Effects of Naturally Occurring Isoflavones on Prostaglandin E₂ Production

• Abstract
• Abbreviations
• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Preparation of various isoflavones
• Preparation of rat peritoneal macrophages
• Determination of PGE₂ concentrations
• Statistical analysis
• Results
• Effects of various isoflavones on TPA-induced PGE₂ production
• Discussion
• Acknowledgements
• References

Abstract

Previously, we reported that the isoflavones tectorigenin and tectoridin, a glycosylated tectorigenin, isolated from the rhizomes of Belamcanda chinensis have an activity to inhibit prostaglandin (PG) E₂ production in 12-O-tetradecanoylphorbol 13-acetate (TPA)-stimulated rat peritoneal macrophages. The inhibitory effect of tectorigenin is more potent than that of tectoridin. In this study, we investigated the structure-activity relationship of various isoflavones in the inhibition of PGE₂ production in TPA-stimulated rat peritoneal macrophages. The isoflavones examined were isolated from the rhizomes of B. chinensis, and the flowers and the rhizomes of Pueraria thunbergiana. The order of potency to inhibit PGE₂ production was as follows; irisolidone, tectorigenin > genistein > tectoridin (tectorigenin 7-glucoside), glycitein > daidzein. Kakkalide (irisolidone 7-xylosylglucoside), glycitin (glycitein 7-glucoside), daidzin (daidzein 7-glucoside), puerarin (daizein 8-glucoside), and genistin (genistein 7-glucoside) showed no significant inhibition. These findings indicated that 6-methoxylation and 5-hydroxylation increase the potency to inhibit PGE₂ production and 7-O-glycosylation decreases the inhibitory activity.

Abbreviations

COX:cyclooxygenase

PG:prostaglandin

PTK:protein tyrosine kinase

TPA:12-O-tetradecanoylphorbol 13-acetate

Abstract

Previously, we reported that the isoflavones tectorigenin and tectoridin, a glycosylated tectorigenin, isolated from the rhizomes of Belamcanda chinensis have an activity to inhibit prostaglandin (PG) E₂ production in 12-O-tetradecanoylphorbol 13-acetate (TPA)-stimulated rat peritoneal macrophages. The inhibitory effect of tectorigenin is more potent than that of tectoridin. In this study, we investigated the structure-activity relationship of various isoflavones in the inhibition of PGE₂ production in TPA-stimulated rat peritoneal macrophages. The isoflavones examined were isolated from the rhizomes of B. chinensis, and the flowers and the rhizomes of Pueraria thunbergiana. The order of potency to inhibit PGE₂ production was as follows; irisolidone, tectorigenin > genistein > tectoridin (tectorigenin 7-glucoside), glycitein > daidzein. Kakkalide (irisolidone 7-xylosylglucoside), glycitin (glycitein 7-glucoside), daidzin (daidzein 7-glucoside), puerarin (daizein 8-glucoside), and genistin (genistein 7-glucoside) showed no significant inhibition. These findings indicated that 6-methoxylation and 5-hydroxylation increase the potency to inhibit PGE₂ production and 7-O-glycosylation decreases the inhibitory activity.

Abbreviations

COX:cyclooxygenase

PG:prostaglandin

PTK:protein tyrosine kinase

TPA:12-O-tetradecanoylphorbol 13-acetate

Introduction

Recently, we have isolated two isoflavones, tectorigenin and tectoridin, from the rhizomes of Belamcanda chinensis as anti-inflammatory constituents [1], and shown that they inhibit prostaglandin (PG) E₂ production in rat peritoneal macrophages stimulated by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the endomembrane Ca²⁺-ATPase inhibitor thapsigargin [2]. Herbal medicines are considered to be important for the treatment of several diseases [3]. Among the biologically active compounds in the herbal medicines, flavonoids including isoflavones have a wide array of biological activities including anti-inflammatory [4] and anti-tumor [5] effects and activity to reduce hepatic injury [6].

