Antioxidative and Anti-Inflammatory Phytochemicals and Related Stable Paramagnetic Species in Different Parts of Dragon Fruit.

In this study, we investigated the antioxidant and anti-inflammatory phytochemicals and paramagnetic species in dragon fruit using high-performance liquid chromatography (HPLC) and electron paramagnetic resonance (EPR). HPLC analysis demonstrated that dragon fruit is enriched with bioactive phytochemicals, with significant variations between each part of the fruit. Anthocyanins namely, cyanidin 3-glucoside, delphinidin 3-glucoside, and pelargonidin 3-glucoside were detected in the dragon fruit peel and fresh red pulp. Epicatechin gallate, epigallocatechin, caffeine, and gallic acid were found in the dragon fruit seed. Additionally, 25-100 mg × L-1 of dragon fruit pulp and peel extracts containing enrichment of cyanidin 3-glucoside were found to inhibit the production of reactive oxygen species (ROS), reactive nitrogen species (RNS), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in cell-based studies without exerted cytotoxicity. EPR primarily detected two paramagnetic species in the red samples. These two different radical species were assigned as stable radicals and Mn2+ (paramagnetic species) based on the g-values and hyperfine components. In addition, the broad EPR line width of the white peel can be correlated to a unique moiety in dragon fruit. Our EPR and HPLC results provide new insight regarding the phytochemicals and related stable intermediates found in various parts of dragon fruit. Thus, we suggest here that there is the potential to use dragon fruit peel, which contains anthocyanins, as a natural active pharmaceutical ingredient.

Maillard Reaction Intermediates and Related Phytochemicals in Black Garlic Determined by EPR and HPLC Analyses.

The Maillard reaction intermediates and related phytochemicals in garlic (Allium sativum L.), which was heated for various lengths of time, using X-band (9 GHz) electron paramagnetic resonance (EPR) and high performance liquid chromatography (HPLC) were investigated. Non-spin-trap and non-destructive EPR detected the total reaction intermediates (radicals). The g-value of the signal was 2.004. The signal with a peak-to-peak linewidth (ΔHpp) was approximately 0.67 milli Tesla (mT). The values of the intermediates are suggestive of organic compounds. The garlic darkened in color with the increasing number of heating days. Melanoidin, responsible for darkening of the garlic, was detected at an absorbance of 400 nm. Analysis of the correlation between the EPR intensity and melanoidin absorbance showed a good correlation coefficient (0.98). In addition, 5-hydroxymethyl furfural (5-HMF) and total phenolic compounds increased with the increasing number of heating days. Moreover, trace amount of Fe3+ was observed in the black garlic by EPR. Non-destructive EPR is a useful method for evaluating not only Maillard reaction intermediates, but also the pigment associated with the reaction processes.

EPR spectroscopic investigation of psoriatic finger nails.

BACKGROUND: Nail lesions are common features of psoriasis and found in almost half of the patients. However, there is no feasible spectroscopic method evaluating changes and severity of nail psoriasis. EPR (electron paramagnetic resonance) might be feasible for evaluating nail conditions in the patients of psoriasis. METHODS: Finger nails of five cases with nail psoriasis, (three females and two males) were examined. Nail samples were subjected to the EPR assay. The small piece of the finger nail (1.5 × 5 mm(2)) was incubated in ~50 µM 5-DSA (5-doxylstearic acid) aqueous solutions for about 60 min at 37°C. After rinsing and wiping off the excess 5-DSA solution, the nail samples were measured by EPR. RESULTS: EPR spectra were analyzed using the intensity ratio (Fast/Slow) of the two motions at the peaks of the lower magnetic field. We observed two distinguishable sites on the basis of the EPR results. In addition, the modern EPR calculation was performed to analyze the spectra obtained. The nail psoriasis-related region is 2~3 times higher than that of the control. CONCLUSION: The present EPR results show that there are two distinguishable sites in the nail. In the case of nail psoriasis, the fragile components are 2~3 times more than those of the control. Thus, the EPR method is thought to be a novel and reliable method of evaluating the nail psoriasis.

