or ethanol over a wide range of concentration. The negative control tube contained 100 μL of blood, 25 μL of DHR, and the sample. The rest of the assay was similar to the standard DHR assay protocol involving lysing the red blood cells and fixing cells with immunoprep (Beckman Coulter) solution followed by flow cytometric analysis. The ROS level obtained from a different set of experiments was normalized to percentage by comparison to the situations with the positive and negative controls. 2.5. Lipopolysaccharide (LPS) Stimulation Assay for the Determination of Anti-inflammatory Property. To evaluate the inflammatory response, the LPS assay was conducted in accordance with the procedure reported earlier.9,10 The secretion of one of the pro-inflammatory cytokines, TNF-α, was evaluated after the blood contacted the neat phytochemicals or the membranes. The amount of secreted TNF-α was determined and marked by using Bio-Plex Pro Assay (Bio-Rad Laboratories, Hercules). The samples were analyzed by utilizing the Luminex 200 instrument (Luminex, Austin, TX, USA) supported by Luminex XYP platform and data analysis software, Luminex xPONENT 3.1. To compare among various sets of experimental results, the amount of TNF-α population was normalized by that of the positive and negative controls and converted into percentage.

Figure 2. Cell viability studies on polymer materials and phytochemicals. Cell viability studies conducted with PBMC for (a) neat PA, PES, and PSf membranes and (b) pure phytochemicals, mangiferin and genistein, solutions illustrating the nontoxic nature as modifying agents. Values are means ± SEM with n = 3.

Cytotoxicity studies were extended to the phytochemically modified membranes. We employed PA/X membranes for demonstration, where X stands for mangiferin (M) or genistein (G). As evident from Figure 3, all the PA/X membranes

3. RESULTS Neat and phytochemically modified PA, PSf, and PES membranes were prepared via coagulation-induced phase separation by immersing the polymer solutions into water bath to form asymmetric porous membranes. Prior to all biocompatibility tests, it is essential to carry out cytotoxicity measurements because any material intended for medical device applications must be noncytotoxic to blood cells when in its pure form or when bound either physically or chemically to a substrate. As can be seen in Figure 2a, the unmodified PA and PSf membranes (i.e., similar to the commercial HD membranes) exhibits excellent cell viability, which is comparable to that of the control. The same observation was made in the PES membrane indicating no harmful effect to the blood cells. Figure 2b illustrates the comparison of cell viability between pure mangiferin and genistein, confirming that the phytochemicals we selected were noncytotoxic to the peripheral blood mononuclear cells (PBMC) in the entire concentration range of the assay. The above finding established the basis for the selection of genistein and mangiferin toward subsequent development of the functional HD membranes for suppressing dialysis induced oxidative stress (DIOS).

Figure 3. Cell viability studies on PA/X membranes. Cell viability studies conducted with PBMC for PA/X membranes (where X represents mangiferin (M) or genistein (G)) illustrate the nontoxic nature of the membranes. Values are means ± SEM with n = 3.

exhibited excellent cell viabilities, which were comparable to the viability of the control. According to our earlier demonstration (neat polymeric membranes and neat phytochemicals showed no harmful effect to the blood cell), it was reasonable to extend to the blending systems that would exhibit no harmful effect to the PBMC. It was estimated that the phytochemical loading in these modified membranes ranges between 100 and 200 μg/ 11995



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cm2 of membrane area. Although the cytotoxicity results of neat modifying agents as well as of modified membranes are very encouraging, it is by no means sufficient in actual biomedical applications. That is to say, passing the cytotoxicity test is merely a minimum necessary criterion for biocompatibility tests and thus we further performed the DHR and LPS assays to determine the antioxidant and anti-inflammatory properties of pure phytochemicals and their modified membranes. Figure 4a shows the results form DHR assay performed with the neat polymeric membranes. The ROS levels have been

genistein showed very promising antioxidant properties, i.e., significant suppression of ROS levels (i.e., about 40% reduction) at a concentration as low as 25 μg/mL. This trend continues to even lower the ROS level to 50% with increasing genistein dosage (Figure 3b). The ROS levels of the various mangiferin- and genisteinmodified polymer membranes were later carried out, and we selected modified PA/X membranes as the representative for the demonstration of the effect of mangiferin and genistein depicted in Figure 5a. Figure 5a shows, on the one hand, that

Figure 4. Antioxidant studies on polymer materials and phytochemicals. DHR assay performed with (a) neat polymeric membranes and (b) DMSO solutions of pure mangiferin and genistein. Values are means ± SEM with n = 5. Asterisks (*) denote statistical significance with p < 0.05.

