Eucommia ulmoides extracts prevent the formation of advanced glycation end products

Hosoo S., Koyama M., Kato M., Hirata T., Yamaguchi Y., Yamasaki H., Wada A., Wada K., Nishibe S., Nakamura K.

Singh R., Devi S., Gollen R.

During the past few decades, there have been numerous studies related to free radical chemistry. Free radicals including reactive oxygen species (ROS) and reactive nitrogen species are generated by the human body by various endogenous systems, exposure to different physiochemical conditions, or pathological states, and have been implicated in the pathogenesis of many diseases. These free radicals are also the common by-products of many oxidative biochemical reactions in cells. When free radicals overwhelm the body's ability to regulate them, a condition known as oxidative stress ensues. They adversely alter lipids, proteins, and DNA, which trigger a number of human diseases. In a number of pathophysiological conditions, the delicate equilibrium between free radical production and antioxidant capability is distorted, leading to oxidative stress and increased tissue injury. ROS which are mainly produced by vascular cells are implicated as possible underlying pathogenic mechanisms in a progression of cardiovascular diseases including ischemic heart disease, atherosclerosis, cardiac arrhythmia, hypertension, and diabetes. This review summarizes the key roles played by free radicals in the pathogenesis of atherosclerosis, diabetes, and dyslipidaemia. Although not comprehensive, this review also provides a brief perspective on some of the current research being conducted in this area for a better understanding of the role free radicals play in the pathogenesis of atherosclerosis, diabetes, and dyslipidaemia.

Nagai R., Shirakawa J., Fujiwara Y., Ohno R., Moroishi N., Sakata N., Nagai M.

Approximately 100 years have passed since the Maillard reaction was first reported in the field of food chemistry as a condensation reaction between reducing sugars and amino acids. This reaction is thought to progress slowly primarily from glucose with proteins in vivo. An early-stage product, called the "Amadori product", is converted into advanced glycation end products. Those accumulate in the body in accordance with age, with such accumulation being enhanced by lifestyle-related diseases that result in the denaturation of proteins. Recent studies have demonstrated that intermediate carbonyls are generated by several pathways, and rapidly generate many glycation products. However, accurate quantification of glycation products in vivo is difficult due to instability and differences in physicochemical properties. In this connection, little is known about the relationship between the structure of glycation products and pathology. Furthermore, the interaction between proteins modified by glycation and receptors for advanced glycation end products is also known to induce the production of several inflammatory cytokines. Therefore, those inhibitors have been developed over the world to prevent lifestyle-related diseases. In this review, we describe the process of protein denaturation induced by glycation and discuss the possibility of using the process as a marker of age-related diseases.

Hirata T., Ikeda T., Fujikawa T., Nishibe S.