Elucidated lipid structures of various human stratum corneum investigated by electron paramagnetic resonance spectroscopy.

BACKGROUND: Electron paramagnetic resonance (EPR) in conjunction with a slow-tumbling simulation is useful for defining stratum corneum (SC) lipid structure. The objectives of this investigation were to evaluate and elucidate the lipid structure of various human SC as a function of the depth. METHODS: The SC from mid-volar forearms of human volunteers and a cadaver was stripped consecutively from one to three times using a glass plate coated with a cyanoacrylate resin. Spin probes were used to monitor SC ordering. EPR spectra were analyzed by an EPR slow-tumbling simulation. Keratin solution from human epidermis was mixed with 5-doxylstearic acid (5-DSA) aqueous solution. RESULTS: EPR analyses of 5-DSA and CHL probes showed immobilized and mobile spectra, respectively. The simulation for 5-DSA spectra showed differences in ordering values of the SC as a function of depth. EPR of keratin/5-DSA showed mobile spectral pattern. In addition, EPR of CHL showed a weak and a broad signal. CONCLUSIONS: The keratin/5-DSA results imply that the most likely location of 5-DSA probe is in SC lipid. CHL probe may not easily permeate the SC. The EPR results along with the simulation analysis provide elucidated SC lipid structure.

Structural Analysis of the Stratum Corneum using EPR and EPR Imaging with Stable Spin Probes.

X-band (9.4 GHz) electron paramagnetic resonance (EPR) and electron paramagnetic resonance imaging (EPRI) were used for elucidating structural aspects of the stratum corneum (SC). We found that psoriasis vulgaris (PV)-SC has a less-ordered structure than that of the control SC, indicating the abnormal architecture of PV-SC. Different spectra were observed for the PV-SC. The three-line spectral pattern suggests that the 5-doxylstearic acid (5-DSA) is mobile or less rigid in the SC. The simulated order parameter (S0) value obtained for 5-DSA in the SC was approximately 0.20. The statistical analysis suggests that the value of the PV-SC is significantly smaller than that of the control (p < 0.01). Thus, we suggest that this EPR assay is of great use for evaluating SC function. In addition, the EPRI of various SC samples provides a useful image concerning SC status. A strong red image was observed for the PV skin. No red lesion region was observed in the control. The EPR images differentiated various sizes and number distribution concerning the disordered states in the SC.

Detection of Antioxidant Phytochemicals Isolated from Camellia japonica Seeds Using HPLC and EPR Imaging.

In this study, we investigated the formation of stable radicals and compounds related to antioxidants in Camellia japonica seeds using high performance liquid chromatography (HPLC) and X-band electron paramagnetic resonance imaging (EPRI). The C. japonica seed coat extracts exhibited antioxidant activity in both in vitro and cell-based studies. The extracts inhibited reactive oxygen and reactive nitrogen species production in cell-based studies. HPLC chromatograms indicated that hydrophilic antioxidant compounds-namely, gallic acid, gallocatechin, epigallocatechin, caffeine, catechin, epicatechin, and epicatechin gallate-were found in the methanolic extract. Lipophilic antioxidant compounds-including α-tocopherol, γ-tocopherol, ß-tocopherol, δ-tocopherol, α-tocotrienol, γ-tocotrienol, and δ-tocotrienol-were found in the hexane extract. EPRI primarily detected paramagnetic species in seed coats. These radical species were stable organic radicals based on the peak-to-peak line width and g-values. The signals from these stable radicals were strong and stable with a g-value of 2.002. Noninvasive EPRI of the radicals present in C. japonica seeds indicated that the stable radicals were essentially located in the seed coats. The EPRI of the cotyledon demonstrated that additional radicals were localized at an apex of the cotyledon. The results indicated that the stable radicals detected by EPRI and the hydrophilic and lipophilic antioxidant compounds analyzed by HPLC were related to antioxidant reactants and products.

