Hydroalcoholic extract based-ointment from Punica granatum L. peels with enhanced in vivo healing potential on dermal wounds.

The present study reports for the first time, the in vivo wound healing potential of Punica granatum L. peels. A 5% (w/w) methanolic extract based-ointment was formulated and evaluated for its wound healing in guinea pigs. The ointment was applied in vivo on the paravertebral area of twelve excised wounded models once a day for 10 consecutive days. The ointment significantly enhanced the wound contraction and the period of epithelialization as assessed by the mechanical (contraction rate, tensile strength), the biochemical (increasing of collagen, DNA and proteins synthesis) and the histopathological characteristics. Such investigation was encouraged by the efficiency of the methanolic extract as antimicrobial and antioxidant. Indeed, the extract showed antioxidant activity as strong as natural and synthetic compounds (Trolox, BHA, Quercetin). Furthermore, the extract exhibited significant antibacterial and antifungal activity against almost all tested bacteria: Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Klebsiella pneumoniae, Salmonella anatum, Salmonella typhimurium, Streptococcus pneumoniae, and fungi Candida albicans, Candida glabrata, Trichopyton rubrum and Aspergillus niger. The formulated ointment might well find use as skin repair agent without hazard to human health based on these results and on the fact that it has been well established that the extracts of pomegranate used in conditions similar to those applied by traditional medicine, showed no toxic effects.

Optic cup enlargement followed by reduced optic nerve head circulation after optic nerve stimulation.

PURPOSE: To investigate changes in optic nerve head (ONH) circulation, visual evoked potentials (VEPs), and ONH cupping after stimulation of the optic nerve. METHODS: Electrodes were fixed above the optic chiasma in rabbits under general anesthesia. Screw-type electrodes for VEP recording were fixed on the dura. ONH circulation, intraocular pressure (IOP), and blood pressure (BP) were measured after the passage of a current of 0.1 mA for 0.1 second (weak stimulation), 1 mA for 1 second (moderate), 5 mA for 10 seconds (strong), or 25 mA for 10 seconds (severe). Normalized blur (NB), indicative of tissue blood flow and velocity, was measured in the ONH after each stimulation, by using a laser speckle circulation analyzer. Changes in VEP and ocular fundus were also recorded. The ratio of cup area (CA) to disc area (DA) was measured before and 4 weeks after stimulation. After all experiments, the ONH was histologically examined. RESULTS: Weak stimulation increased NB in ONH for 10 minutes, whereas strong or severe stimulation significantly decreased NB for a longer time, in a dose-dependent manner. BP showed no significant change, except with severe stimulation. IOP was not significantly changed. VEP amplitude was reduced 30 minutes after strong stimulation. The CA-to-DA ratio was significantly increased 4 weeks after strong stimulation. In some rabbits, disc hemorrhage occurred, followed by enlargement of disc cupping, with slight gliosis. CONCLUSIONS: Electrical stimulation of the optic nerve changed ONH circulation and VEPs and increased disc cupping. This technique warrants further investigation as an experimental model for normal-tension glaucoma.

Antipruritic and antidermatitic effect of extract and compounds of Impatiens balsamina L. in atopic dermatitis model NC mice.

We examined the effects of a 35% ethanol extract (IB) from the petals of Impatiens balsamina L. and the principal active compounds from IB on chronic and serious pruritus and the development of dermatitis using NC mice, a model of atopic dermatitis. IB at 100 mg/kg significantly inhibited serious scratching behaviour in the NC mouse with established dermatitis when administered i.v. 1 h before, or p.o. 24 h before the measurement. A 10 microg/kg dose of kaempferol 3-rutinoside and 2-hydroxy-1,4-naphthoquinone (lawsone) isolated from IB also inhibited scratching behaviour in the NC mouse with established dermatitis. When 4-week-old NC mice with no symptoms were administered orally 100 mg/kg/day of IB until 13 weeks of age, protection was also noted against scratching behaviour during the development of dermatitis. IB was effective for the prevention and treatment of atopic dermatitis.