Chemistry Eucommia ulmoides Oliver is a single species of the genus Eucommia which is the only genus in the family Eucommiaceae. The bark has been traditionally used in analgesics, tonics, and hypotensives in China. Moreover, the Chinese medical book “Bencao gangmu” describes fresh Eucommia leaf (EL) as being “consumed as a vegetable.” The product “Tochu-cha,” which is available in the Japanese markets and used in beverages, is roasted EL containing the iridoid glycosides: geniposidic acid and asperuloside, and a caffeic acid derivative: chlorogenic acid as its major ingredients. Eight iridoids, together with 14 known compounds were isolated from Eucommia leaf extract (ELE). The detailed investigation of the constituents of Eucommia green leaf powder (EGLP) led to the isolation of six iridoids, five flavonoids, and chlorogenic acid. Three additional new iridoids were obtained, two of which (eucomoside B and eucomoside C) were isolated by the condensation of geniposidic acid and amino acids (phenylalanine and tryptophan, respectively), whereas the third (eucomoside A) was determined to be an acetal compound with a linkage from C-3 of the iridoid to C-2 of the glucose unit, each being the first such natural compound described. Bioactivities Spontaneously hypertensive rats fed ELE exhibited a dose-dependent antihypertensive effect. Moreover, follow-up studies have confirmed that ELE decreases systolic blood pressure in humans after long-term intake. These antihypertensive effects are presumed to be due to the activity of a so-called EL glycoside, which contains geniposidic acid as a major component. The extract from ELs containing EL glycoside is used as the food supplement in the food for specified health uses (FOSHU). Metabolic syndrome (MS)-like rats fed ELE or EGLP exhibited dose-dependent antiobesity and anti-MS effects. Both forms of ELs minimized increases in body weight and visceral fat in a dose-dependent manner. Concomitantly, an increase in plasma adiponectin levels and a suppression of plasma resistin and tumor necrosis factor α levels were demonstrated. The chronic administration of asperuloside isolated from ELs suppressed increases in body weight, white adipose tissue (WAT) weight, plasma triglyceride levels, and free fatty acid levels in a mouse model. Further, the chronic administration of asperuloside enhanced the basal metabolic rate in rats fed a high-fat diet (HFD) and significantly decreased respiratory quotientsmore than that in the HFD control group, suggesting an acceleration of lipid metabolism. Real-time PCR studies showed that ELE and asperuloside enhanced metabolic function in several organs, including the diminution of adenosine 5′-triphosphate production in WAT, the acceleration of β-oxidation in the liver, and an increase in the use of ketone bodies/glucose in skeletal muscle, all of which may exert antiobesity effects under HFD conditions due to novel mechanisms. To our knowledge, this is the first study showing the antiobesity effects of iridoid asperuloside. These findings are expected to result in new health applications of EL and the development of EL as a FOSHU.

Zhang Q., Su Y., Zhang J.

Fujikawa T., Hirata T., Hosoo S., Nakajima K., Wada A., Yurugi Y., Soya H., Matsui T., Yamaguchi A., Ogata M., Nishibe S.

AbstractEucommialeaves (Eucommia ulmoidesOliver) contain chlorogenic acid (a caffeic acid derivative) and geniposidic acid and asperuloside (ASP), iridoid glucosides used in beverages. We used a metabolic syndrome rat model, produced by feeding a 35 % high-fat diet (HFD), to examine potential anti-obesity and anti-metabolic syndrome effects and mechanisms of chronic administration of ASP. These effects were compared withEucommialeaf extract (ELE), the positive control, which exhibits anti-obesity effects. A total of six rats were studied for 3 months in five groups. ASP suppressed body weight, visceral fat weight, food intake and circulating levels of glucose, insulin and lipids, and increased the plasma adiponectin level in rats on a HFD. These effects are similar to those of ELE, except for the influence on the plasma glucose level. RT–PCR studies showed that ASP (like ELE with known anti-obesity effects) diminished isocitrate dehydrogenase 3α, NADH dehydrogenase flavoprotein 1 (Comp I) mRNA and fatty acid synthase levels (white adipose tissue), increased carnitine palmitoyltransferase 1α and acyl-CoA dehydrogenase, very-long-chain mRNA levels (liver), and increased Glut4, citrate synthase, isocitrate dehydrogenase 3α, succinyl CoA synthase, peroxisomal 3-ketoacyl-CoA thiolase, dihydrolipoamide succinyl transferase and succinate dehydrogenase mRNA levels (skeletal muscle) under HFD conditions. Interestingly, ASP administration resulted in significantly increased mRNA levels of uncoupling protein 1 (UCP1) in the brown adipose tissue of HFD-fed rats; ELE did not affect the expression of UCP1. The increased expression of UCP1 may be negated by many ingredients other than ASP in the ELE. These findings suggest that chronic administration of ASP stimulates anti-obesity and anti-metabolic syndrome activity in HFD-fed rats across several organs, similar to ELE administration; thus, ASP may be an important ingredient of ELE.

Shimasaki S., Kubota M., Yoshitomi M., Takagi K., Suda K., Mera K., Fujiwara Y., Nagai R.

