ANTIOXIDANT COMPOSITIONS

TECHNICAL FIELD OF THE INVENTION

This invention relates to an antioxidant for use in human and animal nutrition as well as cosmetics.

BACKGROUND ART

Bioflavonoids are polyphenols widely found in vascular plants. These polyphenol plant molecules are strong scavengers of free radicals and chelate heavy metals. Some of the molecules also inhibit the allergic and inflammatory reaction, stimulate protein synthesis and influence enzyme systems in animals. High concentrations of the bioflavonoids are found in a variety of plant materials and can thus easily be extracted from certain materials.

There is a general movement away from the use of chemotherapeutic synthetic and antibiotic feed additives in animal production. The reason for this is the general negative effect of the chemotherapeutic, synthetic and antibiotic feed additives on the ecosystem. There is also pressure from the customer who is demanding animal products produced without the use of chemotherapeutic, antibiotic and synthetic substances. Animal nutritionists thus have to search for substitute feed additives, in order to keep on producing animals in an economically viable way.

Natural products like live microbial cultures and plant extracts have to be researched and developed and made available to industry in order to combat the negative effect of intensive animal production systems.

Vitamin A and E are examples of natural substances which have a positive effect on animal production. These antioxidative vitamins play an important role in the immune reaction, detoxifying in body, in vivo scavenging of free radicals, preventing mycotoxicosis etc. The vitamins are however expensive feed additives and fortification of animal fees is not always economically viable.

The blackwattle tree including, amongst other species used in tannic acid production, Acacia mearnsii , was identified by the Applicant as a bioflavonoid rich plant source with the potential of being used as a natural feed additive. Large quantities of the bark are currently produced on a commercial scale in South Africa

It is therefore an object of this invention to provide an extract of Acacia mearnsii which exhibits anti-oxidant effects and finds application in raw materials and finished animal feeds; prevents radical chain damage to vitamins, exhibits vitamin protective activity in vivo in order to prevent oxidation and depletion of vitamins A and E in poultry and rats; and act as free radical scavenger in cosmetic products as a preventative of free radical damage to the skin.

DISCLOSURE OF THE INVENTION

According to a first aspect of the invention, an ethyl acetate extract of the bark of the blackwattle tree and specifically,/\cac/a meamsii tree is provided as an anti-oxidant composition.

The invention also provides for the addition of the blackwattle extract to impart antioxidant properties to the raw materials of animal feedstuffs as well as the final product of animal feedstuffs. The extract may be added in powdered or liquid form.

The invention further provides the use of Acacia mearnsii extract to reduce or prevent free radical chain damage to vitamins in vitro in poultry and rats, to prevent oxidation and depletion of vitamins.

The invention further provides for the use of the Acacia mearnsii extract as a medicament for the prevention of free radical damage to the skin.

The Acacia mearnsii extract is preferably prepared by a process comprising the steps of milling dried bark chips into a fine powder in a hammer mill using 0.5 mm sieve, adding the dry powder (50Og in 10 litres) to distilled water and maintaining at room temperature (about 25 ⁰ C) under nitrogen for approximately four days, out of direct sunlight, with occasional agitation; filtering the aqueous mixture and extracting the filtrate

with one litre of ethyl acetate with vigorous agitation before allowing the extracted mixture to settle and partition into organic and aqueous phases over a 24 hour period, separating the organic phase and drying with anhydrous sodium sulphate before evaporating off the ethyl acetate under vacuum with temperature being maintained below 50 degrees Celsius; mixing the dark syrup - like residue with 1 :1 acetone- methanol solution and then evaporating to form a red-brown powder.