Testosterone 5alpha-reductase inhibitor bisnaphthoquinone derivative from Impatiens balsamina.

The 35% EtOH extract of aerial parts of Impatiens balsamina L. has been investigated for activity against testosterone 5alpha-reductase. Activity-guided fractionation led to the identification of the bisnaphthquione derivative named impatienol (1), 3-hydroxy-2-¿[3-hydroxy-1,4-dioxo (2-naphthyl)] ethyl¿ naphthalene-1, 4-dione, which exhibited significant testosterone 5alpha-reductase inhibitory activity. This 5alpha-reductase inhibitory compound has been previously synthesized, but this is the first report of its isolation from a natural source.

Beneficial effects of omega-3 fatty acid treatment on the recovery of cardiac function after cold storage of hyperlipidemic rats.

Cardiac effects of omega-3 polyunsaturated fatty acids (PUFAs) were studied in female Wister rats fed a standard diet (control [C] diet) or a high-cholesterol (HC) diet. Subgroups of rats from these groups were treated with eicosapentaenoic acid-E (EPA) or docosahexaenoic acid-95E (DHA) for 5 weeks. Although plasma total cholesterol (TC) and triglyceride (TG) levels were higher in each group fed the HC diet versus each group fed the C diet, EPA administration with the HC diet (HC + EPA) significantly (P < .05) reduced these levels. An isolated working-heart preparation was used to determine cardiac function. Cardiac output (CO) was lower in rats fed the HC diet and HC + DHA versus any of the groups fed the C diet (P < .05). In addition, left ventricular (LV) maximum differentiation of pressure-time curve (dp/dt) was lower in the rats fed the HC diet versus any of the C diet groups (P < .05). After evaluation of cardiac function in each rat, the heart was stored in a histidine-tryptophan-ketoglutarate solution for 8 hours at 4 degrees C. The heart was then reperfused, and recovery of cardiac function was evaluated. No significant differences were observed for post-preservative cardiac function within the C diet groups. However, within the HC diet groups, HC + EPA significantly (P < .05) improved the recovery of cardiac function. In addition, HC + DHA also significantly (P < .05) improved the recovery of coronary flow (CF) and LV dp/dt. No significant differences were observed for plasma TC and TG concentrations in the C diet groups. EPA administration significantly decreased cardiac levels of palmitic, oleic, and linoleic acids in the HC diet groups. No significant differences were observed for cardiac levels of free fatty acids (FFAs) within the C diet groups. Cardiac EPA and DHA levels were significantly (P < .05) elevated in EPA- or DHA-treated rats compared with the other diet-fed rats. Cardiac EPA levels were also elevated in DHA-treated rats compared with untreated rats (P < .05). These results suggest that EPA attenuates coronary and myocardial preservation injuries through an increase in serum lipids and an accumulation of myocardial FFAs resulting from a HC diet.

Screening method for PAF antagonist substances: on the phenolic compounds from Impatients balsamina L.

A blood pressure monitoring system was developed for studying antagonist effects against platelet activating factor (PAF), a chemical mediator of anaphylactic hypotension. Using this system, we were able to determine the inhibitory effects of a 35% ethanol extract (IB) from the petals of Impatiens balsamina L. against PAF-induced hypotension and the active principal compounds from IB. One mechanism of the antianaphylactic effects of IB was characterized as PAF-antagonist effects.

Antipruritic dinaphthofuran-7,12-dione derivatives from the pericarp of Impatiens balsamina.

Dinaphthofuran-7,12-dione derivatives named balsaminones A (1) and B (2) were isolated from the pericarp of Impatiens balsamina L. together with the known compound 2-methoxy-1,4-naphthoquinone (3). Their structures were elucidated by spectral techniques. These compounds have significant antipruritic activity.

Osteopathy in broiler chicks fed toxic mimosine in Leucaena leucocephala.

