Inhibitory effect of an ellagic acid-rich pomegranate extract on tyrosinase activity and ultraviolet-induced pigmentation

A pomegranate extract (PE) from the rind containing 90% ellagic acid was tested for its skin-whitening effect. PE showed inhibitory activity against mushroom tyrosinase in vitro, and the inhibition by the extract was comparable to that of arbutin, which is a known whitening agent. PE, when administered orally, also inhibited UV-induced skin pigmentation on the back of brownish guinea pigs. The intensity of the skin-whitening effect was similar between guinea pigs fed with PE and those fed with L-ascorbic acid. PE reduced the number of DOPA-positive melanocytes in the epidermis of UV-irradiated guinea pigs, but L-ascorbic acid did not. These results suggest that the skin-whitening effect of PE was probably due to inhibition of the proliferation of melanocytes and melanin synthesis by tyrosinase in melanocytes. PE, when taken orally, may be used as an effective whitening agent for the skin.     

Lightening Effect on Ultraviolet‐Induced Pigmentation of Guinea Pig Skin by Oral Administration of a Proanthocyanidin‐Rich Extract from Grape Seeds

Antioxidants such as vitamins C and E have been reported to inhibit the progression of ultraviolet (UV) radiation‐induced pigmentation in the skin of hairless mice. However, little is known of the lightening effect of proanthocyanidin, a powerful polyphenolic antioxidant, on UV‐induced pigmentation of the skin. We investigated the lightening effect of oral administration of a proanthocyanidin‐rich grape seed extract (GSE) using guinea pigs with UV‐induced pigmentation. These pigmented guinea pigs were fed diets containing 1% GSE or 1% vitamin C (w/w) for 8 weeks. GSE‐feeding had an apparent lightening effect on the guinea pigs’ pigmented skin. Histologic evaluation demonstrated a decrease in the number of 3,4‐dihydroxyphenylalanine (DOPA)‐positive melanocytes as well as 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG)‐positive, Ki‐67‐positive, proliferating cell nuclear antigen (PCNA)‐positive melanin‐containing cells in the basal epidermal layer of the UV‐irradiated skin in GSE‐fed guinea pigs. In contrast, these parameters did not change in the skin of vitamin C‐fed or control guinea pigs. GSE inhibited the activity of mushroom tyrosinase and also inhibited melanogenesis without inhibiting the growth of cultured B16 mouse melanoma cells. In conclusion, we demonstrated that oral administration of GSE is effective in lightening the UV‐induced pigmentation of guinea pig skin. This effect may be related to the inhibition of melanin synthesis by tyrosinase in melanocytes and the reactive oxygen species (ROS)‐related proliferation of melanocytes.     

Lightening effect on ultraviolet-induced pigmentation of guinea pig skin by oral administration of a proanthocyanidin-rich extract from grape seeds

Antioxidants such as vitamins C and E have been reported to inhibit the progression of ultraviolet (UV) radiation-induced pigmentation in the skin of hairless mice. However, little is known of the lightening effect of proanthocyanidin, a powerful polyphenolic antioxidant, on UV-induced pigmentation of the skin. We investigated the lightening effect of oral administration of a proanthocyanidin-rich grape seed extract (GSE) using guinea pigs with UV-induced pigmentation. These pigmented guinea pigs were fed diets containing 1% GSE or 1% vitamin C (w/w) for 8 weeks. GSE-feeding had an apparent lightening effect on the guinea pigs' pigmented skin. Histologic evaluation demonstrated a decrease in the number of 3,4-dihydroxyphenylalanine (DOPA)-positive melanocytes as well as 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive, Ki-67-positive, proliferating cell nuclear antigen (PCNA)-positive melanin-containing cells in the basal epidermal layer of the UV-irradiated skin in GSE-fed guinea pigs. In contrast, these parameters did not change in the skin of vitamin C-fed or control guinea pigs. GSE inhibited the activity of mushroom tyrosinase and also inhibited melanogenesis without inhibiting the growth of cultured B16 mouse melanoma cells. In conclusion, we demonstrated that oral administration of GSE is effective in lightening the UV-induced pigmentation of guinea pig skin. This effect may be related to the inhibition of melanin synthesis by tyrosinase in melanocytes and the reactive oxygen species (ROS)-related proliferation of melanocytes.     