Nondestructive Evaluations of Melanin-related Compounds in the Skin Using Electron Paramagnetic Resonance and Permeability Measurements.

Melanin-related compounds in paraffin-embedded tissue specimens of basal cell carcinoma (BCC), seborrheic keratosis (SK), malignant melanoma (MM), and nevus pigmentosus (NP) were nondestructively investigated using permeability measurements (light penetration into pigmented lesion), X-band (9.4 GHz) electron paramagnetic resonance (EPR), and EPR imaging (EPRI). The paramagnetic species in BCC, SK, MM, and NP specimens were analyzed using intensity, linewidth, spectral pattern, and X-band EPRI. The EPR spectra of BCC, SK, and NP showed a single line pattern. The EPR signal intensities of the BCC, SK, and NP samples corresponded to the permeability values that are directly related to pigment color tone, except for MM. The correlation coefficient between EPR and permeability was supported by the high degree of linear relation in the range. We further analyzed MM and speculated that MM contains an additional signal of the pheomelanin radical. In MM and NP samples, two-dimensional (2D) EPRI revealed paramagnetic species distribution and different magnitudes. The paramagnetic (radical) species are directly related to the pigmented lesion site. To conclude, spectroscopic analyses suggest that pheomelanin-related compounds may exist in malignant melanoma.

X-band EPR Spectroscopic Investigation of Seborrheic Keratosis.

Seborrheic keratosis (SK) is a common, noncancerous growth on the skin. However, the paramagnetic species (radicals) in pigmented SK have not been investigated yet. X-band (9 GHz) electron paramagnetic resonance (EPR) and EPR imaging (EPRI) were used to nondestructively investigate paraffin-embedded SK. Paramagnetic species in SK specimens were analyzed using linewidth, spectral pattern, and X-band EPRI. The EPR spectra of the SK showed a single line pattern. The EPR results revealed that the peak-to-peak linewidths (ΔHpp) of paraffin-embedded SK samples were 0.58 ± 0.02 mT. The g-value was 2.004 for those samples using EPR standards. EPR signal intensities of the SK samples reasonably corresponded to those for permeability values that are directly related to pigment color tone. Moreover, the two-dimensional (2D) EPRI of the SK showed the distribution of paramagnetic species in the samples with different magnitudes for the first time. The distribution corresponds to the pigmented region. We established that the paramagnetic species was melanin radicals, based on the EPR results obtained in addition to in vivo oxidation of melanin pigments. The present results suggest that EPR and 2D EPRI techniques can be useful for the radical characterization and evaluation of various types of SK.

Investigation of Pigments in Thai Purple Rice Using Electron Paramagnetic Resonance Imaging and HPLC.

Paramagnetic species (radicals) related pigments in Thai purple rice were investigated by electron paramagnetic resonance (EPR), X-band (9 GHz) EPR imaging (EPRI), and HPLC. The location and distribution of the paramagnetic species in purple and white rice were determined by EPR and EPRI. EPR primarily detected three paramagnetic species in purple rice, which were identified as organic radicals, Mn2+, and Fe3+ based on the g-values and hyperfine components of the EPR signals. Noninvasive two-dimensional (2D) EPRI revealed that these stable radicals are primarily located in the pigmented region of purple rice, while very few radicals were observed in the interior of the rice. HPLC revealed that the major compounds were cyanidin-3-O-glucoside and peonidin-3-O-glucoside. EPR, EPR imaging, and HPLC results indicate that the stable radicals contain the radical state of anthocyanins and are mostly found within the pigmented embryo region of purple rice. They could be either associated with antioxidant activities or could be one of the products of their oxidative decomposition.

Investigation of Paraffin-embedded Basal Cell Carcinoma Using Electron Paramagnetic Resonance.