Further studies of mimosine toxicity in broiler chicks were done to clarify a possibility of osteopathy. The mineral content and density of femur and the strength, ductility, and toughness for the index of mechanical properties significantly decreased in the 1% mimosine group, compared with those in the control and restricted groups. The stiffness had a decreasing tendency in the 1% mimosine group. Consequently, it was concluded that chicks fed ad libitum a 1% mimosine diet for 12 days developed osteopathy. The bone mineral density and the strength of the restricted group were lower than those of the control group, and those of the 1% mimosine group were still lower than those of the restricted group. Contents of pyridinoline and deoxypyridinoline in the excrement were significantly higher in the restricted group than those in the control group, but the contents in the 1% mimosine group were significantly lowest among the groups. Osteopathy in chicks fed mimosine, therefore, seemed to be done by loss of appetite and changing to a low turnover of bone caused by mimosine.

Influences of dietary omega-3 polyunsaturated fatty acids on the recovery of cardiac and renal functions after preservation in hyperlipidemic rats.

BACKGROUND: The effects of a soybean oil diet and a high-cholesterol oil (HC) diet, and an HC diet with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) supplementation, on basal and postpreservative cardiac function of the hearts and on postpreservative renal function of the kidneys from older rats were examined. METHODS: Groups 1 through 4 of 100-week-old rats were fed either soybean oil, HC, HC with EPA, or HC with DHA, respectively, for 12 weeks. Blood was collected for analysis of plasma fatty acids, and the heart and left kidney were removed from the rat. In experiment 1, the heart was perfused on a Langendorff apparatus. After evaluation of the cardiac function of each rat, the heart was stored in histidine-tryptophan-ketoglutarate solution for 8 hr at 4 degrees C. The heart was reperfused and the recovery of cardiac function was evaluated. The coronary perfusate during reperfusion was collected to measure 6-keto prostaglandin F1alpha and thromboxane B2. Coronary flow (CF) perfused with Krebs-Henseleit bicarbonate (KHB) solution containing 5-hydroxytryptamine (5-HT) and nitroglycerin were evaluated in the Langendorff mode with atrial pacing (330 beats/min). In experiment 2, the excised left kidney was immediately flushed and preserved with University of Wisconsin solution for 8 hr at 4 degrees C. The kidney was then reperfused with KHB solution and renal function was evaluated. RESULTS: The plasma and cardiac EPA levels in group 3 were significantly higher than the levels found in the other groups. The plasma and cardiac ratios of EPA to arachidonic acid were significantly higher in groups 3 and 4 than in groups 1 and 2. There were no significant differences in basal cardiac function among any of the diet-fed rats. The percentage values of the recovery of aortic flow, cardiac output (CO), and left ventricular max dp/dt in group 3 and CO in group 4 were significantly higher than in group 2. In addition, the recovery of CF in group 3 tended to be higher than in group 2 (P=0.07). The percentage values of the recovery of aortic flow, CF, CO, and left ventricular max dp/dt in group 1 were significantly lower than in the other dietary groups. CF reperfused with KHB solution containing 5-HT was significantly higher in group 3 than in groups 1 and 2. CF reperfused with KHB solution containing 5-HT was significantly higher in group 4 than in group 1. CF reperfused with KHB solution containing nitroglycerin in group 3 tended to be higher than in groups 1 and 2 (P=0.07). The thromboxame B2 concentrations in the coronary perfusate during reperfusion in groups 3 and 4 were significantly lower than in groups 1 and 2. Fractional sodium reabsorption in group 3 was significantly higher than in group 2. Inulin clearance in groups 3 and 4 was significantly higher than in group 1. The postpreservative urinary flow in group 3 was significantly higher than in groups 1 and 2. The urinary flow was significantly higher in group 4 than in group 1. CONCLUSIONS: These results suggest that EPA administration may attenuate preservation and reperfusion injury and improve the recovery of cardiac and renal functions in hyperlipidemic and older rats. DHA administration may also show beneficial effects on kidney preservation in hyperlipidemic rats.

Accumulation of 1-o-alkyl-2,3-diacylglycerols in cultured rat keratinocytes.