He K., Li X., Chen X., Ye X., Huang J., Jin Y., Li P., Deng Y., Jin Q., Shi Q., Shu H.

Traditional Chinese medicine (TCM) has been used for treating complex chronic diseases owing to their fewer side-effects, better patient tolerance and relatively less cost. The present work was carried out to study the anti-diabetic efficacy and mechanisms of 34 TCMs.Streptozotocin (STZ)-diabetic mice were orally administrated with corresponding herbal solution once a day for 4 weeks. At the end of experiment, the level of plasma glucose, malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and the serum aldose reductase (AR) were determined, the effects of TCM extract on α-glucosidase and angiotensin-converting enzyme (ACE) in vitro were also evaluated.13 out of the 34 herbs showed a statistically significant plasma glucose lowering action compared with the diabetic control group. Biochemical analysis revealed that Atractylodes macrocephala, Codonopsis pilosula, Dioscorea opposite, Flos lonicerae and Pueraria lobata may retard the progression of diabetes via reduce the blood glucose level and prevent the increase of AR activity. Other tested herbs, such as Ramulus cinnamomi, Cinnamomum cassia, and Eucommia ulmoides, showed the antidiabetic ability by either prevent the decrease in SOD activity or suppress the increase of MDA. Zymologic assay reveals that Pueraria lobata and Anemarrhena asphodeloides showed the highest inhibition against α-glucosidase and ACE respectively. Interestingly, the post-treatment glucose levels and AR activity were positively correlated with kidney/body weight of 34 herbs treated diabetic mice (p = 0.02, 0.04 respectively).Several potential antidiabetic herbs derived from Chinese traditional pharmacopeia such as Dioscorea opposite, Pueraria lobata, Codonopsis pilosula and Ramulus cinnamomi, have been found to exert a beneficial action on diabetes and diabetic complications via multi-mechanisms.

METORI K., OHASHI S., TAKAHASHI S., TAMURA T.

We evaluated the effects of Du-Zhong leaf extract on the serum and liver lipids in rats fed a high-fat diet supplemented with animal fat, cholesterol and cholate. The Du-Zhong leaf extract suppressed significantly the high-fat diet-induced increases in total serum cholesterol, serum triacylglycerol and hepatic triacylglycerol but not the total hepatic cholesterol. The Du-Zhong leaf extract also suppressed the high-fat diet induced increases in very-low density lipoprotein and low density lipoprotein without affecting high density lipoprotein cholesterol. These results suggest that Du-Zhong leaf extract may be beneficial for the regulation of hyperlipidemia.

Kobayashi Y., Hiroi T., Araki M., Hirokawa T., Miyazawa M., Aoki N., Kojima T., Ohsawa T.

Tea made from Eucommia ulmoides leaves is widely consumed as a health food, since recent studies have revealed various pharmacological effects of the tea, e.g. a hypotriglyceridaemic effect. This study was conducted to elucidate the mechanisms underlying the plasma triglyceride-lowering effect of E. ulmoides leaves.Rats were divided into four groups: a normal group, a group fed a high-fat/high-fructose diet (untreated group) and two groups fed a high-fat/high-fructose diet and E. ulmoides tea (4 or 20 g L(-1) extract, treated groups). Plasma triglyceride concentrations were reduced in treated groups in a dose-dependent manner compared with the untreated group. DNA microarray analysis revealed that genes involved in hepatic α-, β- and ω-oxidation, mainly related to the peroxisome proliferator-activated receptor α and δ signalling pathway, were up-regulated in the treated group. High-performance liquid chromatography analysis showed that E. ulmoides leaves contain three phytochemicals that make up 60 mg g(-1) of the material and are likely to be the active components.This study indicates that the promotion of fatty acid oxidation, probably by the action of phytochemicals, participates in the ameliorative effect of E. ulmoides leaves on hypertriglyceridaemia. These findings provide the scientific evidence for the functionality of E. ulmoides.

Hirata T., Kobayashi T., Wada A., Ueda T., Fujikawa T., Miyashita H., Ikeda T., Tsukamoto S., Nohara T.