Previously, we isolated a series of isoflavones from Pueraria thunbergiana, whose rhizomes have been used as a herbal medicine for the treatment of colds, and whose flowers have been used for the treatment of diabetes mellitus. PGE₂ is produced mainly by macrophages and causes pain and fever and increases microvascular permeability. In patients with diabetes mellitus, PGE₂ decreases glucose-induced insulin secretion from β-islets and worsens their condition [7], [8].

In the present study, we examined the effect of isoflavones isolated from the rhizomes and flowers of P. thunbergiana on the TPA-induced PGE₂ production in rat peritoneal macrophages. In addition, we compared the effect of these isoflavones with that of tectorigenin and tectoridin, and analyzed the structure-activity relationship of these isoflavones.

Materials and Methods

Preparation of various isoflavones

Puerarin (4′,7-dihydroxyisoflavone 8-β-D-glucopyranoside, colorless powder, m.p. 187 °C, purity > 95 %), daidzin (4′-hydroxyisoflavone 7-O-β-D-glucopyranoside, pale yellowish powder, m.p. 232 - 235 °C, purity > 90 %) and daidzein (4′,7-dihydroxyisoflavone, pale yellowish powder, m.p. 316 - 318 °C, purity > 95 %) were isolated from the rhizomes of Pueraria thunbergiana Benth according to the method described by Hayakawa et al. [9]. Kakkalide (5-hydroxy-4′,6-dimethoxyisoflavone 7-β-D-xylopyranosyl-(1→6)-O-β-D-glucopyranoside, pale yellowish powder, m.p. 251 °C, purity > 98 %), irisolidone (5,7-dihydroxy-4′,6-dimethoxyisoflavone, colorless needles, m.p. 188 - 190 °C, purity 99 %), glycitin (4′-hydroxy-6-methoxyisoflavone 7-O-β-D-glucopyranoside, colorless powder, m.p. 210 °C, purity > 95 %) and glycitein (4′,7-dihydroxy-6-methoxyisoflavone, yellowish powder, m.p. 175 - 180 °C, purity > 95 %) were isolated from the flowers of P. thunbergiana according to the method described by Kurihara and Kikuchi [10], [11], and Park et al. [12]. Tectorigenin (4′,5,7-trihydroxy-6-methoxyisoflavone, pale yellowish needles, m.p. 230 °C, purity > 98 %) and tectoridin (4′,5-dihydroxy-6-methoxyisoflavone 7-O-β-D-glucopyranoside, pale yellowish needles, m.p. 257 - 258 °C, purity > 98 %) were isolated from the rhizomes of Korean Belamcanda chinensis Leman (Iridaceae) [1]. Genistein (4′,5,7,-trihydroxyisoflavone) and genistin (4′,5-dihydroxyisoflavone 7-O-β-D-glucopyranoside) were purchased from Fujicco (Hyogo, Japan). The chemical structures of the isoflavones examined are shown in Fig. [1].

Preparation of rat peritoneal macrophages

A solution of soluble starch (Wako Pure Chemical Ind., Osaka, Japan) and bacto peptone (Difco Laboratories, Detroit, MI), 5 % each, that had been autoclaved at 120 °C for 15 min was injected intraperitoneally into male Sprague-Dawley rats (400 - 500 g, specific pathogen-free, Charles River Japan Inc., Kanagawa, Japan) at a dose of 5 ml per 100 g body weight. Four days later, the rats were sacrificed and the peritoneal cells were harvested [13].