The present study was undertaken to identify the chemical structure of neutral lipid accumulated in cultured rat keratinocytes and to address their metabolism. Neutral lipid of similar mobility with alkyldiacylglycerol was isolated from cultured rat keratinocytes by thin layer chromatography. The long-chain diols derived from the neutral lipids were identified as 1-alkylglycerol based on the mass spectra of their nicotinylidene derivatives. Thus these neutral lipids were identified as 1-o-alkyl-2,3-diacylglycerols (ADAG). Addition of rat serum elevated the level of ADAG with increasing trend of linoleic acid concentration in this fraction. [14C]Acetate added to the confluent plates was incorporated into alkyl- and acyl-chains of ADAG with incubation in 24 h, and remained un-metabolized up to 72 h. This, however, is not the case for the label incorporation into phospholipid and triacylglycerol. Radioactivities of these two lipid fractions appeared to reach the maximum in 24 h, and thereafter decreased to 72 h with a similar decay curve. Incorporation of [14C]acetate into phospholipid and ADAG was significantly depressed, and that into triacylglycerol and free cholesterol was increased by the supplementation of the medium with rat serum. In concomitance with the accumulation of ADAG, the concentration of ethanolamine-plasmalogen increased in the cultured keratinocytes. The results of the present study first showed the elevated level of ether lipid synthesis in the proliferating primary culture of rat keratinocytes.

Dietary effects of corn oil, oleic acid, perilla oil, and evening [corrected] primrose oil on plasma and hepatic lipid level and atherosclerosis in Japanese quail.

To compare the atherogenecity of different fats and oils, a total of forty, 40-day-old male Japanese quails were fed one of the following diets for three months: basal diet (control), a diet-containing 15% corn oil (CO) and 2% cholesterol (CH), a diet-containing 15% oleic acid (OL) and 2% CH, a diet-containing 15% perilla oil (PE) and 2% CH, a diet-containing 15% evening [corrected] primrose oil (PR) and 2% CH. A higher plasma cholesterol concentration was found in the birds in the CO and OL groups, whereas the PE and PR groups showed a much lower level of plasma cholesterol than the CO and OL groups. In proportion to the increased plasma cholesterol, both CO and OL groups showed narrowing of the lumen of the ascending aorta and its large branches due to marked lipid-rich intimal thickening. Ultrastructural changes in the ascending aorta and its large branches were correlated with the degree of intimal thickening. The major foam cell types were macrophages and fibroblastic cells. The PE and PR groups showed the fewest lipid-rich intimal thickening lesions in their ascending aorta and its large branches. These findings suggest that the alpha-linolenic acid contained in perilla oil is less atherogenic than oleic and linoleic acid, and gamma-linolenic acid contained in evening [corrected] primrose oil has a tendency to decrease the plasma lipid level.

Selective use of L-valine and L-isoleucine for the biosynthesis of branched-chain fatty acids in rat skin.

Iso- and anteiso-fatty acids are detected in more than trace amounts in rat skin surface lipid. The terminal portion of even carbon number iso- and anteiso-fatty acids are synthesized respectively from valine (Val) and isoleucine (Ile) by essentially the same reaction sequences established for straight chain fatty acids. This paper describes the stereospecific biosynthesis of these branched chain fatty acids (BCFAs) and alcohols (BCFALs) in rat skin. Dependence of the concentration of these BCFAs on dietary L-Val and L-Ile was studied. Concentrations of even carbon number iso- and anteiso-fatty acid increased respectively with dietary L-Val and L-Ile. The saturation dose appeared to be 2% for L-Val and 1% for L-Ile. Supplementation of the diet with 2% D-Val, however, did not affect the concentration of even carbon number iso-fatty acid in rat skin surface lipid despite a comparable serum Val level to that of the 2% L-Val group. A similar experiment using 1% DL-Ile found that L-isomer, but not D-isomer, in the circulation was used for the biosynthesis of anteiso-fatty acids. This view was applicable to the incorporation of D-Val and DL-Ile into related BCFALs.

Precursor role of branched-chain amino acids in the biosynthesis of iso and anteiso fatty acids in rat skin.