In an alternative form of the invention the Acacia mearnsii extract is prepared by a more cost effective process comprising the steps of: milling dried bark chips into a fine powder in a hammer mill using 0.5 mm sieve, adding the dry powder (50Og in 10 litres) to distilled water and maintaining at room temperature (about 25 ⁰ C) under nitrogen for approximately four days, out of direct sunlight, with occasional agitation; filtering off the water and putting aside one litre thereof, mixing the remaining nine litres with approximately one kilogram of vermiculite and drying under reduced pressure at temperatures not exceeding 50 degrees Celsius; the one litre set aside is then extracted three times with ethyl acetate as described above, and the total mass of red-brown powder (organic extract) calculated with the total concentration of this vermiculite extract being expressed as percentage organic extract present (m/m); milling the dried vermiculite to a fine powder in a hammer mill with a 0.5 mm sieve.

The invention also provides a composition containing the blackwattle. The composition may be a food or food product.

The composition may also be a dietary supplement, such as a nutraceutical or other nutritional composition.

Alternatively, the composition may be a pharmaceutical composition comprising the extract described above, admixed with one or more pharmaceutically acceptable excipients.

In a further aspect, the invention provides the use of an Acacia mearnsii extract as a nutraceutical, such as a dietary supplement, or as an active ingredient in the preparation of medical or functional foods and beverages.

The invention is further described with reference to the following examples. However, it should be appreciated that the invention is not limited to these examples.

EXAMPLE 1

Two models were used to determine if bioflavonoids present in the blackwattle bark extracts might be capable of delaying oxidation process. The first model is the 1, i-diphenyl-2-picrylhydrazyl (DPPH) model and is widely used to determine the free radical scavenging capacity of antioxidants. The second model the poly-unsaturated fatty acid model, directly determines if antioxidants prevent poly-unsaturated fatty acids from being oxidized.

1, 1-diphenyl-2-picrylhydrazyl (DPPH) is a stable free radical with a deep-violet colour due to the extra electron. When this electron is taken over by another molecule in the free radical chain the deep-violet colour change brownish with resultant decrease in optical density. If an antioxidant is mixed with the DPPH free radical solution it will take over the extra electron. By measuring the optical density of this electron takeover by an antioxidant against time, one can get a good idea of the capacity of the antioxidant. This is a good model for measuring the free radical scavenging (antioxidant) capabilities of an antioxidant. Alpha - tocopherol a natural antioxidant is widely used as reference when determining the free radical scavenging capacity.

The PUFA (poly-unsaturated fatty acid) - model is a modified and still unstandardized method for evaluating antioxidants directly in feed samples. The method is based on the interpretation of the fatty acid composition of a lipid rich feed sample after undergoing controlled oxidation. Antioxidant capacity in lipid rich feed samples is usually determined by measuring the degradation products (hydroperoxides, aldehydes etc.) of the fatty acid oxidation process. The PUFA model however uses the substrate most susceptible to oxidation (poly-unsaturated fatty acids) as indication of antioxidant capacity and not end products.

MATERIALS AND METHODS

MATERIALS

DPPH - model; 1,1-diphenyl-2-picrylhydrazyl (DPPH), catechin, rutin and quercetin were obtained from Sigma USA. The natural Acacia meamsii organic and water plant extracts were prepared according to the methods described above and contained the same physical and chemical properties. Ethanol was used as solvent in preparing the reagents and a Hitachi U3200 spectrophotometer with 1 cm absorption cells were used for monitoring the optical density.

PUFA - MODEL; A high fat content mixture of raw materials were obtained from a local feed company, the mixture consisted of 60% full fat soya, 20% fish meal and 20% carcass meal. The homogen mixtures were milled through a 0.5 mm sieve in order to obtain small oxidisable feed particles. A standard oxygen bomb, normally used to determine the gross energy of feedstuffs, was used to oxidize the feed samples in a standardized environment. Endox a commercial antioxidant was obtained from Feed Ad SA (Kemin), and consist of ethoxyquin, butyl hydroxyanhydrone (BHA) and citric acid. The natural Acacia mearnsii organic and water extracts were produced using the same method as described above and contained the same physical and chemical properties.

