Introduction

Sacoglottis gabonensis stem bark is commonly used as palm wine additive in some rainforest communities of Southern Nigeria. Laboratory studies have shown that the extract bergenin (Fig 1) may possess antioxidant properties (Ekong and Ejike 1974, Madusolumuo, 1993; Okechukwu 1998). Ekong and Ejike reported that bergenin isolated from the bark extract had antioxidant properties towards yeast alcohol metabolism. A similar observation was made with the crude extract by Okechukwu (1998) who demonstrated that it was a non-competitive inhibitor of yeast alcohol and aldehyde dehydrogenases. Madusolumuo (1993) studied the effect of the bark extract in mammalian system and found that pretreatment with the additive protected the rat liver from acetaminophen- induced cytotoxicity.

We have recently examined the antioxidant activity of the crude extract in the mammalian system in detail using 2,4-dinitrophenyl hydrazine as the experimental peroxidant. The crude extract was found to effectively inhibit experimental lipid peroxidation in the liver, brain and red blood cells as determined by the tissue concentrations of three stable intermediates of the lipid peroxidation pathway, lipid hydroperoxide, lipid hydroperoxide carbonyl and lipid aldehyde (Maduka and Okoye 2002a). The crude extract was subsequently shown to exert a sparing effect on tissue antioxidant vitamins, ascorbic acid and vitamin E, protecting them against the 2,4-DNPH-induced depletion in the liver, brain and red blood cell, and to remove the inhibitory effect of 2,4-DNPH on the activity of the antioxidant enzyme, superoxide dismutase (Maduka and Okoye 2002b).

Figure 1

Figure 1: Structure of bergenin (2,1-benzopyrone derivative of isocoumarin, molecular weight C H O).

This paper reports on a study on the effect of bergenin isolated from S. gabonensis on 2,4-DNPH experimental lipid peroxidation and tissue ascorbic acid, primarily for the purpose of ascertaining whether or not it contributes to the observed antioxidant effect of the crude extract on lipid peroxidation.

Materials And Methods

Isolation of Bergenin

Animal Treatment and Tissue Sample Collection

Biochemical Analyses of Samples

Results

The results are summarized on Tables 1-3. As can be seen from Tables 1 and 2, administration of DNPH expectedly induced lipid peroxidation in the three tissues, the values of the two intermediates determined (lipid hydroperoxide carbonyl and lipid aldehydes) being statistically higher in DNPH-treated rats (Group 3) than in the corresponding saline controls (Group 2). In both cases, pretreatment with bergenin significantly reduced the level of DNPH-induced peroxidation; the level of each intermediate was significantly lower in rats given DNPH after pretreatment with bergenin (Group5) than in non-pretreated rats given DNPH (Group 3). However, pretreatment with bergenin did not completely prevent oxidative damage by DNPH. As can be seen from Table 2, the concentration of the peroxidation intermediate, lipid aldehyde in each tissue was significantly higher in bergenin-pretreated rats given DNPH (Group 5) than in bergenin controls (Group 4). For lipid hydroperoxide carbonyl (Table 1), the difference was significant only in the case of the liver.

Figure 2

Table 1: Effect of bergenin on the level of lipid peroxidation product, lipid hydroperoxide (as determined by malondialdehyde) in the rat liver, red blood cells and brain during 2,4-DNPH-induced peroxidation.

Figure 3

Table 2: Effect of bergenin on the level of lipid peroxidation product, fatty acid aldehydes in rat tissues after 2, 4-DNPH-induced perxidation

Tissue ascorbic acid content (Table 3) was generally lower in DNPH -treated rats (Group 3) than in the corresponding saline controls (Group 2) but the difference was significant only in the case of the red blood cell, suggesting marked DNPH-induced depletion. Ascorbic acid content of each of the three tissues was significantly higher in rats given DNPH after pretreatment with bergenin (Group 5) than in non-pretreated rats given DNPH (Group 3), an indication that bergenin protected against DNPH-induced depletion of liver, brain and red blood cell ascorbic acid. Also in each case, tissue ascorbic acid content of rats given only bergenin (Group 4) was significantly higher than in water controls (Group 1), suggesting that bergenin also protects against endogenous oxidants.