Figure 5. Antioxidant studies on phytochemically modified polymer membranes. Effect of (a) PA/X membranes (where X represents mangiferin (M) or genistein (G)) and (b) PSf/G and PES/G membranes on ROS levels measured by DHR assay. Values shown are mean ± SEM obtained from three experiments (n = 5). Asterisks (*) represent statistical significance in comparison with positive control results.

carried out and converted into percentage based on the positive and negative controls. It can be noticed that the blood contact with unmodified PA membranes has led to a slight increase in the ROS level; the increase in ROS level was found to be as high as 8% compared to that of the positive control. On the one hand, the PSf membrane did little or no change to the ROS level as compared to the case of the control. On the other hand, neat PES membrane shows its ability to reduce the ROS levels for about 45% as compared to the case of the positive control. A similar study was extended to the mangiferin/DMSO and genistein/DMSO solutions as a function of modifier concentration. Figure 4b exhibits ROS level of blood upon contacting with pure mangiferin and genistein solutions. Despite extensive literature evidence supporting the antioxidant properties of mangiferin,22−24 we were astounded to observe a systematic increase in oxygen radicals up to 200 μg/mL mangiferin. A much higher mangiferin concentration (e.g., 500 μg/mL, data not shown) was required to see any noticeable reduction in ROS levels, which becomes impractical. Unlike mangiferin,

mangiferin-modified PA membrane increased the ROS levels, which is not surprising when one combines the findings that neat PA membrane and pure mangiferin raise the ROS to a higher level. On the other hand, a dose-dependent suppression of oxygen radicals was shown from genistein-modified PA membranes. As explained earlier, the unmodified PA membrane exhibits an increase of oxygen radicals by a few percent. However, with the addition of genistein in the PA membrane, the ROS level was found to decline to 46% at the 70/30 PA/G composition. This 54% reduction of the oxygen radicals is close to that of the results of the pure genistein system shown in Figure 3. However, when the genistein concentration in the PA/G membranes was above 20 wt %, the membrane became unstable and very brittle and crumbled. Considering the fact of membrane integrity, we later tested the membranes containing no more than 20 wt % of the phytochemical. 11996



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property can be unambiguously attributed to glucose loading. Therefore, it is reasonable to infer that it is the glucose unit in mangiferin that raises the level of intracellular oxygen radicals. It may be postulated that the antioxidant property of mangiferin may be improved if the glucose unit were cleaved from the xanthone backbone. To further prove our hypothesis, we utilized genistin, xanthone, and tetrahydroxyxanthone to compare with genistein and mangiferin to determine the effect of the glucose unit on each molecule. As shown in Figure 1, genistin has the same isoflavoid backbone as genistein, and one hydroxyl group is replaced by a glucose unit. Xanthone is the basic backbone of mangiferin without any hydroxyl group or glucose unit, whereas tetrahydroxyxanthone has the same xanthone backbone with four hydroxyl groups. Figure 7 shows the ROS levels of the blood after contacted with various neat phytochemicals. As mentioned before, on the one hand, mangiferin could raise up the ROS levels about 30% after contacting with blood, and genistein showed its ability to reduce the ROS levels about 40% when the concentration was up to 200 μg/mL. Xanthone, on the other hand, did not alter the ROS levels in the whole range of concentrations; i.e., the ROS levels remained at 100%. Tetrahydroxyxanthone fails to suppress the ROS levels at lower concentrations in the blood, but it exhibited the ability to reduce the ROS level to 78% with a higher dose of 200 μg/mL. Genistin, which has a glucose unit on its backbone, was shown to increase the ROS levels in the whole range of concentrations. Additional to the antioxidant property, we also determined the anti-inflammatory response of the phytochemicals. On the basis of neat phytochemicals, we measured the levels of one of the pro-inflammatory cytokines generation, i.e., TNF-α, in the whole blood after contacting with various reagents. Figure 8a exhibits the TNF-α levels in the blood during incubation with genistein, genistin, and glucose. It can be noticed that the TNFα levels perform a dose-dependent reduction with the increase concentration of genistein in blood. The TNF-α level was reduced to 42% as compared to that for the positive control. On the other hand, the neat glucose elevates the TNF-α level in the blood. For the glycoside, genistin, it can be observed that the TNF-α levels show no significant inhibitory effect in the in vitro studies. Figure 8b shows the comparison of TNF-α levels in blood after contacting with mangiferin, tetrahydroxyxanthone, and xanthone. One can easily discern that mangiferin exhibits a marginal effect in suppressing the generation of TNF-α. Tetrahydroxyxanthone can reduce the TNF-α level better than mangiferin, whereas the effect of xanthone is between those of mangiferin and tetrahydroxyxanthone.