The rats were treated in accordance with procedures approved by the Animal Ethics Committee of the Graduate School of Pharmaceutical Sciences, Tohoku University, Japan. The peritoneal cells were suspended in Eagle’s minimal essential medium (EMEM, Nissui, Tokyo, Japan) containing 10 % calf serum (ICN Pharmaceuticals, K.K.,Tokyo, Japan), penicillin G potassium (18 μg/ml) and streptomycin sulfate (50 μg/ml) (Meiji Seika, Tokyo, Japan) at a density of 1.5 × 10⁶ cells/ml. One hundred microliters of the cell suspension were poured into each well of a 96-well plastic tissue culture plate (Nippon Becton Dickinson, Tokyo, Japan), and incubated for 2 h at 37 °C. The wells were then washed three times with the medium to remove non-adherent cells, and the adherent cells were further incubated for 20 h at 37 °C. After three washes, the adherent cells were used for the subsequent experiments. More than 95 % of the adherent cells were identified as macrophages [13].

Determination of PGE₂ concentrations

Macrophages (1.5 × 10⁵ cells) were incubated for 8 h at 37 °C in 100 μl of medium containing TPA (48.6 nM) (Sigma, St. Louis, MO, USA) with or without isoflavones (Fig. [1]). After incubation, the 96-well plate was centrifuged at 1500 × g and 4 °C for 5 min, and the conditioned medium was collected. PGE₂ concentrations in the supernatant fraction were measured by radioimmunoassay [13]. PGE₂ antiserum was purchased from PerSeptive Diagnostics (Cambridge, MA, USA).

Statistical analysis

The statistical significance of the results was analyzed by Dunnett’s test for multiple comparisons and Student’s t-test for unpaired observations.

Results

Effects of various isoflavones on TPA-induced PGE₂ production

When rat peritoneal macrophages were incubated at 37 °C in medium containing TPA (48.6 nM), the PGE₂ concentration in the conditioned medium was increased time-dependently at 4 to 24 h (data not shown). Therefore, the effects of isoflavones on TPA-induced PGE₂ production were examined at 8 h (Fig. [2]). Daidzein showed weak but significant inhibition (IC₅₀; > 30 μM). Daidzin and puerarin showed no significant inhibition. Glycitein exhibited moderate inhibitory activity (IC₅₀; 25 μM), but glycitin had no inhibitory activity. Genistein showed more activity (IC₅₀; 15 μM) than glycitein, while genistin had no inhibitory activity. Among the isoflavones examined, tectorigenin, a positive control [2], and irisolidone were the most potent inhibitors (IC₅₀; 3.0 μM and 2.9 μM, respectively). Tectoridin had less inhibitory activity (IC₅₀; > 30 μM) than tectorigenin, and kakkalide showed no inhibitory activity. The IC₅₀ values for PGE₂ production of these isoflavones are shown in Fig. [1]. In non-stimulated rat peritoneal macrophages, these isoflavones showed no significant inhibition of PGE₂ production (data not shown).

Discussion

We have reported that tectorigenin and tectoridin isolated from the rhizomes of B. chinensis inhibit the TPA-induced production of PGE₂ [1] by inhibiting the expression of cyclooxygenase (COX)-2 protein [2]. The inhibitory effect of tectorigenin was much more potent than that of tectoridin (tectorigenin 7-glucoside). It was also shown that daidzin (daidzein 7-glucoside) and puerarin (daidzein 8-glucoside) had no inhibitory activity although daidzein exhibited significant inhibition. In addition, other glycosylated isoflavones such as glycitin, genistin, and kakkalide showed weaker inhibitory activity than each of their aglycones, glycitein, genistein, and irisolidone, respectively. These findings strongly indicated that the glycosylation of isoflavones reduces the inhibitory effect. As to flavones, it is reported that glycosylation tends to lower the inhibitory effect of flavones on COX in cell culture [14]. Also in sheep seminal vesicle COX, glycosylated flavones showed no direct inhibitory activity [15]. In both flavones and isoflavones, glycosylation decreases biological activity. The mechanism by which glycosylation does this remains to be elucidated.