Basal cell carcinoma (BCC) in paraffin-embedded specimens was investigated by nondestructively electron paramagnetic resonance (EPR) and X-band (9.45 GHz) EPR imaging (EPRI). A histopathological examination of specimens showed the melanin contents and revealed that they were predominantly of the nodular types. A single-line EPR pattern was observed in the BCC specimens, and the spectra of the samples were analyzed using linewidth and spectral pattern parameters. The eumelanin-related radical was observed in paraffin-embedded BCC specimens. The stability of the radical was supported by sepia pigment experiments. The g-value and peak-to-peak linewidths (ΔHpp) were 2.0046 and 0.61 ± 0.02 mT (mean ± SEM), respectively. Strong EPRI signals correspond to areas of strong pigmentation in the samples. Thus, histopathological examination, EPR, and EPRI of BCC specimens showed that the radical distribution reflects on the spatial distribution of pigments in the samples.

Free Radical Scavenging Capability of Various Defatted Sesame Seed Cakes and Hulls Using EPR Compared with In Vitro Testing and HPLC Analysis.

The free radical scavenging activities of black and white sesame seed hulls and the powder of black and white sesame seed cakes were investigated using noninvasive continuous wave electron paramagnetic resonance (EPR) and antioxidant assays. With black sesame seed hulls and the powder of black sesame seed cakes, EPR detected the very strong single-line signal intensities that correspond to the stable organic radicals, while the spectrum of the white sesame seed hulls and the white sesame seed cakes showed no signal. The in vitro antioxidant activities of black and white sesame seed cake extract were evaluated by DPPH free radical scavenging activity, hydrogen peroxide scavenging activity, and ferric reducing antioxidant power (FRAP) assay. The results indicated that the extract from black sesame seed cake possessed a greater DPPH radical inhibitory activity and hydrogen peroxide inhibitory activity than white sesame seed cake extract, with IC50 values of 0.847 ± 0.011 mg/mL and 0.338 ± 0.007 mg/mL, respectively. Black sesame seed cake extract also showed a strong reducing power with a FRAP value of 1.307 ± 0.037 mM Fe (II)/g of extract weight and an EC1 value of 0.683 ± 0.002 mg/mL. The main compounds from the black and white sesame seed cake extracts were analysed using high-performance liquid chromatography (HPLC). The results revealed that the main compounds in black and white sesame seed cake extracts were in a group of water-soluble lignans, mainly sesaminol triglucoside and sesaminol diglucoside. However, sesaminol diglucoside was found in large amounts in the black sesame seed cake extract, while it was found in a very small amount in the white sesame seed cake extract. Therefore, these results demonstrated considerable antioxidant capacity of the sesame seed, especially in the black strain.

Sucrose radical-production cross-section regarding heavy-ion irradiation.

We investigated the sucrose radical-production cross-section induced by heavy-ion irradiation. L-alanine was also used in order to compare radical yield and cross-section. The stable free radicals after irradiation were analyzed by electron paramagnetic resonance (EPR). The radical yield obtained by the irradiated samples had a logarithmic correlation with the LET (linear energy transfer). Quantitative EPR analyses showed that radical productions for sucrose and L-alanine vary both by different particle irradiation and the LET under the same absorbed dose. Furthermore, the cross-sections of radical productions for samples were calculated. Both cross-sections for C ions irradiation under LET 30 keV/microm at 50 Gy dose were approximately 3.0x10(-9) microm(2), taking account of the molecular areas of the samples. The values of the cross-sections imply that multiple ionizing particles involve producing stable radicals.

EPR and HPLC Investigation of Pigments in Thai Purple Rice.