For the observation of the effect of genistein on reducing the ROS level in the PA system, we further conducted the DHR assay with the genistein-modified PSf and PES membranes. The results shown in Figure 5b exhibited that the genistein modification in both PSF and PES membranes could help reduce the ROS level of blood. PSf/G membranes could reduce 27% of the ROS level after contacting with blood. PES/G membranes were observed to have better reduction on the ROS level, i.e., 57% as compared to that of the control. The PES/G membranes exhibited better antioxidant property related to PSf/G membranes. This result was not surprising. The neat PES showed its stronger ability to suppress the ROS level compared to that of the neat PSf membrane. With the addition of genistein in the membranes, PES/G membranes showed better antioxidant property is understandable. We were puzzled by the observed opposite trends of genistein versus mangiferin as pure materials or modification agents in regard to oxygen radical generation despite encouraging conclusions made in literature regarding the excellent antioxidant properties of mangiferin.18,19 The unexpected poor performance of mangiferin in the ROS suppression in the present study may be reconciled as follows. By comparing the chemical structures of mangiferin and genistein (Figure 1), one may postulate that the glucose unit in mangiferin might be responsible for the elevated ROS levels. To test the above hypothesis, we carried out the DHR experiments using pure glucose solution and various glucose/ genistein mixtures, and the results are plotted in Figure 6. We

Figure 6. DHR assay comparing the antioxidant properties of pure genistein and glucose with that of genistein/glucose mixtures, which demonstrates the effect of glucose addition on genistein. Note that the genistein concentration of the mixtures was kept exactly the same as that of pure genistein, while allowing the total concentration of the solutions to vary. Values shown are mean ± standard deviation obtained from three experiments (n = 5). Asterisks indicate p < 0.05 and imply statistical significance as compared to the positive control results.

4. DISCUSSION Neat and phytochemically modified PA, PSf, and PES membranes were found to exhibit excellent cell viability without any harmful effect on the blood cells, indicating the membranes reach the minimum requirement for the biomedical applications. The pure PA membranes show its effect to induce the ROS level in the blood after the in vitro DHR tests. It is well-known that the oxygen radical generation occurs due to the prolonged contact of blood with polymer membranes. This finding is very important from the hemodialysis perspective, because typical hemodialysis patients’ blood contacts the HD membrane approximately 3−4 h per session and three sessions per week. As blood interacts with the synthetic polymer surface,

are able to confirm that pure glucose indeed increased the oxygen radical generation in neutrophils as high as 20%. Clinical studies also support the notion of increased ROS generation by leucocytes after oral ingestion of glucose.25 Because genistein had a positive antioxidant effect and glucose had a negative antioxidant effect, a DHR assay experiment was devised by mixing glucose with genistein at various 100/0, 75/ 25, 50/50, and 0/100 genistein/glucose ratios. As evidenced in Figure 6, the ROS level rises with increasing glucose concentration, suggesting that the observed inferior antioxidant 11997



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Figure 7. Antioxidant properties of various phytochemicals. Comparison of antioxidant property determined by using DHR assay of various chemicals, i.e., mangiferin, xanthone, 3,4,5,6-tetrahydroxyxanthone, genistin, and genistein. Values shown are mean ± standard deviation obtained from three experiments (n = 5). Asterisks indicate p < 0.05 and imply statistical significance as compared to the positive control results.