The potent isoflavones, tectorigenin, genistein, and irisolidone, commonly possess a 5-hydroxy residue. The inhibitory effect of daidzein and glycitein, neither of which has a 5-hydroxy residue, was weaker than that of genistein (5-hydroxy-daidzein) and tectorigenin (5-hydroxyglycitein), respectively. These findings strongly suggested that 5-hydroxylation of isoflavones increases the inhibitory effect on the TPA-induced PGE₂ production. As to the inhibitory effect of genistein on protein tyrosine kinase (PTK) activity, Akiyama et al. [16] reported that the 5-hydroxy group in genistein is important for its inhibitory activity. Other isoflavones having a 5-hydroxy group, such as irisolidone and tectorigenin, may also have an inhibitory effect of PTK.

When we compared the inhibitory effect of genistein and daidzein with that of tectorigenin (6-methoxygenistein) and glycitein (6-methoxydaidzein), we found the latter isoflavones had more potent activity. These findings strongly suggested that 6-methoxylation of isoflavones increases the inhibitory effect on the TPA-induced PGE₂ production.

You et al. [17] reported that daidzein and tectorigenin show very weak inhibition of human platelet COX activity in vitro. We clarified that tectorigenin and tectoridin have no inhibitory effect on isolated COX-1 (from sheep seminal vesicles) and COX-2 (from sheep placenta) [2]. Therefore, the inhibitory effect of such isoflavones on TPA-induced PGE₂ production might not be due to the direct inhibition of COX. Recently, we reported that the TPA-induced PGE₂ production is dependent on COX-2 [18] and tectorigenin and tectoridin inhibit the TPA-induced production of PGE₂ by suppressing the expression of COX-2 [2]. Other isoflavones that inhibited the TPA-induced PGE₂ production might have the same mechanism of action.

Genistein is widely used as a PTK inhibitor [16]. Because genistein inhibited the TPA-induced PGE₂ production more than diadzein (5-dehydroxygenistein), activation of PTK might be part of the mechanism behind the TPA-induced PGE₂ production. However, diadzein, which does not inhibit PTK [16], weakly but significantly inhibited the production of PGE₂. This finding suggests that PTK is important but not exclusively responsible for the TPA-induced PGE₂ production. Casein kinase II, which is inhibited by daidzein [19], might be a candidate for one of the kinases participating in the TPA-induced PGE₂ production. It remains to be elucidated whether the isoflavones directly inhibit casein kinase II.

In patients suffering from non-insulin-dependent diabetes mellitus, PGE₂ negatively regulates insulin secretion by glucose [20]. In patients suffering from insulin-dependent diabetes mellitus, one of the autoimmune diseases, PGE₂ might exacerbate destruction of β-islets by the immune system. Therefore, it is possible that the extract of P. thunbergiana restores β-islet function by inhibiting PGE₂ production. The flowers of P. thunbergiana contain irisolidone, one of the strongest inhibitors of TPA-induced PGE₂ production, but the rhizomes of it do not [10]. Therefore, the flower extract of P. thunbergiana might have a stronger inhibitory effect on PGE₂ production than the rhizome extract. Because strong inhibition of PGE₂ production is necessary to increase glucose-induced insulin secretion in non-insulin-dependent diabetes mellitus patients, and suppression of inflammation in β-islets is nesessary for recovery of insulin-dependent diabetes mellitus patients, it might be reasonable to use the flower extract of P. thunbergiana for the treatment of diabetes mellitus.

In conclusion, among the derivatives of isoflavone examined, tectorigenin and irisolidone were the most potent inhibitors of the TPA-induced PGE₂ production. The structure-activity relationship study revealed that for strong inhibition, the 5-hydroxy group, 7-hydroxy group, 6-methoxy group, and 4′-hydroxy or 4′-methoxy group are necessary, while glycosylation of the 7-hydroxy group reduces the inhibitory activity. Tectorigenin and irisolidone might be lead compounds that inhibit PGE₂ roduction in inflammatory cells probably through inhibition of COX-2 protein expression.