Inhibitory Effect of an Ellagic Acid-Rich Pomegranate Extract on Tyrosinase Activity and Ultraviolet-Induced Pigmentation

A pomegranate extract (PE) from the rind containing 90% ellagic acid was tested for its skin-whitening effect. PE showed inhibitory activity against mushroom tyrosinase in vitro, and the inhibition by the extract was comparable to that of arbutin, which is a known whitening agent. PE, when administered orally, also inhibited UV-induced skin pigmentation on the back of brownish guinea pigs. The intensity of the skin-whitening effect was similar between guinea pigs fed with PE and those fed with L-ascorbic acid. PE reduced the number of DOPA-positive melanocytes in the epidermis of UV-irradiated guinea pigs, but L-ascorbic acid did not. These results suggest that the skin-whitening effect of PE was probably due to inhibition of the proliferation of melanocytes and melanin synthesis by tyrosinase in melanocytes. PE, when taken orally, may be used as an effective whitening agent for the skin.     

Hormonal effects of vitamin D3 on epidermal melanocytes

The role of cholecalciferol, 25(OH) D3, and 1,25(OH)2 D3, as modulators of melanocyte function and proliferation has been examined. Topical application of 100 micrograms cholecalciferol to the pinnal epidermis of DBA/2J mice for 5 or 10 days increased the number of L-dihydroxyphenylalanine-positive (DOPA-positive) melanocytes and had a synergistic effect with a low dose of ultraviolet B light (UVB). Application of 1 microgram 1,25(OH)2 D3 had a transient effect on epidermal melanocytes. Addition of cholecalciferol to pure cultures of human melanocytes did not alter their tyrosinase activity (therefore, melanin synthesis) or growth rate even after 72 hours of treatment. However, treatment of similar cultures with 1,25(OH)2 D3 at a concentration equal to or greater than 10(-8) M suppressed tyrosinase activity but did not affect proliferation. The effect of 25(OH) D3 was similar to, but lower in magnitude than, that of 1,25(OH)2 D3. We attempted to demonstrate the presence of specific receptors for 1,25(OH)2 D3 in normal human melanocytes using the monoclonal antibody (Mo Ab) 9A7 gamma raised against the receptor for 1,25(OH)2 D3. Melanocytes were exposed to 9A7 gamma and to a secondary biotinylated Ab and analyzed by the fluorescence activated cell sorter (FACS). An increase in the specific fluorescent signal was constantly observed. By using the immunoblotting technique, we observed a major immunoreactive species that migrated in the 53-kD region in normal melanocytes. The size of this major immunoreactive species was smaller in melanoma cells than in normal melanocytes. This correlates with the finding that the former cells were unresponsive to cholecalciferol, 25(OH) D3, or 1,25(OH)2 D3 treatment. These results predict a direct role for 1,25(OH)2 D3 as an effector of normal melanocyte function.     

Antioxidant vitamin levels do not exhibit negative correlation with the extent of acute myocardial infarction

Serum levels of vitamin E (VE), beta-carotene (BC) and vitamin C (VC) were determined in 50 patients with the first acute myocardial infarction (AMI) before starting thrombolytical treatment. VE and BC were determined by HPLC, VC spectrophotometrically. The reperfused patients were divided according to vitamin concentrations into four groups. The lowest quartile was compared with the rest of the studied population (VE: group with high (H)>15.6 microM>group with low (L), BC: H>0.07 microM>L, VC: H>25 microM>L) in the following parameters: extent of myocardial damage (area under the curves of troponin I, CK-MB during 48 h), arrhythmia and congestive heart failure occurrence, size of ejection fraction, positivity of ventricular late potentials. No significant differences between groups H and L for either VE, BC or VC were found (P 0.05). As no correlation between serum concentrations of vitamins E, C and beta-carotene and the extent and clinical course of AMI was found, the actual vitamin concentrations may be important for prevention of ischemic heart a disease, but they do not play a decisive role in the acute phase of myocardial infarction in humans.     

Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A

The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.     

Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A

The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.     

Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.     

Syndecan‐2 regulates melanin synthesis via protein kinase C βII‐mediated tyrosinase activation

Syndecan‐2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan‐2 in melanogenesis. Syndecan‐2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA‐mediated knockdown of syndecan‐2 was associated with reduced melanin synthesis, whereas overexpression of syndecan‐2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan‐2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan‐2 expression, and this up‐regulation of syndecan‐2 was required for UVB‐induced melanin synthesis. Taken together, these data suggest that syndecan‐2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin‐associated diseases.     

A combination of alpha-tocopherol,vitamin C and N-acetyl cysteine increases unsaturated fatty acid levels in hydrogen peroxide-induced Candida tropicalis (ATCC 13803)

This research was aimed at evaluating the antioxidant effects of combinations of alpha lipoic acid (LA), vitamin C (VC), N-acetyl cysteine (NAC) and alpha-tocopherol (TOC) on lipid level and fatty acid composition of C. tropicalis (ATCC 13803) against hydrogen peroxide toxicity. According to the experimental results, the cell density of C. tropicalis increased significantly in NAC+LA+H2O2, NAC+TOC+ H2O2 and NAC+VC+H2O2 groups (p<0.001) at the end of 48 and 72 h incubation times. The total lipid level in H2O2 and H2O2 + antioxidant-supplemented groups was lower than that of the control group. In the fatty acid composition of C. tropicalis, the palmitic acid level was raised in the NAC group (p<0.05), whereas its level was reduced in the other supplemented groups. While the oleic acid level increased in NAC+TOC+H2O2 and NAC+VC+H2O2 (p<0.001) groups, its level slightly decreased in the H2O2 group. The linolenic acid level was low in all the supplemented groups, but linoleic acid and total mono-unsaturated fatty acid (MUFA) levels were high in these groups compared with the control group. Total polyunsaturated fatty acid level (PUFA) decreased in NAC and H2O2 groups (p<0.01), but its level increased in NAC+LA+H2O2 and NAC+TOC+H2O2 groups (respectively, p<0.01, p<0.001). Total saturated fatty acid level decreased significantly in NAC+TOC+H2O2, NAC+H2O2 and NAC+VC+H2O2 (p<0.001) groups (p<0.01), whereas total unsaturated fatty acid level increased in NAC, NAC+H2O2, NAC+LA+H2O2, NAC+TOC+H2O2 and NAC+VC+H2O2 groups. In conclusion, our data showed that the levels of total unsaturated fatty acid, MUFA and PUFA were raised with the combinations of NAC and TOC, LA and VC in C. tropicalis cells subjected to hydrogen peroxide toxicity.     

The Expression of Tyrosinase,Tyrosinase-Related Proteins 1 and 2 (TRP1 and TRP2), the Silver Protein,and a Melanogenic Inhibitor in Human Melanoma Cells of Differing Melanogenic Activities

The expression of various melanogenic proteins, including tyrosinase, the tyrosinase-related proteins 1 (TRP1) and 2 (TRP2/DOPAchrome tautomerase), and the silver protein in human melanocytes was studied in six different human melanoma cell lines and compared to a mouse derived melanoma cell line. Analysis of the expression of tyrosinase, TRP1, TRP2, and the silver protein using flow cytometry revealed that in general there was a positive correlation between melanin formation and the expression of those melanogenic enzymes. Although several of the melanoma cell lines possessed significant activities of TRP2, the levels of DOPAchrome tautomerase in extracts of human cells were relatively low compared to those in murine melanocytes. Melanins derived from melanotic murine JB/MS cells, from melanotic human Ihara cells and HM-IY cells, from sepia melanin, and from C57BL/6 mouse hair were chemically analyzed. JB/MS cells, as well as Ihara cells and HM-TY cells, possessed significant amounts of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derived melanins, this being dependent on the activity of TRP2. Kinetic HPLC assays showed that 5,6-dihydroxyindole (DHI) produced during melanogenesis was metabolized quickly to melanin in pigmented KHm-1/4 cells, whereas DHI was stable in amelanotic human SK-MEL-24 cells. A melanogenic inhibitor that has been purified from SK-MEL-24 cells that suppressed oxidation of DHI in the presence or absence of tyrosinase, but had no effect on DHICA oxidation. The sum of these results suggest that the expression of melanogenic enzymes as well as the activity of a melanogenic inhibitor are critical to the production of melanin synthesis in humans.     