peroxidase, which act as the first line of defense against oxidative damage to cells. Such defense mechanisms deteriorate in HD patients and over a period of time, the excessive production of oxygen radicals overpowers the antioxidant function, and thus eventually causing dialysis-induced oxidative stress (DIOS) to HD patients.26 PSf membrane had little or no effect on the ROS level as compared to the effect of the control. Pure PES membrane has a greater ability to reduce the ROS levels than the positive control, which implies that PES can be the best material among the three for making biocompatible HD membranes. According to our results, it was surprisingly found that pure mangiferin and the mangiferin-modified membrane showed pro-oxidant property that induced the ROS level in the blood after the in vitro tests. The present finding is at odds with the study by Garciá et al.22 who observed the suppression trend of mangiferin and who reported that mangiferin can reduce the production of NO in peritoneal macrophages that were stimulated with LPS and Interferon-γ resulting reduction in extracellular ROS production by macrophages. There are two possible reasons for the differences in our finding verus Garciá et al.22 The first reason comes from the difference in methodologies that were employed in these two studies, i.e., intracellular oxygen generation by the DHR technique of the present work versus extracellular ROS levels measured by Garciá et al.22 The DHR assay probes the changes occurring during the early stage of electron transport as opposed to the extracellular ROS levels, which provides information at some later stages. The second difference may be due to different cell lines that were used in the determination of ROS levels; i.e., Garciá et al. utilized peritoneal macrophages derived from mouse as opposed to the current study, which used neutrophils from human blood. On the other hand, genistein and the genistein-modified membranes showed excellent property on reducing the production of the ROS after contact with blood. According to Peterson et al.,27 genistein has been shown to inhibit the priming events of high level ROS production especially in human polymorphonuclear cells cultured in vitro. The reported

Figure 8. Anti-inflammatory properties of various phytochemicals. The TNF-α level measured by Luminex after incubating with LPS stimulated whole blood for 24 h. (a) Effects of genistein, glucose, and genistin can be seen in a dose-dependent manner. (b) Values are means ± SEM with n = 4. Asterisks (*) denote statistical significance with p < 0.05; double asterisks (**) denote statistical significance with p < 0.1.

the neutrophils undergo an oxidative burst resulting in the formation of highly potent oxygen radicals and, therefore, oxidative stress builds up in the patient’s body. The radicals thus generated are quickly neutralized by enzymatic antioxidant molecules such as superoxide dismutase and glutathione 11998