Monoester fraction of rat skin surface lipid has been shown to contain more than trace amounts of branched-long-chain fatty acids (BCFAs) of the iso and anteiso series. These BCFAs are biosynthesized using either branched-chain amino acids (BCAAs) or branched-chain alpha-keto acids (BCKAs), or using both of them as precursor. This study has been carried out to address which precursor, BCAAs or BCKAs in the circulation, are mainly utilized for biosynthesis of BCFAs. Dietary supplement of [14C]-valine and isoleucine-induced sharp rise of serum concentration of these two amino acids and their respective alpha-keto acids, and elevated the levels of related BCFAs and branched-chain fatty alcohols in the monoester fraction. A larger proportion of label in the total skin surface lipid was found in the monoester fraction in which fatty acid and alcohol accounted for approx. 80% of total radioactivity. Incorporation of intravenously administered [14C]-BCAAs and BCKAs into the monoester fraction revealed that BCAAs were far better as precursors than BCKAs for BCFA biosynthesis in rat skin. Among three BCAAs, leucine differed from valine or isoleucine in that this amino acid was primarily utilized for production of straight-chain fatty acids rather than for production of related BCFA.

Serum-free culture of rat keratinocytes.

Procedures for the serum-free culture of rat keratinocytes have been established. Basal cells prepared from epidermis of newborn rat were stored in liquid nitrogen and used for primary culture. Among the available media, MCDB 153, developed originally for human keratinocyte (HK) culture, was the best for the development of serum-free formulation. To grow rat keratinocytes, bovine serum albumin was arbitrarily substituted for the macromolecule supplements needed for HK culture, i.e. fetal bovine serum protein or bovine pituitary extract. Qualitative and quantitative adjustment of supplements was thereafter made to support rapid cell growth. Satisfactory cell growth was achieved in the optimized medium of MCDB 153 supplemented with growth factors and amino acids: insulin (10 micrograms/ml), hydrocortisone (0.1 microgram/ml), epidermal growth factor (25 ng/ml), calcium chloride (0.2 mM), histidine (0.23 mM), isoleucine (0.05 mM), tryptophane (0.015 mM), threonine (1.25 mM), tyrosine (0.031 mM), alanine (4.08 mM), and albumin (2 mg/ml). This optimized culture system was superior to the original HK culture condition for rapid growth of rat keratinocytes. Under our condition, cells grew as a monolayer, becoming confluent, but without stratification, and were passaged 2 to 3 times without any changes in morphology. The serum-free formulation allows us to control more accurately the concentrations of biomolecules in the medium including lipids and hormones, and therefore will be suitable for the study focusing on lipid metabolism or hormonal regulation of rat keratinocytes.

Serum-free culture of rat keratinocytes.

Procedures for the serum-free culture of rat keratinocytes have been established. Basal cells prepared from epidermis of newborn rat were stored in liquid nitrogen and used for primary culture. Among the available media, MCDB 153, developed originally for human keratinocyte (HK) culture, was the best for the development of serum-free formulation. To grow rat keratinocytes, bovine serum albumin was arbitrarily substituted for the macromolecule supplements needed for HK culture, i.e. fetal bovine serum protein or bovine pituitary extract. Qualitative and quantitative adjustment of supplements was thereafter made to support rapid cell growth. Satisfactory cell growth was achieved in the optimized medium of MCDB 153 supplemented with growth factors and amino acids: insulin (10 µg/ml), hydrocortisone (0.1 µg/ml), epidermal growth factor (25 ng/ml), calcium chloride (0.2 mM), histidine (0.23 mM), isoleucine (0.05 mM), tryptophane (0.015 mM), threonine (1.25 mM), tyrosine (0.031 mM), alanine (4.08 mM), and albumin (2 mg/ml). This optimized culture system was superior to the original HK culture condition for rapid growth of rat keratinocytes. Under our condition, cells grew as a monolayer, becoming confluent, but without stratification, and were passaged 2 to 3 times without any changes in morphology. The serum-free formulation allows us to control more accurately the concentrations of biomolecules in the medium including lipids and hormones, and therefore will be suitable for the study focusing on lipid metabolism or hormonal regulation of rat keratinocytes.