Transplantation of Melanocytes Obtained from the Skin Ameliorates Apomorphine-Induced Abnormal Behavior in Rodent Hemi-Parkinsonian Models

Tyrosinase, which catalyzes both the hydroxylation of tyrosine and consequent oxidation of L-DOPA to form melanin in melanocytes, is also expressed in the brain, and oxidizes L-DOPA and dopamine. Replacement of dopamine synthesis by tyrosinase was reported in tyrosine hydroxylase null mice. To examine the potential benefits of autograft cell transplantation for patients with Parkinson’s disease, tyrosinase-producing cells including melanocytes, were transplanted into the striatum of hemi-parkinsonian model rats or mice lesioned with 6-hydroxydopamine. Marked improvement in apomorphine-induced rotation was noted at day 40 after intrastriatal melanoma cell transplantation. Transplantation of tyrosinase cDNA-transfected hepatoma cells, which constitutively produce L-DOPA, resulted in marked amelioration of the asymmetric apomorphine-induced rotation in hemi-parkinsonian mice and the effect was present up to 2 months. Moreover, parkinsonian mice transplanted with melanocytes from the back skin of black newborn mice, but not from albino mice, showed marked improvement in the apomorphine-induced rotation behavior up to 3 months after the transplantation. Dopamine-positive signals were seen around the surviving transplants in these experiments. Taken together with previous studies showing dopamine synthesis and metabolism by tyrosinase, these results highlight therapeutic potential of intrastriatal autograft cell transplantation of melanocytes in patients with Parkinson’s disease.     

Radicals and melanomas

The synthesis of melanin involves the oxidation of phenolic substrates by the enzyme tyrosinase. In vertebrates tyrosinase is present only in specialized cells (melanocytes), where it catalyses the oxidation of tyrosine and certain diphenolic intermediate products to quinones which polymerize to give rise to melanin. This specialized metabolic pathway provides a possible approach to the specific chemotherapy of malignant tumours of pigment cells (malignant melanoma). Certain analogues of tyrosine are oxidized by tyrosinase generating reactive orthoquinones with cytotoxic potential. One such analogue, 4-hydroxyanisole, has been investigated as a possible specific melanocytotoxic precursor. The parent compound inhibits DNA synthesis but exhibits little general toxicity, while the tyrosinase oxidation products are highly toxic to cells. The mechanism of this toxicity may involve semiquinone radicals. Encouraging initial results have been obtained from clinical pilot studies using intra-arterial infusion of hydroxyanisole in patients with localized recurrences of malignant melanoma.     

Investigation of the regulation of pigmentation in alpha-melanocyte-stimulating hormone responsive and unresponsive cultured B16 melanoma cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to alpha MSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1 degree) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to alpha MSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro alpha MSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1 degree cells. alpha MSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1 degree cells to produce melanin in response to alpha MSH is not due to a lack of alpha MSH receptors or cAMP response to alpha MSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1 degree and F1 cells.     

Investigation of the Regulation of Pigmentation in α-Melanocyte-Stimulating Hormone Responsive and Unresponsive Cultured B16 Melanoma Cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to αMSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1°) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to αMSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro αMSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1° cells. αMSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1° cells to produce melanin in response to αMSH is not due to a lack of αMSH receptors or cAMP response to αMSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1° and F1 cells.     