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increasing the expression of NADPH and the generation of ROS. According to in vitro ferric reducing antioxidant power assay,32 mangiferin was demonstrated to have antioxidant property, showing the ability to scavenge the free radicals or superoxide anion to reduce the ROS levels. However, mangiferin exhibited pro-oxidant activity in the in vitro antioxidant DHR assay, which might be due to the presence of the glucose unit of itself. In our previous study, genistein was used as a modifying agent in polymer membranes to provide anti-inflammatory property.9,10 We found that pure genistein can suppress the production of one of the pro-inflammatory cytokines, i.e., TNFα, from LPS-induced human blood monocytes. Pure genistein is known to modulate the pro-inflammatory cytokines by several pathways. First, genistein is an effective inhibitor of several enzyme activities33 such as protein tyrosine kinase, protein kinase C, and protein kinase A. The inhibition of these protein kinases by genistein results in reducing the generation of pro-inflammatory cytokines.34 Second, genistein can also reduce the LPS activation of nuclear factor-κB (NF-κB),35 which influences the production of pro-inflammatory cytokines. As reported in a previous study,36 the levels of TNF-α increase with the addition of pure glucose. The proinflammatory cytokines, i.e., IL-1β, IL-6, and TNF-α, reacted upon the glucose challenge; especially the levels of IL-1β and TNF-α increased rapidly responding to the glucose intake. Although the glucose exhibits a dose-dependent relation with pro-inflammatory cytokines, which are directly related to inflammatory response, glucose, however, is an important component in blood maintained in a very narrow range to provide energy for cells in human bodies. The levels of glucose in the blood are monitored by the cells in the pancreas’s Islets of Langerhans and are regulated by hormones, primarily glucagon and insulin. In a healthy person, the glucose level is under control. However, the levels of cytokines related to glucose become risk factors to patients who suffer from diabetes, impaired glucose tolerance (IGT), insulin resistance, metabolic syndrome, hyperglycemia, or even septicemia, when often the patient needs to go under hemodialysis. Nakamura et al. has reported that a high level of IL-6 in blood was correlated with hyperglycemia and with difficulties in glucose control for the patient suffering from septicemia.37 Furthermore, IL-6 has been reported that it can cause significant increases in plasma glucagon levels or induce peripheral resistance to insulin action to induce dose-dependent increases in blood glucose.38 Morohoshi et al. have reported that both IL-6 and TNF-α levels and immunoreactivity increased with a higher concentration of glucose in blood in vitro.39 Their results suggest that hyperglycemia may cause hyperfibrinogenemia in diabetic patients via IL-6-dependent and TNF-dependent mechanism. In other words, the relation between glucose and inflammatory response are highly associated with cytokine levels. We suspected that the glucose unit of genistin would affect the ant-inflammatory property and induce the generation of TNF-α. However, our result suggested that a low concentration of genistin did not show significant effect in altering the TNF-α level as compared to the case with the control, which is consistent with previous findings.40,41 Because genistin, unlike genistein, is not recognized as a tyrosine kinase inhibitor, genistin has little or no effect on the generation of TNF-α in lower concentration ranges. On one hand, Wang et al.41 reported that a higher dose of genistin in blood would induce

levels of ROS inhibition by genistein solution (approximately 50% in the concentration range 15−100 μg/mL27) agree well with the present finding. Upon activation of the cells, the membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) and cytosolic components of the enzyme assemble in the membrane and form the active enzyme. NADPH oxidase catalyzes the reduction of O2 to superoxide anion (O2•−), which then rapidly dismutates to hydrogen peroxide (H2O2). This series of events is referred to as the electron transport chain. Subsequently, H2O2 may be converted by the enzyme myeloperoxidase into highly reactive compounds such as hypochlorous acid (HOCl).26,28 Therefore, for a neutrophil to undergo oxidative burst, a functionally intact NADPH oxidase is critical. In this case, genistein was successfully able to inhibit the expression of NADPH, which is the first step of the electron transport chain to form superoxide anion and subsequent dismutation to H2O2. By virtue of genistein’s ability to prevent NADPH expression, long-term oral administration of genistein has been recommended to improve the health of endothelial cells.29 In a nut shell, the cell viability results displayed in Figure 2 combined with antioxidant properties of modifying agents in Figure 4 indicate that it may be possible to fabricate better antioxidant membranes with genistein modification. The unexpected poor performance of mangiferin in suppressing ROS levels of blood in the present study drew our attention. We hypothesized that the glucose unit in the mangiferin backbone might be responsible for the elevated ROS levels. We confirmed that pure glucose indeed increased the ROS level as high as 20% related to the positive control. Moreover, the genistein/glucose mixtures showed relatively poorer antioxidant effect with the increase of concentration of glucose. To further prove our hypothesis, genistin, xanthone, and tetrahydroxyxanthone were used to compare with genistein and mangiferin to determine the effect of the glucose unit on each molecule. The results are consistent with our hypothesis that the presence of a glucose unit will affect the ROS level by increasing the generation of oxygen radicals in the blood. Clinical studies also support the notion of increased ROS generation by leucocytes after oral ingestion of glucose.25 The in vitro and in vivo studies25,30 suggested that the glucose could increase the ROS generation produced by leucocytes and mononuclear cells. Furthermore, glucose would increase the p47phox, which is a major protein component of NADPH oxidase, because glucose could be an important modulator of p47phox gene expression. The increase of p47phox would result in increasing expression of NADPH oxidase and further inducing the generation of ROS. It has also been reported that high blood glucose levels in patients who suffer from diabetes might activate protein kinase C and result in generation of NADPH oxidase. The ROS production would increase through the activation of NADPH oxidase. However, according to the studies by Lee et al.,31 it was demonstrated that a series of soybean isoflavones and their glycosides did not show detectable pro-oxidant activity. Genistin even showed a higher radical scavenging rate than genistein determined by using DPPH assay. Although the DPPH assay suggested that genistin had superior radical scavenging property relative to genistein, we found the opposite trend in the ROS reduction from the in vitro experiment that we conducted. The results implied that the genistin could scavenge free radicals and be considered an antioxidant agent. However, the glucose unit, known as the glycone, might affect when it contacted with blood, resulting in 11999



