Inhibitory Effect of Prenylated Flavonoid in Euchresta japonica. and Artocarpus heterophyllus. on Lipid Peroxidation by Interaction of Hemoglobin and Hydrogen Peroxide

Abstract

We present the results of an in vitro. investigation of the inhibitory effect of the prenylated flavonoid, 5,7,4′-trihydroxy-6,8-diprenylisoflavone, isolated from Euchresta japonica. Benth (Luguminosae) and Artocarpus heterophyllus. Lam. (Moraceae), on lipid peroxidation by interaction of hemoglobin and hydrogen peroxide. This inhibitory effect was stronger than that of genistein, a non-prenylated isoflavone. The result demonstrates that prenylated flavonoid has more antioxidative effect than non-prenylated flavonoid.

Keywords:

• Artocarpus heterophyllus. Lam
• Euchresta japonica. Benth
• hemoglobin
• hydrogen peroxide
• inhibitory effect
• lipid peroxidation
• prenylated flavonoid
• 5,7,4′-trihydroxy-6,8-diprenylisoflavone

Introduction

Lipid peroxidation (LPO) is induced by the interaction of hemoglobin and hydrogen. Such LPO is involved in atherosclerosis (Berliner & Heinecke, 1996) and liver disease (Jaeschke et al., 2002). It was recognized that some flavonoids have inhibitory effects on LPO by oxygen free radical (Heim et al., 2002). We showed that some isoflavones, such as biochanin A, daidzein, formononetin, and genistein, have inhibitory effects on lecithin peroxidation by the interaction of hemoglobin and hydrogen peroxide (Toda & Shirataki, 1999). There are no prenyl groups in the chemical structures of biochanin A, daidzein, formononetin, and genistein. Shirataki et al. (1982) isolated prenylated flavonoid, 5,7,4′ -trihydroxy-6,8-diprenylisoflavone, from Euchresta japonica. Benth (Leguminosae) and Artocarpus heterophyllus. Lam. (Moraceae), which has been used as an antidote or an analgesic.

In this paper, we investigated the inhibitory effect of 5,7,4′ -trihydroxy-6,8-diprenylisoflavone on LPO, which was induced by the interaction of hemoglobin and hydrogen peroxide.

Materials and Methods

Reagents

Butylated hydroxytoluene (BHT), genistein, lecithin (from egg yolk), mannitol, thiobarbituric acid (TBA), α-tocopherol, and trichloroacetic acid (TCA) were obtained from Wako Pure Chemicals (Osaka, Japan).

Extraction and isolation of 5,7,4′-trihydroxy-6,8-diprenylisoflavone from Euchresta japonica.

The air-dried roots of Euchresta japonica. and Artocarpus heterophyllus. were collected in Mt. Ichifusa, Kumamoto prefecture, Japan, in April 1981 and identified by Dr. Yoshiaki Shirataki, Department of Pharmaceutical Science, Josai University, Japan. A voucher specimen was deposited in the Department of Pharmaceutical Science, Josai University, Japan, by Dr. Yoshiaki Shirataki. Cut root was extracted with methanol. The methanol extract was fractioned repeatedly by silica gel column chromatography, with benzene–ethyl acetate mixture as the eluent. They afforded 5,7,4′ -trihydroxy-6,8-diprenylisoflavone, identified on the basis of melting point and spectral comparison by Dr. Yoshiaki Shirataki, Department of Pharmaceutical Science, Josai University, Japan (Shirataki et al., 1982).

Inhibitory test on lecithin peroxidation

Lecithin peroxidation induced by the interaction of hemoglobin and hydrogen peroxide was performed by the following method. The test sample was added to the reaction mixture containing 2.5 mM lecithin, 15 µM hemoglobin, and 15 µM hydrogen peroxide in 50 mM Tris-HCl buffer (pH 7.4) in a total volume of 1 ml. The mixture was incubated at 37°C for 1 h. To the reaction mixture was added 2 ml of TBA reagent that contained 0.37% TBA, 15% TCA, 0.04% BHT, and 2% ethanol. This mixture was heated at 100°C for 15 min. The solution was centrifuged at 3000 rpm for 10 min. The absorbance of the supernatant at 535 nm was determined as malondialdehyde (MDA) (Burge & Aust, 1978). The inhibitory ratio of the test sample was evaluated by the following equation: where MD_ab is absence of test sample and MDA_pr is presence of test sample.

Inhibitory ratios of each sample were tested with five experiments.

Statistical analysis

Values were expressed as the mean ± standard error of five experiments. The results were analyzed by the non-parametric analysis of variation (ANOVA) Scheffe f.-test.

Results and Discussion

Table 1 shows that the inhibitory ratio of 5,7,4′ -trihydroxy-6,8-diprenylisoflavone is 80.7 ± 2.4% at a concentration of 250 µM and increased in a concentration-dependent manner.

Table 1 Inhibitory effect of 5,7,4′-trihydroxy-6,8-diprenylisoflavone on LPO by interaction of ferrous ion and hydrogen peroxide in vitro.

Test sample	Concentration (µM)	Inhibitory ratio (%)
5,7,4′-trihydroxy-6,8-diprenylisoflavone	0.25	49.2 ± 0.9^a.,b.,c.
	2.5	56.2 ± 1.2^d.,e.,f.
	25	64.5 ± 1.9^g.,h.,i.
	250	80.7 ± 2.4^j.,k.,l.
Genistein	0.25	40.5 ± 3.0^a.
	2.5	48.6 ± 3.1^d.
	25	55.7 ± 1.9^g.
	250	67.9 ± 0.5^j.
Mannitol	0.25	0.4 ± 0.1^b.
	2.5	9.7 ± 0.3^e.
	2.5	17.6 ± 3.0^h.
	250	25.7 ± 4.3^k.
α-Tocopherol	0.25	28.4 ± 5.4^c.
	2.5	47.3 ± 3.7^f.
	25	95.7 ± 3.4^i.
	250	98.2 ± 5.3^j.

Values are means ± SE (n = 5).

^{a,b,c,d,e,f,g,h,i,j.}p < 0.01.

The inhibitory ratios of genistein as a non-prenylated isoflavone, mannitol as hydroxy radical scavenger, and α-tocopherol as an antioxidant, are 67.9 ± 0.5%, 25.7 ± 4.3%, and 98.2 ± 5.3% at a concentration of 250 µM (Table 1).

The 50% inhibitory concentration (IC₅₀) of 5,7,4′-trihydroxy-6,8-diprenylisoflavone, genistein, and α-tocopherol are 0.12, 3.62, and 3.07 µM, resepectively.

Genistein is 5,7,4′ -trihydroxyisoflavone. 5,7,4′-Trihydroxy-6,8-diprenylisoflavone has two hydroxy groups at 6,8-position (Fig. 1). Isoflavonoids have been shown to have antioxidative effects (Harper et al., 1999). It has been suggested that genistein may have antioxidant activity on LPO (Ferretti et al., 2004). Prenylated flavonones, cycloheterophullin, and artonins A and B, isolated from Artocarpus heterophyllus. Lam., have powerful antioxidant properties (Ko et al., 1998). Genistein has been demonstrated to have antioxidative effects on oxidative stress such as LPO by oxygen free radical (Toda & Shirataki, 1999). The current results demonstrate that a prenylated flavonoid has more antioxidative effect than non-prenylated flavonoids.

Figure 1 Chemical structures of 5,7,4′ -trihydroxy-6,8-diprenylisoflavone and genistein

Our results are consistent with this previous observation. Further studies on the relationship between herbs containing prenylated isoflavonoids and antioxidative effects are in progress in our laboratory.