Coordinated mRNA and Protein Expression of Human LAMP-1 in Induction of Melanogenesis After UV-B Exposure and Co-Transfection of Human Tyrosinase and TRP-1 cDNAs

In order to better understand the cascade of melanogenic events in melanocytes, this report has introduced our two recent approaches for the expression of melanogenesis/or melanosome-associated genes and encoded proteins in melanocytes (melanoma cells) after repeated exposure to UV -B and after cotransfection of two human genes, i.e., tyrosinase and tyrosinase-related protein-1 (TRP-1). Repeated exposure of UV B (2.5–5.0 mJ/cm²) caused not only upregulation of tyrosinase and TRP-1 genes but also coordinated increase in the gene and protein synthesis expression of Lamp-1 (lysosome-associated membrane protein-1). When COS-7 kidney cells and amelanotic melanoma (C32 and SKMEL-24) and melanotic melanoma (G361 and SK-MEL-23) cells were exposed to cotransfection of human tyrosinase and TRP-1 cDNAs, there was also an increased expression of Lamp-1 mRNA and protein along with tyrosinase activation and new melanin synthesis. Importantly, single transfectants of human tyrosinase cDNA revealed marked cellular degeneration, whereas this degeneration was not seen in single transfectants of TRP-1 cDNA or cotransfectants of human tyrosinase and TRP-1 cDNAs, indicating that TRP-1 prevented, along with Lamp-1, programmed death of melanocytes after transfection of tyrosinase gene. The coordinated expression of TRP-1 and Lamp-1 was further confirmed by antisense oligodeoxynucleotide hybridization experiment against Lamp-1 gene, showing the decreased expression of TRP-1 as identified by three different types of anti-TRP-1 monoclonal antibodies. We propose therefore that human tyrosinase and TRP-l, when activated or expressed together, will coordinate to upregulate the mRNA expression and protein synthesis of Lamp-1. The Lamp-1 molecules will, in turn, cover the inner surface of melanosomal membrane, together with TRP-1 molecules, thus protecting the melanosomal membrane from toxic melanin intermediates generated during melanogenesis in the presence of active tyrosinase. In contrast, the expression of other lysosome-related proteins, e.g., β-galactosidase and CD63 is not stimulated in new melanogenesis.     

Effects of triple antioxidant combination (vitamin E, vitamin C and alpha-lipoic acid) with insulin on lipid and cholesterol levels and fatty acid composition of brain tissue in experimental diabetic and non-diabetic rats

The aim of this research was to examine the effects of a triple antioxidant combination (vitamins E (VE) and C (VC) plus alpha-lipoic acid (LA)) on the total lipid and cholesterol levels and the fatty acid composition of brain tissues in experimental diabetic and non-diabetic rats. VE and LA were injected intraperitoneally (50 mg/kg) four times per week and VC was provided as a supplement dissolved in the drinking water (50 mg/kg). In addition, rats in the diabetes 1 and D+VELAVC groups were given daily by subcutaneous insulin injections (8 IU/kg), but no insulin was given to rats in the diabetes 2 group. The results indicate that the brain lipid levels in the D+VELAVC, diabetes 1 and diabetes 2 groups were higher than in the control group (P<0.01). Total lipid was also higher in the non-diabetic rats treated with LA and VC. Total cholesterol was higher in the diabetes 1 and diabetes 2 groups (P<0.05) than in controls. Cholesterol levels were similar in the D+VELAVC and LA groups but lower in the VC, VE and VELAVC groups of non-diabetic rats (P<0.05 and P<0.01). In respect of fatty acid composition, palmitic acid levels were lower in the diabetes 2 and non-diabetic VE groups than the control group (P<0.05), but higher in the non-diabetic LA group (P<0.05). Oleic acid (18:1 n-9) levels were lower in the diabetic and non-diabetic groups than the control group (P<0.01), but higher in the non-diabetic LA group. Arachidonic acid (20:4 n-6) levels were similar in the diabetes 1, D+VELAVC and control groups (P>0.05) but higher in the non-diabetic VE, VC, LA and VEVCLA groups (P<0.05) and lower in the diabetes 2 group (P<0.05). Docosahexaenoic acid (22:6 n-3) was elevated in the diabetes 2 and VEVCLA groups (P<0.01, P<0.05). In conclusion, the current study confirmed that treatment with a triple combination of VE, VC and LA protects the arachidonic acid level in the brains of diabetic and non-diabetic rats.