METHODS

DPPH -model; The method described by Blois, 1958 and Breugnot, 1991 was used. A 0.5mM DPPH solution were also used in this study as described by literature. Antioxidants evaluated were quercetin, rutin and catechin, the three monomelic flavonoids generally used in various studies. The organic and water Acacia mearnsii extracts were also evaluated in order to compare the natural flavoid rich plant extracts antioxidant capacity against the pure synthetic flavonoids. Concentrations of flavonoids and plant extracts were expressed as percentage in order to obtain comparable results on a one to one basis. The reason for this is the fact that the exact molecular mass and ratio of flavonoids present in the plant extract are not known and may vary from plant to plant. The antioxidant capacity of the pure flavonoids were determined using different concentrations. From this data a concentration range were identified to give an average optical density decrease which can easily be plotted and interpreted. The following

solutions with concentrations falling in the above range were made up. Concentrations of pure synthetic flavonoids and the organic extract were the same (0.33% m/v). The water extract is dried in vermiculite and thus not soluble because of the vermiculite carrier and accordingly, a water solution (water infused with Acacia mearnsii bark) containing 1.7% m/v total flavonoids was used in order to obtain a result from the water extract fraction of the bark. A 100 mM (4.3% m/v) alpha - tocopherol solution was used as reference substance for determining antioxidant capacity. Ethanol was used as solute and the method completed in a semidark environment to prevent any oxidation. One ml of the DPPH solution is put into a 1 cm crystal cuvette. Ten microliters of the above antioxidant solutions is then put into the DPPH filled cuvette, and optical density (516 nm) measured over five minutes. The absorption spectra of each solution is then plotted against time with the alpha-tocopherol plot as reference.

PUFA - model; The method used is selfdesigned and not standardised. The objective of using this method was to see how the flavonoids react under practical conditions to oxidation. Four batches (5Og) of the lipid rich homogenous raw material mixture was mixed with two percent m/v Endox, two percent m/v A. mearnsii organic extract and four percent m/v of the A.mearnsii water extract. The two percent inclusion rate is much higher than concentrations currently used in practical situations, it was decided however to use the higher concentrations because the rate (100% oxygen at six time atmospheric pressure) of oxidation applied to a highly oxidisable mixture is much higher than under normal conditions. The concentration of the water extract with vermiculite as carrier were four percent because of the relatively low flavonoid content in this plant extract. It was however included in the study to see if the water extract had any antioxidant capabilities after being dried in the vermiculite. A control mixture (5Og) was also used with no antioxidants added. Each of these 50 g samples were put in the oxygen bomb, flushed with 100% oxygen and then loaded with 100% oxygen to a pressure of 600 kPa. The bomb was then put in a 9O ⁰ C oil bath for 30 minutes and cooled after which the sample were kept at room temperature (25 ⁰ C) for 3 days in open bottles. Total lipid and fatty acid analysis was done using thin layer and gas chromatography (Kock & Ratledge, 1993).

RESULTS AND DISCUSSION

DPPH - model; The decrease in optical density of a 0.5 mM DPPH solution mixed with the synthetic monomeric flavonoids quercetin, rutin and catechin in shown in Figure 1a.

Catechin was the strongest scavenger of the DPPH radical with an optical density of 0.28 mm after 30 seconds. Quercetin was the second strongest scavenger of the DPPH radical with rutin and alpha - tocopherol in the same scavenging order. The three flavonoids end point optical density after 300 seconds were in the same order at 0.1 nm in comparison with the 0.2 nm of the reference substance, alpha - tocopherol. This showed that the speed by which the DPPH radicals were scavenged was in the order catechin > quercetin > rutin and the overall free radical scavenger capacity also in the same order.

The magnitude by which the Acacia mearnsii organic and water extract scavenged the DPPH radicals can be seen in Figure 1b (decrease in optical density). The scavenging speed and end point optical density of both extracts were of the same magnitude. The plant extracts scavenged the DPPHB radicals quicker than the flavonol flavonoids quercetin and rutin, but slower than catechin. The optical density of the plant extracts after 30 seconds were about 0,49 nm in comparison with the 0.28 nm and 0.5 nm of catechin and quercetin respectively. After 60 seconds the plant extracts had a optical density of 0.28 nm in comparison with the 0.39 nm of quercetin and the 0.18 nm of catechin. The plant extracts endpoint optical density after 300 seconds were lower than the monomeric flavonoids and it looks as if the plant extracts might have a greater free radical scavenging capacity than the monomeric flavonoids.