We investigated the pigments in Thai purple rice using electron paramagnetic resonance (EPR), X-band (9 GHz) EPR imaging (EPRI), and HPLC. The location and spatial distribution of the paramagnetic species in purple and white rice were determined by EPR and EPRI. EPR primarily detected three paramagnetic species in purple rice, which were identified as stable radicals, Mn2+, and Fe3+ based on the gvalues and hyperfine components of the EPR signals. Subsequent noninvasive two-dimensional (2D) EPRI revealed that these stable radicals are primarily located in the pigmented region of purple rice, while very few radicals were observed in the interior of the rice. HPLC revealed that the major compounds were cyanidin-3-O-glucoside and peonidin-3-O-glucoside. Scavenging activities, EPR, and EPR imaging results indicate that the stable radicals contain the radical state of anthocyanins and are mostly found within the pigmented embryo region of purple rice. They could be either associated with scavenging activities or could be one of the products of their oxidative decomposition.

Melanin Radicals in Paraffin-embedded Melanoma Investigated Using Surface-type Dielectric Resonator for X-band EPR.

We investigated melanin radicals in paraffin-embedded malignant melanoma (MM) using a surface-type dielectric resonator for X-band electron paramagnetic resonance (EPR) and analyzed the radical species. The surface-type resonator's performance was examined using 5 - 10 µL of 0.1 mM 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) aqueous solution in a 1.0-mm (i.d.) glass capillary as well as 1,1-diphenyl-2-picrylhydrazyl (DPPH) powder. The surface-type detection has approximately two times poorer S/N ratio than commercial insertion-type detection. A sample of the paraffin-embedded MM specimen was used for the radical detection. We obtained an EPR spectrum of melanin radicals in the paraffin-embedded melanoma sample (size ∼3 × 4 × 3 mm). A single line (∼0.64 mT peak-to-peak line-width) with a small shoulder was observed and was identified as a pheomelanin-related radical. The pheomelanin radical can be directly related to the MM. Thus, the present results were a good indication for noninvasive measurement, as well as for detailed analyses of melanin radicals in human MM.

Spin-probe investigations of head group behavior in aqueous dispersions of a nonionic amphiphilic compound.

Head group behavior of nonionic amphiphilic compound, (poly(oxyethylene) hydrogenated castor oil, HCO), in aqueous dispersions were investigated by EPR (electron paramagnetic resonance) in conjunction with a modern slow-tumbling simulation. The aliphatic spin probes, 5-doxylstearic acid (5-DSA) and 3beta-doxyl-5alpha-cholestane (CHL), were used to obtain fluidity of the surface region of the membrane. The order parameter (S (0)) using the simulation for 5-DSA and CHL in the region were approximately 0.4 and 0.2, respectively. The ordering results suggest that the head group region of the membrane is somewhat fluid. The rotational diffusion coefficients (R ( perpendicular) approximately 1/(6tau(R))) for the probes were 3.4 x 10(7) and 7.1 x 10(7) s(-1), respectively. Activation energies, calculated using the temperature dependence of diffusion coefficients, were 18 and 17 kJ/mol for the probes. The EPR results imply that the CHL probe in the HCO membrane has quite different behavior in comparison with that of PC (phosphatidylcholine) from egg. Thus, the present EPR analyses have provided quantitative insight into the surface region of the amphiphilic membrane.

Investigating Pigment Radicals in Black Rice Using HPLC and Multi-EPR.

We investigated the location and distribution of paramagnetic species in black and white rice using electron paramagnetic resonance (EPR), X-band (9 GHz) EPR imaging (EPRI), and HPLC. EPR primarily detected two paramagnetic species in black rice, which were identified as a stable radical and Mn2+ species, based on the g values and hyperfine components of the EPR signals. The signal from the stable radical appeared at g ≈ 2.00 and was relatively strong and stable. Subsequent noninvasive two-dimensional (2D) EPRI revealed that this stable radical was primarily located in the pigmented region of black rice, while very few radicals were observed in the rice interior. Pigments extracted from black rice were analyzed using HPLC; the major compound was found to be cyanidin-3-glucoside. EPR and HPLC results indicate that the stable radical was only found within the pigmented region of the rice, and that it could either be cyanidin-3-glucoside, or one of its oxidative decomposition products.

Investigating the Distribution of Stable Paramagnetic Species in an Apple Seed Using X-Band EPR and EPR Imaging.