The anti-hypertensive effect of Eucommia leaves has been confirmed clinically, and the study of their anti-obesity properties has advanced. However, the compounds involved in their anti-obesity effect have not been fully elucidated. In this Letter, we examined the anti-obesity effect of Eucommia green leaf extract (EGLE) divided into five fractions with high porous polystyrene gel and of the compounds isolated, geniposidic acid, asperuloside and chlorogenic acid, respectively. A metabolic syndrome-like clinical model in mice was generated by feeding a 40% high-fat diet to examine the anti-obesity effects of chronic administration of test substance. After 4 weeks, body weight, white adipose tissue weight, plasma triglyceride levels and total cholesterol levels in the model mice were significantly inhibited by the 30% MeOH fraction (containing much higher levels of asperuloside than the other fractions), and these effects were similar to those of EGLE. Chronic administration of isolated asperuloside in Eucommia leaves suppressed increases in model mouse body weight, white adipose tissue weight, plasma triglyceride levels and free fatty acids levels. These results suggest that asperuloside in Eucommia leaves has important anti-obesity effects.

Fujikawa T., Hirata T., Wada A., Kawamura N., Yamaguchi Y., Fujimura K., Ueda T., Yurugi Y., Soya H., Nishibe S.

Eucommia bark (Eucommia ulmoidesOliver) has been used as an herbal medicine, and more recently, the plant's leaves have been widely used to prepare tea which may have anti-obesity properties. We used a metabolic syndrome-like rat model, produced by feeding a 35 % high-fat diet (HFD), to examine potential anti-obesity and anti-metabolic syndrome effects and mechanisms of chronic administration of Eucommia leaf as an extract or green leaf powder. Eighty rats were studied for 3 months in ten groups. Both forms of Eucommia leaves minimised increases in body weight and visceral fat in a dose-dependent fashion. Increases in plasma levels of TAG and NEFA, and insulin resistance secondary to HFD were lessened by both forms of Eucommia leaf. Concomitantly, an increase in plasma adiponectin levels and suppression of plasma resistin and TNF-α levels were confirmed. Real-time PCR studies showed that both forms of Eucommia leaf enhanced metabolic function across several organs, including diminishing ATP production (white adipose tissue), accelerating β-oxidation (liver) and increasing the use of ketone bodies/glucose (skeletal muscle), all of which may exert anti-obesity effects under HFD conditions. These findings suggest that chronic administration of either form of Eucommia leaves stimulates the metabolic function in rats across several organs. The anti-obesity and anti-metabolic syndrome activity in this rat model may be maintained through secretion and regulation of adipocytokines that depend on the accumulation of visceral fat to improve insulin resistance or hyperlipaemia.

Horii Y., Tanida M., Shen J., Hirata T., Kawamura N., Wada A., Nagai K.

Eucommia ulmoides Oliver leaf extracts (ELE) have been shown to exert a hypolipidemic effect in hamsters. Therefore, it was hypothesized that ELE might affect lipid metabolism via changes in autonomic nerve activities and causes changes in thermogenesis and body weight. We examined this hypothesis, and found that intraduodenal (ID) injection of ELE elevated epididymal white adipose tissue sympathetic nerve activity (WAT-SNA) and interscapular brown adipose tissue sympathetic nerve activity (BAT-SNA) in urethane-anesthetized rats and elevated the plasma concentration of free fatty acids (FFA) (a marker of lipolysis) and body temperature (BT) (a marker of thermogenesis) in conscious rats. Furthermore, it was observed that ID administration of ELE decreased gastric vagal nerve activity (GVNA) in urethane-anesthetized rats, and that ELE given as food reduced food intake, body and abdominal adipose tissue weights and decreased plasma triglyceride level. These findings suggest that ELE stimulates lipolysis and thermogenesis through elevations in WAT-SNA and BAT-SNA, respectively, suppresses appetite by inhibiting the activities of the parasympathetic nerves innervating the gastrointestinal tract, including GVNA, and decreases the amount of abdominal fat and body weight via these changes.