The results showed that the Acacia mearnsii extracts reacted similar to the monomeric flavonoids in their pattern of scavenging the DPPH free radical. One can thus conclude that although other plant metabolites present in the plant extracts might have influenced the scavenging pattern, the scavenging of free radicals by the plant extracts are due to the presence of flavonoids in the A. mearnsii plant extracts. The plant extracts also compare very well to the monomeric flavonoids in their capacity of scavenging free radicals. The plant extracts and monomeric flavonoids were in relation

to the reference antioxidant, alpha - tocopherol very strong scavengers of the DPPH free radical. It also appears that the strong scavenger capacity of the plant extracts were because of the catechin - flavonoid dimer present in the plant extracts, there catechin was the strongest monomelic flavonoid scavenger of the DPPH free radicals.

PUFA - MODEL; The fatty acid concentration of the control raw material mixture and the mixture mixed with Endox and the two plant extracts were expressed as a percentage of total lipids and can be in Figure 2. The 16 and 18 carbon fatty acids spectrum were determined and were subdivided according to degree of saturation. The poly unsaturated fatty acids were determined by pooling the 18.2 and 18.3 fatty acid concentration and were used as a parameter of degree of oxidation.

The PUFA concentration of samples containing Endox, organic extract and water extract were 36.04, 50.65 and 40.58 percent respectively, higher than the control sample with no additives. This showed that the two percent organic extract added to the raw material mixture were the strongest inhibitor of PUFA degradation through oxidation. The 16.0 fatty acid concentration being the shortest saturated fatty acid determined were 19.31 , 20.30 and 14.85 percent respectively lower than the control sample. This showed that the organic extract sample had the lowest concentration of the 16.0 fatty acid and is thus sample with the lowest oxidation damage to its unsaturated fatty acids. The water extract with vermiculite as carrier, although at a higher concentration, also protected the PUFA's from being oxidised. The reason for this might be the fact that this extract contains a lot of the complex tannin molecules not soluble in the ethyl acetate solution used for the extraction of the organic extract. These results showed that the flavonoid rich A.mearnsii plant extracts have the ability to operate as antioxidant under commercial conditions.

The DPPH - model showed that the monomeric flavonoids as well as the A.mearnsii plant extracts are in relation too alpha - tocopherol, very active scavengers of the DPPH free radical. It also showed that the scavenger capacity of the plant extracts are probable due to the flavonoid content of the plant extracts. The results further suggested that the strong scavenger effect of the plant extracts might be because of the catechin - flavonoid dimer present in the plant extract, there catechin was the strongest scavenger of the three monomeric flavonoids tested.

The PUFA - model showed that the A. mearnsii plant extracts can be used under normal conditions in raw materials and finished feeds.

Vitamin/ antioxidant protective effect

Two experiments (rats and broilers) were done to determined the vitamin/antioxidant protective effect of A. mearnsii. Four treatments (diets), control, synthetic flavonoids, organic A.mearnsii plant extract and water A. mearnsii plant extract were used in both experiments. The type and inclusion rate of bioflavonoids were the same for both experiments and can be seen in Figure 3. The pure monomelic laboratory refined flavonoids were obtained from Sigma USA and the organic and water A. mearnsii plant extracts produced as described above and had the same chemical and physical properties as described.

EXAMPLE 2 - EXPERIMENT WITH RATS

Fourty Sprague Dawley rats (five males and five females in each treatment) were fed a normal rat growth diet from weaning onwards for eight weeks. The rat growth pellets were milled and the bioflavonoids mixed in the concentrations given in Figure 3. Diets control, synthetic flavonoids, organic A. mearnsii plant extract and water A.mearnsii plant extract were given ad lib each day by making a wet paste which is more palatable than a dry mash. The left overs were thrown away and a fresh paste mixed every morning.