This study investigated the location and distribution of paramagnetic species in apple seeds using electron paramagnetic resonance (EPR) and X-band (9 GHz) EPR imaging (EPRI). EPR primarily detected two paramagnetic species per measured seed. These two different radical species were assigned as stable radicals and Mn2+ species based on the g values and hyperfine components. The signal from the stable radical was noted at g ≈ 2.00 and was strong and relatively stable. The subsequent noninvasive EPRI of the radical present in each seed revealed that the stable radicals were located primarily in the seed coat, with very few radicals observed in the cotyledon of the seed. These results indicate that the stable radical species were only found within the seed coat, and few radical species were found in other seed parts.

EPR imaging and HPLC characterization of the pigment-based organic free radical in black soybean seeds.

We investigated the location and distribution of paramagnetic species in dry black, brown, and yellow (normal) soybean seeds using electron paramagnetic resonance (EPR), X-band (9 GHz) EPR imaging (EPRI), and HPLC. EPR primarily detected two paramagnetic species in black soybean. These two different radical species were assigned as stable organic radical and Mn2+ species based on the g values and hyperfine structures. The signal from the stable radical was noted at g ≈ 2.00 and was relatively strong and stable. Subsequent noninvasive two-dimensional (2D) EPRI of the radical present in black soybean revealed that the stable radical was primarily located in the pigmented region of the soybean coat, with very few radicals observed in the soybean cotyledon (interior). Pigments extracted from black soybean were analyzed using HPLC. The major compound was found to be cyanidin-3-glucoside. Multi-EPR and HPLC results indicate that the stable radical was only found within the pigmented region of the soybean coat, and it could be cyanidin-3-glucoside or an oxidative decomposition product.

X-band Electron Paramagnetic Resonance Investigation of Stable Organic Radicals Present under Cold Stratification in 'Fuji' Apple Seeds.

We investigated stable organic radicals formed in response to cold stratification in 'Fuji' apple seeds using X-band (9 GHz) electron paramagnetic resonance (EPR) technique. This technique primarily detected two paramagnetic species in each seed. These two different radical species were assigned as a stable organic radical and Mn2+ species based on the g values and hyperfine components. Signal from the stable radicals was noted at a g value of about 2.00 and was strong and relatively stable. Significant radical intensity changes were observed in apple seeds on refrigeration along with water supplementation. The strongest radical intensity and a very weak Mn2+ signal were also observed for the seeds kept in moisture-containing sand in a refrigerator. Noninvasive EPR of the radicals present in each seed revealed that the stable radicals were located primarily in the seed coat. These results indicate that the significant radical intensity changes in apple seeds under refrigeration for at least 90 days followed by water supplementation for one week, can be related to cold stratification of the seeds.

Characterization of Melanin Radicals in Paraffin-embedded Malignant Melanoma and Nevus Pigmentosus Using X-band EPR and EPR Imaging.

Continuous wave electron paramagnetic resonance (CW EPR) and X-band (9 GHz) EPR imaging (EPRI) were used to nondestructively investigate the possible differentiation between malignant melanoma (MM) and nevus pigmentosus (NP) melanin radicals in paraffin-embedded specimens. The EPR spectra of both samples were analyzed using linewidth, spectral pattern, and X-band EPRI. The CW-EPR spectra of the MM showed an additional signal overlap. Eumelanin- and pheomelanin-related radicals were observed in the MM specimens. The EPR results revealed that the peak-to-peak linewidths (ΔHpp) of paraffin-embedded MM and NP samples were 0.65 ± 0.01 and 0.69 ± 0.01 mT, respectively. The g-value was 2.005 for both samples. Moreover, the two-dimensional (2D) EPRI of the MM showed different signal intensities at the different tumor stages, unlike the NP, which displayed fewer variations in signal intensity. Thus, the present results suggest that EPR and 2D EPRI can be useful for characterization of the two melanin radicals in the MM and for determination of their size and concentration.