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the TNF-α levels. On the other hand, Zhao et al.42 reported that genistin can reduce the secretion of TNF-α through oral administration because genistin can be biologically converted to genistein.43,44 It has been reported that mangiferin can modulate the gene expression of cytokines, i.e., TNF-α, that regulate macrophage activity and participate directly in patenting anti-inflammation activities.24 The TNF-αmRNA levels in inflammatory macrophages stimulated by LPS inhibited by mangiferin further accomplish the reduction of TNF-α. Furthermore, TNF-α also plays a key role in regulating the production of other proinflammation cytokines, such as IL-1 and IL-6.45 In other words, the formation of TNF-α and inflammation response involving macrophage activation can be blocked by the presence of mangiferin. Our finding reveals consistency related to other’s studies.46 Xanthone and tetrahydroxyxanthone have been shown to have potent anti-inflammatory activities; e.g., xanthone suppresses the generation of TNF-α- and TNF-αinduced intercellular adhesion moledule-1 (ICAM-1). The reduction of ICAM-1 can block the primary step during inflammation, i.e., the adhesion of leukocytes to the endothelium.47 On the other hand, tetrahydroxyxanthone can inhibit the TNF-α production via inhibition of ROS production.48 All three xanthone compounds, mangiferin, xanthone, and tetrahydroxyxanthone, are capable of garnering the anti-inflammation activity by suppressing the TNF-α production. However, due to the presence of the glucose unit in the mangiferin structure, mangiferin is inferior in reducing the TNF-α level as compared to the abilities of xanthone and tetrahydroxyxanthone.

tetrahydroxyxanthone can significantly reduce the TNF-α levels related to the control. Due to the presence of glucose on the structures, genistin and mangiferin showed poorer performance in TNF-α inhibition.

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AUTHOR INFORMATION

Corresponding Author

*Thein Kyu. E-mail: [email protected]. Phone: +1-330-9726672. Fax: +1-330-972-3406. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support of this work by the Ohio Soybean Council is gratefully acknowledged. REFERENCES

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5. CONCLUSIONS In summary, the cytotoxic and antioxidant properties of PA, PSf, and PES membranes modified with mangiferin and genistein were evaluated in vitro using fresh human blood. All the unmodified and modified membranes have been shown to be noncytotoxic to the PBMC. Pure PA membranes raised the levels of reactive oxygen species (ROS), which might lead to DIOS. PSf membrane showed no significant change in ROS levels, whereas pure PES membrane was found to have the excellent ability to reduce the ROS levels. However, PES raised the cytokine levels, suggesting the need for further modification. Pure genistein exhibited dose-dependent reduction of the ROS levels in blood, which might be attributed to the NADPH inhibition by genistein. On the other hand, mangiferin raised the intracellular ROS levels. Due to the lack of antioxidant activity in pure mangiferin, mangiferin-modified membranes showed no suppression of intracellular oxygen radical generation, whereas the genistein-modified membranes exhibited significant suppression of ROS in a dose-dependent manner. Of particular interest is that PES/G membranes have the best performance in reduction of the ROS levels among the three materials we tested. It was hypothesized and subsequently verified experimentally that the presence of glucose in mangiferin indeed hinders its antioxidant capability. It was also found that pure glucose indeed increased the ROS level in the blood. By comparing the phytochemicals with or without a glucose unit, we concluded that the chemicals without glucose, such as genistein, xanthone, and tetrahydroxyxanthone, exhibited better performance in reducing ROS level than the glycosides, i.e., genistin and mangiferin. We further investigate the effect of glucose to the inflammatory responses. We found that genistein and 12000



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