After eight weeks the male and female rats ere decapitated and liver and blood samples taken from each rat. The blood samples were centrifuged to separate plasma from the cells and the liver samples (+/-one gram) homogenized in phosphate buffer. This whole process took place in semi-darkness in order to prevent any degradation of vitamins by light. The samples up to the stage of analysis were also kept on ice. Alpha - tocopherol, retinol and retinol ester concentrations were determined in the liver and plasma samples by using High Performance Liquid Chromatography (HPLC) (Nierenberg and Nann, 1992; Van Jaarsveld et al, 1992). Protein content was measure by the method Lowry et al, 1951).

Differences in average vitamin concentration between male and female rats were detected and the results therefore expressed separately. The alpha - tocopherol, retinol and retinol ester concentration of male and female rats receiving the control diet, diet supplemented with synthetic flavonoids, diet supplemental with the organic A. meamsii extract and diet supplemented with the water A. meamsii extract can be seen in Figure 5 and Figure 6. The concentration of liver vitamins were expressed an nmol/mg protein and in the plasma as nmol/ml.

Alpha - tocopherol; No significant (p-0.05) difference were detected between male liver alpha - tocopherol levels for different dietary treatments. The organic A. meamsii dietary treatment however at 17.9% increase in male liver alpha - tocopherol levels in relation with the control although not significant. The alpha - tocopherol concentration in male liver of synthetic flavonoid and water A.mearnsii dietary treatments were in the same order as the control treatment. In female livers significant difference (p-0.05) were detected between the synthetic flavonoid treatment and the control. The synthetic flavonoid, organic A. meamsii and water A. meamsii dietary treatments showed respectively at 45.1, 17.8 and 14.2% increase in liver alpha - tocopherol levels.

No significant (p-0.05) differences between dietary treatments were found for male plasma alpha - tocopherol levels. The synthetic flavonoid treatment showed a 20.5% increase in plasma alpha - tocopherol level and the organic and water A.mearnsii treatments a decrease of 1.4 and 11.3% respectively. No significant differences (p- 0.05) were found between female plasma alpha - tocopherol levels for different dietary treatments. A definite plasma alpha - tocopherol increasing tendency were showed for the synthetic flavonoid (18.9%), organic - (12.3%) and water (14.8%) A. meamsii plant extract treatments, although none significant higher.

The liver and plasma alpha - tocopherol concentration of female rats were about 20-30% higher compared to the males. This differences might be due to differences in growth rate which will lead.

Vitamin concentration in liver (a) and plasma (b) of rats receiving a control diet and diets supplemented with synthetic flavonoids and A. meamsii plant extracts to different vitamin E needs at a specific time and a faster or slower depletion for a specific

sex. Human females were found to preferentially transport vitamin E in plasma and lymph with high density lipoproteins and human males with low density lipoproteins (Combs, 1992). There may thus be difference in vitamin E metabolism between male and females of similar species.

Retinol; A significant difference (p-0.05) in male liver retinol level were found between the control and synthetic flavonoid dietary treatments. A lowering tendency of male liver retinol level in relation with the control were found for the synthetic flavonoid (21.2%), organic - (16.7%) and water (18.4%) A. mearnsii plant extract treatments. Significant differences (p-0.05) were also found in female liver retinol levels between the control and the A.mearnsii plant extracts. The liver female retinol levels of the synthetic flavonoid, organic - and water A. mearnsii plant extracts were respectively 9.4, 31.0 and 26.4% lower than the control treatment levels.

