In recent years, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used in the research on advanced glycation end-products (AGEs), enabling the measurement of several types of AGEs. However, there still remain many unknowns regarding how AGE accumulate in tissues during aging and age-related diseases, and the relationship between each biological tissue and AGE accumulation. We have established a method for the determination of AGEs in various tissues using mice as an animal model and have quantified AGEs in various soft tissues, turning out to be difficult to quantify AGEs in bone tissue, which is a hard tissue, unlike soft tissue processed easily. In this study, we attempted to quantify AGEs in mouse femur using LC-MS/MS. As a result, N^ε-(carboxymethyl)lysine (CML) and N^δ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) were clearly detected in mouse femur, and the amount of CML in femur was significantly higher than that in the crystalline lens. These results confirm that AGE content is high in tissues such as bone, where metabolism and inflammatory reactions occur actively through the action of osteoblasts and osteoclasts. The fact that AGEs in hard tissues as well as soft tissues of mice can now be quantified using LC-MS/MS is expected to make it more effortless to study AGEs in rodents in the future.

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Accumulation of advanced glycation end products (AGEs) in skin tissues due to glycative stress is one of the factors that accelerate skin aging, including decreased elasticity and loss of texture. Suppression of glycative stress is called anti-glycation or glycation care. For anti-glycation of the skin, there are treatments from the inside of the body such as diet and approaches from the outside of the body such as skin care preparations. Furthermore, anti-glycation includes the suppression of postprandial hyperglycemia, suppression of glycation reactions, and decomposition and excretion of AGEs. Various plant materials have been shown to have anti-glycation effects. However, the glycation reaction in the body that leads to the formation of AGEs is a complex multi-pathway reaction, and it is thought that a multi-component approach may be useful. This study was conducted to evaluate the anti-glycation effects of five plant extract complexes (sea buckthorn fruit, Chinese blackberry tea, tea plant leaves, loquat leaves, and rosemary leaves) that can be used in skin care formulations. To evaluate the anti-glycation effects of plant extract complex (PEC) on skin, we examined its inhibitory effects on glycation reaction, AGE cross-link cleavage, inhibitory formation of glycated protein cross-link, glycated protein cross-link cleavage, and antioxidant activity. As results, PEC inhibited the production of fluorescent AGEs in protein glycation models of human serum albumin (HSA), collagen, and keratin. PEC also inhibited the production of pentosidine, N^ε-(carboxymethyl)lysine (CML), 3-deoxyglucosone (3DG), and glyoxal (GO) in a keratin-glucose glycation model. Furthermore, PEC has AGE cross-link-cleavage effect, inhibits protein cross-link formation in a lysozyme-glucose glycation cross-linking model, and degrades proteins dimerized by glycation. PECs may be useful in preventing glycation of skin by suppressing AGE accumulation in skin proteins and preventing functional deterioration caused by the formation of glycated protein cross-links.

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Ribose produces advanced glycation end-products (AGEs) more rapidly than glucose. It has also been reported that N^ε-(carboxymethyl)arginine (CMA) is formed in collagen, which is the most abundant protein in organisms. In this study, AGEs produced by the reaction of ribose with gelatin, a soluble collagen, were evaluated by immunochemical methods using anti-AGEs antibodies, mass spectrometry, and fluorescence intensity measurements. Measurement by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that N^ε-(carboxymethyl)lysine (CML), N^δ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1) and CMA were significantly increased in the ribose-gelatin sample compared to the gelatin alone sample after a 7-day incubation at 37°C. The fluorescence intensity at excitation and emission of 370 nm and 440 nm, respectively, was also significantly increased, whereas the AGE structure detected was unknown. In addition, the pericarp hot water extract ofTrapa bispinosa (TBE) and/or lutein were added to this reaction system to evaluate the inhibitory effect of AGE formation. As a result, inhibition of CML, MG-H1, and CMA formation was observed at ≥ 5 μg/mL of TBE in ELISA and LC-MS/MS. However, no similar inhibitory effect was detected in fluorescence measurements, suggesting that TBE does not inhibit the formation of fluorescent AGEs. Since the inhibition of AGE formation by lutein was not confirmed, it may be a difficult compound to evaluate by in vitro studies. This study showed that ELISA and LC-MS/MS measurement can detect three types of AGEs formation in the ribose-gelatin system and that they are useful for screening compounds, e.g., TBE, that inhibit AGE formation.