Inhibition of ATPase activity in pea plasma membranes by fungal suppressors from Mycosphaerella pinodes and their peptide moieties.

The effects of two suppressors of the defense reactions of host plants, which had been purified from the pea pathogen Mycosphaerella pinodes, as well as the effects of peptide moieties, on the ATPase activity in pea plasma membranes were examined in vitro. One of the suppressors, Supprescin B, inhibited the ATPase activity in a non-competitive manner, but the other suppressor, Supprescin A, did not. Supprescin A was observed to reduce the inhibitory effect of Supprescin B. A tripeptide, Ser-Ser-Gly, and a hexapeptide, Ser-Ser-Gly-Asp-Glu-Thr, which were the respective peptide moieties of Supprescin A and B, inhibited the ATPase activity in a competitive manner. Supprescin B and fragments of the hexapeptide, such as Asp-Glu-Thr and Gly-Asp-Glu, inhibited not only the ATPase activity but also the acid phosphatase activity of plasma membranes in vitro. These results indicate that the acidic amino-acid residues of the "Asp-Glu" moiety seem to act as inhibitors of the phosphatase activity. Thus, the peptide moiety of Supprescin B consists of at least two functional elements.

Organization of the genes encoding chalcone synthase in Pisum sativum.

To analyze the regulation of defense-related genes by signal molecules produced by phytopathogens, we isolated genes that encode chalcone synthase (CHS) in Pisum sativum. We have obtained seven independent genomic clones that contain at least seven classes of CHS genes, identified by the hybridization analysis to CHS cDNA and by the restriction mapping analysis. Two of the genomic clones (clone 5 and 6) each contain two CHS genes in a tandem repeat. The nucleotide sequence analysis of CHS genomic clone 5 revealed that PsCHS1 and PsCHS2 were corresponding genes of the CHS cDNA clones, pCC6 and pCC2, respectively, as reported earlier. Both genes are interrupted by a single intron of 88 nucleotides with identical sequences, although exonic sequences and 5'-flanking sequences are divergent. Nucleotide sequences of the introns in five other classes of CHS genes showed that three classes had an intron of 87 nt with a striking homology to each other, but that the intron of the other two classes of CHS genes showed heterogeneity both in size and nucleotide sequence. 5'-upstream regions of PsCHS1 and PsCHS2 did not show sequence homology except the 31 bp identical sequence that contains the CCTACC motif resembling the box-1 sequence. Both PsCHS1 and PsCHS2 genes are shown to be induced by fungal elicitor by a primer extension analysis and a transient transformation analysis using pea protoplasts prepared from suspension cultured-cells.

Correlation between serum concentrations of the branched chain amino- and alpha-keto-acid and the distribution of the branched-chain fatty acids on rat skin surface.

This study described the relationship between the concentration of the branched-chain fatty acid (BCFA) in rat skin surface lipid and the serum level of the branched-chain amino acid (BCAA) and branched-chain alpha-keto acid (BCKA). The concentrations of the BCFAs in the monoester fraction of the skin surface lipid, and BCAAs and BCKAs in the serum were analyzed in rats fed varying amounts of protein (0 to 40%) and different types of BCAAs (3%) for 2 weeks. The serum concentrations of BCAA were proportional to the protein level by day 10 of the feeding period. This dose response was not sustained by day 14 at the end of the feeding. Protein level dependence was not so evident in the concentration of BCKA. The concentrations of BCFA, even carbon number iso-acid in particular, increased in linear proportion to protein intake in the skin surface lipid. Supplementation of valine and isoleucine to the diet at a 3% level specifically raised the concentration of the respective BCAA and corresponding BCKA in serum, and related BCFA in the skin surface lipid. Addition of leucine, however, did not affect the related BCFA concentration in spite of elevated concentration of leucine and its alpha-keto acid. A good linear correlation between the average concentration of the substrates in the circulation and the concentrations of the product BCFA on the skin surface was thus obtained for valine, isoleucine and their respective alpha-keto acid. This relationship did not appear to hold up for leucine and its alpha-keto acid.