No significant difference (p-0.05) in male plasma retinol levels between different dietary treatments were found. Male plasma retinol levels of the synthetic flavonoid, organic - and water A. meamsii plant extract were in relation to the control respectively 4.9, 24.9 and 4.0% lower. The female plasma retinol concentration of the organic A. mearnsii plant extract treatment were significant (p-0.05) lower than the control. The female plasma retinol concentration of the synthetic flavonoid, organic - and water A. meamsii plant extract treatments were in relation to the control, respectively 7.5, 31.6 and 23.0% lower.

The female liver and plasma retinol concentration of the control groups were respectively 6 and 30% lower than the male control group. Reasons for the difference in retinol status between sexes might be similar to those discussed for alpha - tocopherol, but is as yet unknown.

Retinol ester; No retinol esters were found in plasma of rats, this correlates well with literature which state that retinol is transported by binding to a plasma protein and not as an ester (Combs, 1992). The male liver retinol ester concentration of the water A. meamsii plant extract treatment were significant (p-0.05) lower compared to the control treatment. The male lifer retinol ester concentration of the synthetic flavonoid, organic - and water A. mearnsii plant extract treatment were in relation to the control, respectively

17.6, 31.1 and 59.4% lower. The female liver retinol ester concentration of both the A. mearnsii plant extracts were significant (p-0.05) lower compared to the control treatment. The female liver retinol ester concentration of the synthetic flavonoid, organic - and water A. mearnsii treatments were in relation to the control, respectively 13.3, 50.7 and 65.4% lower.

The female liver retinol ester concentration of the control treatments were 21% higher than the male liver retinol ester concentration.

Conclusion

No definite differences in alpha - tocopherol levels were found in male rat livers between the different dietary treatments. In contrast a definite increase in female liver alpha - tocopherol levels were found for the three flavonoid enriched diets. Male plasma alpha - tocopherol levels like the liver levels also did not differ very much between treatments. The female plasma alpha - tocopherol levels showed the same increasing tendency as liver concentrations for the flavonoid enriched dietary treatments compared to the control treatment. The synthetic flavonoid dietary treatment had the highest alpha - tocopherol status in the liver and plasma compared to the A. mearnsii plant extract dietary treatments which were in the same order.

The three flavonoid enriched dietary treatments lead to an overall decrease in male and female liver retinol concentration. The decrease of female liver retinol levels were more outspoken then the male liver retinol levels. The decrease in plasma retinol levels of male and females followed the same pattern as for liver retinol levels. Again the decrease in female plasma levels were more outspoken that those of the males. The A. mearnsii plant extract treatments had the lowest plasma and liver retinol status in the females.

The flavonoid enriched treatments had a decrease in liver retinol ester levels of both sexes. The A. meamsii treatments had the lowest retinol ester status in the liver and showed a decrease of up to 59%.

Something of interest observed in this study is the difference in vitamin A and E status between male and female Sprague Dawley rats. Enrichment of rat diets with flavonoids also did not have any effect on the alpha - tocopherol status of male rats compared to female rats. Movement in retinol and retinol ester status were however seen in male rats. The reason for the fact that no movement in alpha - tocopherol levels in males were detected is unknown.

The study shows that enrichment of rat diets with synthetic and natural flonoids lead to an increase in alpha - tocopherol status of female rats and a decrease in retinol and retinol ester status of male and female rats. The supplementation of rat diets with synthetic flavonoids lead to a more pronounced increase in alpha - tocopherol status compared to the natural flavonoids. In contrast the supplementation of rat diets with A. mearnsii plant extracts lead to a more pronounced decrease in retinol and retinol ester status of male and female rats.

EXAMPLE 3 - Experiment with chickens

Four hundred day old Cobb broilers (mixed sexes) were fed to a starter mash from day one to day 21 and a grower mash from day 22 to slaughter. Diet composition and formulation specifications can be seen in Figure 7 No therapeutic agents and vaccines were used in this broiler experiment. Both the starter and growth/finisher diets were split in four and experimental diets mixed to the specifications of Figure4. The four treatments were randomly assigned to 12 pens to obtain three replicates per treatment. After 43 days four individual broilers from each group, total of 12 individuals from each diet (six males and six females) were slaughtered. Blood and liver samples were taken and stored on ice. The blood was centrifuged to separate plasma and cells and the liver were homogenized in phosphate buffer. The whole process of sampling and sample preparation were similar to methods used in above. Alpha - tocopherol, retinol and retinol ester concentrations were determined in the plasma and liver samples by using HPLC techniques (Nierenberg and Bann, 1992; Van Jaarsveld et al, 1992). Protein content was measured by the method of Lowry et al, (1951).