Balakumar P., Rohilla A., Krishan P., Solairaj P., Thangathirupathi A.

Thiamine, known as vitamin B(1), plays an essential role in energy metabolism. Benfotiamine (S-benzoylthiamine O-monophoshate) is a synthetic S-acyl derivative of thiamine. Once absorbed, benfotiamine is dephosphorylated by ecto-alkaline phosphatase to lipid-soluble S-benzoylthiamine. Transketolase is an enzyme that directs the precursors of advanced glycation end products (AGEs) to pentose phosphate pathway. Benfotiamine administration increases the levels of intracellular thiamine diphosphate, a cofactor necessary for the activation transketolase, resulting in the reduction of tissue level of AGEs. The elevated level of AGEs has been implicated in the induction and progression of diabetes-associated complications. Chronic hyperglycemia accelerates the reaction between glucose and proteins leading to the formation of AGEs, which form irreversible cross-links with many macromolecules such as collagen. In diabetes, AGEs accumulate in tissues at an accelerated rate. Experimental studies have elucidated that binding of AGEs to their specific receptors (RAGE) activates mainly monocytes and endothelial cells and consequently induces various inflammatory events. Moreover, AGEs exaggerate the status of oxidative stress in diabetes that may additionally contribute to functional changes in vascular tone control observed in diabetes. The anti-AGE property of benfotiamine certainly makes it effective for the treatment of diabetic neuropathy, nephropathy and retinopathy. Interestingly, few recent studies demonstrated additional non-AGE-dependent pharmacological actions of benfotiamine. The present review critically analyzed the multifaceted therapeutic potential of benfotiamine.

Jin X., Amitani K., Zamami Y., Takatori S., Hobara N., Kawamura N., Hirata T., Wada A., Kitamura Y., Kawasaki H.

Eucommia ulmoides Oliv. leaf is a traditional Chinese medicine that exhibits an anti-diabetic action. This study was designed to investigate whether long-term administration of Eucommia ulmoides Oliv. leaves extract (ELE) ameliorates pre-diabetic state of insulin resistance and abnormal perivascular innervation in the hyperinsulinemic state.ELE at doses of 500 and 1000mg/kg was administered orally once daily for 4 weeks in fructose-drinking rats (FDRs). Plasma levels of insulin, blood glucose levels, and perivascular innervation were assessed using biochemical and immunohistochemical methods.FDR showed significant increase in plasma levels of insulin, an index for insulin resistance (Homeostasis Model Assessment ratio-HOMA-IR) and systolic blood pressure (SBP), but not blood glucose levels, as compared with control rats. Immunohistochemical study showed significantly greater density of tyrosine hydroxylase (TH)-like immunoreactivity (LI)-containing nerves and significantly lower density of calcitonin gene-related peptide (CGRP)-LI-containing nerves in mesenteric arteries of FDR than those in control. A 4-week treatment with ELE (500 and 1000mg/kg, p.o.) significantly decreased plasma levels of insulin and HOMA-IR without affecting blood glucose levels and significantly lowered SBP in FDR. ELE treatment in FDR resulted in significant increase in CGRP-LI never fiber density and significant decrease in TH-LI never fiber density in mesenteric arteries of FDR.These results suggest that long-term ELE treatment effectively prevents insulin resistance development and ameliorates abnormal perivascular innervation in FDR.

Wang H., Li Y., Zhang L., Lu M., Li C., Li Y.

Sugawa H., Ikeda T., Tominaga Y., Katsuta N., Nagai R.

Ribose undergoes auto-oxidation and oxidative cleavage between C-2 and C-3 to generate GO and enhance CML accumulation.

Nakashima K., Miyashita H., Yoshimitsu H., Fujiwara Y., Nagai R., Ikeda T.