Acknowledgements

This work was supported in part by the Joint Research Program under the Japan-Korea Basic Scientific Cooperation Program (JSPS 147, KOSEF 996-0700-003-2), and by the Grant-in-Aid for Scientific Research (11470481) from the Ministry of Education, Science, Sports and Culture of Japan.

References

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  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
• 19 Higashi K, Ogawara H. Daidzein inhibits insulin- or insulin-like growth factor-1-mediated signaling in cell cycle progression of Swiss 3T3 cells.  Biochimica et Biophysica Acta. 1994;  1221 29-35
  PubMedSearch in Google Scholar
• 20 Giugliano D, Ceriello A, Saccomanno F, Quatraro A, Paollisso G, D'Onofrio F. Effects of salicylate, tolbutamide, and prostaglandin E₂ on insulin responses to glucose in noninsulin-dependent diabetes mellitus.  Journal of Clinical Endocrinology & Metabolism. 1985;  61 160-6
  PubMedSearch in Google Scholar

Prof. Dr. Kazuo Ohuchi

Laboratory of Pathophysiological Biochemistry

Graduate School of Pharmaceutical Sciences

Tohoku University

Aoba Aramaki

Aoba-ku

Sendai

Miyagi 980-8578

Japan

Email: ohuchi-k@mail.pharm.tohoku.ac.jp

Fax: +81-22-217-6859

References

• 1 Shin K H, Kim Y P, Lim S S, Lee S, Ryu N, Yamada M, Ohuchi K. Inhibition of prostaglandin E₂ production by the isoflavones tectorigenin and tectoridin isolated from the rhizomes of Belamcanda chinensis .  Planta Medica. 1999;  65 776-7
  PubMedSearch in Google Scholar
• 2 Kim Y P, Yamada M, Lim S S, Lee S H, Ryu N, Shin K H, Ohuchi K. Inhibition by tectorigenin and tectoridin of prostaglandin E₂ production and cyclooxygenase-2 induction in rat peritoneal macrophages.  Biochimica et Biophysica Acta. 1999;  1438 399-407
  PubMedSearch in Google Scholar
• 3 De Smet P A. The role of plant-derived drugs and herbal medicines in healthcare.  Drugs. 1997;  54 801-40
  CrossrefPubMedSearch in Google Scholar
• 4 Yasukawa K, Takido M, Takeuchi M, Nakagawa S. Effect of chemical constituents from plants on 12-O-tetradecanoylphorbol-13-acetate-induced inflammation in mice.  Chemical & Pharmaceutical Bulletin. 1989;  37 1071-3
  PubMedSearch in Google Scholar
• 5 Elangovan V, Sekar N, Govindasamy S. Chemopreventive potential of dietary bioflavonoids against 20-methylcholanthrene-induced tumorigenesis.  Cancer Letters. 1994;  87 107-13
  CrossrefPubMedSearch in Google Scholar
• 6 Yamazaki T, Nakajima Y, Niho Y, Hosono T, Kurashige T, Kinjo J, Nohara T. Pharmacological studies on Puerariae flos III: protective effects of kakkalide on ethanol-induced lethality and acute hepatic injury in mice.  Journal of Pharmacy & Pharmacology. 1997;  49 831-3
  PubMedSearch in Google Scholar
• 7 Tran P O, Gleason C E, Poitout V, Robertson R P. Prostaglandin E₂ mediates inhibition of insulin secretion by interleukin-1β.  Journal of Biological Chemistry. 1999;  274 31245-8
  CrossrefPubMedSearch in Google Scholar
• 8 Robertson R P. Eicosanoids as pluripotential modulators of pancreatic islet function.  Diabetes. 1988;  37 367-70
  PubMedSearch in Google Scholar