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The present study conducted a trial for a skin application of a test cosmetic product with 34 healthy women aged between 35 to 55 years of age (43.4 ± 0.6) who satisfied the classification of 1, 2, and 3 in the standards for the wrinkle grade assessment in accordance with "Guidelines for Evaluation of Cosmetic Functions". In a half face method for 8 weeks, the test cosmetic sample was applied to one lateral periorbital lines of a face (applied side) twice a day and no treatment was applied over the other periorbital lines (non-applied side). The test product was a skin-care cosmetic that contained diverse constituents such as Ceratonia siliqua L. fruit extract, Polygonum tinctorium leaf/stem extract, and ribosomal constituents. In test results after 8 weeks for the primary endpoint of grade assessments of periorbital wrinkles, the grade on the applied side indicated significant improvements through a visual assessment during a medical examination and a visual assessment of observing case photographic images by medical specialists. However, results of the primary endpoint on the non-applied side also indicated significant improvements. There was no significant difference between two sides. Replica analysis examining periorbital wrinkles did not suggest any significant changes between on applied or non-applied side as well as between before and after test product application. Secondary endpoint of skin viscoelasticity (R2) significantly improved on the applied and non-applied sides after 8 weeks. There was no significant difference of changes in quantity. In the process of the trial, no adverse event was reported and the safety of the test product was confirmed. We conducted a further investigation to explore possibilities that diverse functional ingredients contained in this product were absorbed through percutaneous absorption with an assistance of ribosomal constituents, and exerted effects toward the other side of the face. To verify this hypothesis, further examinations are required to confirm blood concentrations of active components after tests.

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Objectives: "Quality of sleep" plays an important role for the maintenance of homeostasis of the body. The deterioration of sleep quality induces diverse lifestyle-related disorders. The present study with an open-label trial verified the effects of a test product, a bed mattress with a "distinctive 4-layer 3-dimensional structure", regarding sleep quality and menopausal syndrome. Methods: Potential research participants, 38 women who had complaints about sleep quality underwent a questionnaire survey using the Japanese version of the Pittsburgh Sleep Quality Index (PSQI-J) and the Simplified menopausal index (SMI). Among them, 12 women whose scores of PSQI-J and SMI were high (age: 48.2 ± 0.7, PSQIG: 7.8 ± 0.6, SMI: 53.5 ± 3.3) were selected. The test product mattresses (Nishikawa Co., Tokyo, Japan) were used for 8 weeks, and alternations in physical information were examined in an open trial. The present study was conducted with the approval of an ethics committee. Results: Data results suggested significant improvements in PSQI-J scores 8 weeks after the commencement of the trial. The global score of PSQI significantly improved from 7.8 ± 0.6 to 5.4 ± 0.6 (p = 0.004). SMI scores improved significantly from 53.5 ± 3.3 to 42.5 ± 4.5 (p = 0.003). Assessments of hormones indicated no significant differences in estradiol, progesterone, luteinizing hormone, and follicle stimulating hormone. Values of dehydroepiandrosterone-sulfate (DHEA-s) changed from (149.9 ± 76.5 μg/dL) → 4 weeks (124.9 ± 63.9 μg/dL) → 8 weeks (146.9 ± 59.2 μg/dL). No adverse event was reported. Conclusions: Through the usage of the test products, it was suggested that "improvements in sleep quality" resulted in the mitigation of menopausal symptoms, though secretions of menopause-related hormones did not improve. It is considered that appropriate bed mat usage is an effective and safe measure of supplementary guidance for women with severe symptoms of menopause.

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