Results were statistically analysed using the Yukey-Kramer method. Differences between the means were tested at a 95% significance level. Results are given as mean +/- standard erro (S. E.).

Plasma and liver samples of male and female broilers were also analysed apart as in experiment with rats, no difference in average vitamin concentration were however detected between sexes and results were pooled. The liver vitamin concentration are expressed as nmol/mg protein and the plasma vitamin concentration as nmol/ml plasma. The alpha - tocopherol, retinol and retinol ester concentration in liver and plasma of broilers receiving a control diet, diet supplemented with synthetic flavonoids, diet supplemented with the organic A. mearnsii plant extract and diet supplemented with the water A., mearnsii plant extract can be seen in Figure 8. and Figure 9. _*

Alpha - tocopherol; A significant (p-0.05) increase in liver alpha - tocopherol concentration were detected for the water A. mearnsii plant extract treatment. The liver alpha - tocopherol concentration of the synthetic flavonoid, organic - and water A. meamsii plant extract treatments in relation to the control, were respectively 3.4, 40.1 and 88.8% higher. No significant (p-0.05) differences in plasma alpha - tocopherol levels were found between any of the treatments. A very small increase of between two and 10% were found for diets supplemented with the synthetic and natural flavonoids.

Retinol; The liver retinol concentration of organic - and water A. meamsii plant extract treatments were significant (p-0.05) higher than the control and synthetic flavonoid treatments. The liver retinol concentration of the synthetic flavonoid, organic - and water A. mearnsii plant extract treatments in relation to the control were respectively 22.8, 98.4 and 96.9% . higher. No real differences in plasma retinol concentration between treatments were detected.

Retinol ester No significant (p-0.05) differences in liver retinol ester concentration between the control and flavonoid diets were detected, the natural flavonoid treatments did however had a significant ^' (p-0.05) higher level retinol ester status compared to the synthetic flavonoid ;treatment. The retinol ester concentration of the synthetic flavonoid treatment were in relation to the control, 14.8 % lower and the

organic - and water A. mearnsii plant extraction treatments respectively 20.9 and 22.7% higher.

As in the rats, the same increasing alpha - tocopherol status was seen in broilers. No difference between sexes were observed as was the case in rats

CONCLUSION

These results showed that supplementation of broiler diets with the natural A.mearnsii plant extract did have a protective effect on the antioxidant vitamins in vivo. The protective effect of the synthetic flavonoid supplementation were less pronounced. The mechanism of protection is unknowniand will have to be determined by further studies. Possible mechanisms may firstly be the antioxidant working of bioflavonoids because the flavonoids and vitamins E and A moves in similar routes in the body. A second reason may be the fact that the flavonoids have a positive effect on certain metabolic systems resulting in optimal absorption, transport, storage, etc. Bioflavonoids do influence enzyme systems and inhibition or stimulation of catabolic and anabolic enzymes might also have an effect on the vitamin status of animals.

These experiments with rats and broilers showed that supplementation of diets with natural and synthetic flavonoids did have an effect on the antioxidant vitamin status of the broilers. Factors like specie, sex and maturation age did influence the in vivo vitamin metabolism reaction due to flavonoid intake. The experiments further showed that in most cases, synthetic and natural flavonoid rich plant extracts had similar effects on the vitamin metabolism of rats and broilers.

The protection of vitamins A and E in broilers by the natural A. mearnsii plant extracts is of importance because usage of a stable product like the flavoid plant extract as a partial replacement of these vitamins in commercial broiler diets might be of economic importance.