• 9 Hayakawa J, Noda N, Yamada T, Uno K. Studies on physical and chemical quality evaluation of crude drug preparation. II. Analysis of pharmaceutical preparations including Nux vomica extracts by high performance liquid chromatography.  Yakugaku Zasshi. 1984;  104 50-6
  PubMedSearch in Google Scholar
• 10 Kurihara T, Kikuchi M. Studies on the constituents of flowers. I. On the components of flower of Pueraria thunbergiana Benth.  Yakugaku Zasshi. 1973;  93 1201-5
  PubMedSearch in Google Scholar
• 11 Kurihara T, Kikuchi M. Studies on the constituents of flowers. IV. On the components of flower of Viburnum dilatatum Thunb.  Yakugaku Zasshi. 1975;  95 1098-102
  PubMedSearch in Google Scholar
• 12 Park H J, Park J H, Moon J O, Lee K T, Jung W T, Oh S R, Lee H K. Isoflavone glycosides from the flowers of Pueraria thunbergiana .  Phytochemistry. 1999;  51 147-51
  CrossrefPubMedSearch in Google Scholar
• 13 Ohuchi K, Watanabe M, Yoshizawa K, Tsurufuji S, Fujiki H, Suganuma M, Sugimura T, Levine L. Stimulation of prostaglandin E₂ production by 12-O-tetradecanoylphorbol 13-acetate (TPA)-type and non-TPA-type tumor promoters in macrophages and its inhibition by cycloheximide.  Biochimica et Biophysica Acta. 1985;  834 42-7
  PubMedSearch in Google Scholar
• 14 Moroney M-A, Alcaraz M J, Forder R A, Carey F, Hoult J RS. Selectivity of neutrophil 5-lipoxygenase and cyclo-oxygenase inhibition by an anti-inflammatory flavonoid glycoside and related aglycone flavonoids.  Journal of Pharmacy & Pharmacology. 1988;  40 787-92
  PubMedSearch in Google Scholar
• 15 Alcaraz M J, Hoult J R. Actions of flavonoids and the novel anti-inflammatory flavone, hypolaetin-8-glucoside, on prostaglandin biosynthesis and inactivation.  Biochemical Pharmacology. 1985;  34 2477-82
  CrossrefPubMedSearch in Google Scholar
• 16 Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases.  Journal of Biological Chemistry. 1987;  262 5592-5
  PubMedSearch in Google Scholar
• 17 You K M, Jong H G, Kim H P. Inhibition of cyclooxygenase/lipoxygenase from human platelets by polyhydroxylated/methoxylated flavonoids isolated from medicinal plants.  Archives of Pharmacal Research. 1999;  22 18-24
  PubMedSearch in Google Scholar
• 18 Yamada M, Niki H, Yamashita M, Mue S, Ohuchi K. Prostaglandin E₂ production dependent upon cyclooxygenase-1 and cyclooxygenase-2 and its contradictory modulation by auranofin in rat peritoneal macrophages.  Jorunal of Pharmacology & Experimental Therapeutics. 1997;  281 1005-12
  PubMedSearch in Google Scholar
• 19 Higashi K, Ogawara H. Daidzein inhibits insulin- or insulin-like growth factor-1-mediated signaling in cell cycle progression of Swiss 3T3 cells.  Biochimica et Biophysica Acta. 1994;  1221 29-35
  PubMedSearch in Google Scholar
• 20 Giugliano D, Ceriello A, Saccomanno F, Quatraro A, Paollisso G, D'Onofrio F. Effects of salicylate, tolbutamide, and prostaglandin E₂ on insulin responses to glucose in noninsulin-dependent diabetes mellitus.  Journal of Clinical Endocrinology & Metabolism. 1985;  61 160-6
  PubMedSearch in Google Scholar

Prof. Dr. Kazuo Ohuchi

Laboratory of Pathophysiological Biochemistry

Graduate School of Pharmaceutical Sciences

Tohoku University

Aoba Aramaki

Aoba-ku

Sendai

Miyagi 980-8578

Japan

Email: ohuchi-k@mail.pharm.tohoku.ac.jp

Fax: +81-22-217-6859