Figure 4

Table 3: Effect of bergenin on tissue ascorbic acid level during experimental peroxidation.

Discussion

Ekong and Ejike (1974) first reported that bergenin isolated from S.gabonensis bark exhibited antioxidant properties in palm wine metabolism but did not state the site or mode of action. Okechukwu (1998) subsequently demonstrated that the crude extract of the bark inhibited palm wine yeast alcohol dehydrogenase and aldehyde dehydnegenase. The results obtained in this study suggest that bergenin also exhibits antioxidant properties towards experimental peroxidation in the mammalian system. For the observed reduction in the level of experimental peroxidation and in the rate of depletion of tissue ascorbic acid when rats were pretreated with bergenin, are evidences of antioxidant effect.

The initiation stage of lipid peroxidation is a free radical reaction with lipid hydroperoxides-products of the reaction between lipid peroxyl radical and oxygen-being the first stable intermediate (Wills 1987). Lipid hydroperoxides subsequently give rise to lipid aldehydes. The fact that pretreatment with bergenin reduced the levels of these two stable intermediates in experimental lipid peroxidation is an indication that the photochemical exerts its antioxidant action prior to the formation of the first stable intermediate. More work is required to understand the exact site and mode of action as well as the part of the bergenin molecule responsible for the antioxidant activity in a peroxidation reaction. However, in the case of the later, indications from reports on another antioxidant photochemical point to attachment of both polar and lipophilic substituent groups to the coumarin nucleus. For example, Sugiyama et al (1993) has shown that the presence of both polar and lipophilic substituent groups on the nucleus of purpurogallin molecule was responsible for its antioxidant properties against peroxyl radicals in human erythrocytes.

Theoretical mechanistic biochemistry concept has used the theoretical bioactivation of phenyl hydrazine-induced lipid peroxidation as well as the structure of bergenin as models ( Maduka,2005a and Maduka 2005b) to justify the observed antioxidant properties of Sacoglottis gabonensis(Maduka and Okoye,2002,Maduka et.al,2003).Theoretical mechanistic biochemistry relies on the structures of oxidants to predict their reactivities in tissues.From thje structure of bergenin,positions 1,2,3 and 4 are positions of antioxidant properties with positions 1 and 2 being greater than positions 3 and 4.Bergenin could give off any of the protons to react with the free radicals generated during the theoretical bioactivation scheme of phenyl hydrazine.

Ascorbic acid is a reducing agent which plays a general antioxidant role in the body. The observed reduction of its tissue level in experimental peroxidation is therefore, an indication that it participates in the body's antioxidant defences against xenobiotics like DNPH. Furthermore, the fact that the ascorbic acid sparing effect of bergenin was observed even in the absence of the experimental oxidant, DNPH is an indication that bergenin exerts antioxidant action on endogenous oxidizing agents as well.

Since similar antioxidant effect on lipid peroxidation and tissue ascorbic acid has been observed with the crude extract of S. gabonensis stem bark (Maduka 2000, Maduka and Okoye 2002a, 2002b, Maduka et al., 2003) the present data would confirm that bergenin is a majoy component of Sacoglottis gabonensis bark extract responsible for the antiperoxidising properties of the extract. Bergenin appears to act partly by sparing tissue ascorbic acid,a preferred antioxidant in the brain.As already reported,bergenin is a major isolate of Sacoglottis gabonensis(Ogan,1971)

Which is an alcoholic beverage additive in Nigeria.The case being reported in this presentation is a typical case of manipulation of alcohol effects in the tissues described including the brain.

Correspondence to

H.C.C. Maduka Department Of Biological Sciences, The University Of Hull, HU6 7RX, UK. E-mail: chmaduka@yahoo.com

Acknowledgment