Introduction: As inhibitors of advanced glycation end products (AGEs), such as pyridoxamine, significantly inhibit the development of retinopathy and neuropathy in rats with streptozotocin-induced diabetes, treatment with AGE inhibitors is believed to be a potential strategy for the prevention of aging, age-related diseases, and lifestyle-related diseases, including diabetic complications. In the present study, the MeOH extract of Epimedii Herba (EH; aerial parts of Epimedium spp.) was found to inhibit the formation of Nε-(carboxymethyl)lysine (CML) and Nω-(carboxymethyl) arginine (CMA) during the incubation of collagen-derived gelatin with ribose.Materials and methods: EH was purchased from Uchida Wakan-yaku Co., and a MeOH extract was prepared. Several steps of column chromatography purified the extract. Each fraction was tested for inhibitory activity by ELISA using monoclonal antibodies for CML and CMA.Results: After activity-guided fractionation and purification by column chromatography, three new prenylflavonoids [named Koreanoside L (1), Koreanoside E1 (2), and Koreanoside E2 (3)] and 40 known compounds (4–43) were isolated from EH, and their inhibitory effects against CML and CMA formation were tested. Among these, epimedokoreanin B (8), epimedonin E (21), epicornunin B (22), and epicornunin F (24) inhibited the formation of both CML and CMA, with epimedokoreanin B (8) having the most potent inhibitory effect among the isolated compounds. To obtain the structure–activity relationships of 8, the phenolic hydroxy groups of 8 were methylated by trimethylsilyl-diazomethane to afford the partially and completely methylated compounds of 8. Prenyl derivatives of propolis (artepillin C, baccharin, and drupanin) were used in the assay.Discussion: As only 8 showed significant activity among these compounds, the catechol group of the B ring and the two prenyl groups attached to the flavanone skeleton were essential for activity. These data suggest that 8 could prevent the clinical complications of diabetes and age-related diseases by inhibiting AGEs.

Nakashima Y., Yamamoto N., Tsukioka R., Sugawa H., Ohshima R., Aoki K., Hibi T., Onuki K., Fukuchi Y., Yasuda S., Nagai R., Kinoshita H.

In this study, we aimed to identify starter strains of lactic acid bacteria (LAB) to produce soymilk yogurt that can decrease the risk of diabetes mellitus (DM) and its complications. The inhibition assays of the dipeptidyl peptidase IV (DPP- IV) and advanced glycation end products (AGEs) formation were conducted using 70% ethanol (EtOH) extraction of freeze-dried soymilk yogurt. Soymilk yogurt produced using Weissella confusa TOKAI 2 showed the highest inhibition rate in the α-amylase inhibition assay, while soymilk yogurt produced using Lactiplantibacillus plantarum TOKAI 17 showed the highest inhibition rates in the α-glucosidase and DPP-Ⅳ inhibition assays, respectively. Soymilk yogurt produced using Streptococcus macedonicus TOKAI 4 showed the highest inhibitory activities in N ω -(carboxymethyl) arginine (CMA) and N ε -(carboxymethyl) lysine (CML) formation, respectively. Furthermore, soymilk yogurt prepared using Lb. plantarum TOKAI 17 and Lactiplantibacillus pentosus TOKAI 35 exhibited higher proportions of aglycone isoflavones than soymilk and showed a higher fluorescent AGEs inhibitory activity than that of soymilk ( p < 0.05). Interestingly, the inhibition assay assessing fluorescent AGEs formation using isoflavones indicated that aglycone-type isoflavones, such as genistein and daidzein, had higher inhibitory activities than that of glycosides ( p < 0.05). These results indicate that soymilk yogurt produced using Lb. plantarum TOKAI 17 and Lb. pentosus TOKAI 35 had inhibitory effects on α-glucosidase, DPP-Ⅳ, and AGEs formation in vitro , and that aglycone isoflavones might inhibit the formation of fluorescent AGEs.

Zhao Y., Tan D., Peng B., Yang L., Zhang S., Shi R., Chong C., Zhong Z., Wang S., Liang Q., Wang Y.

Eucommia ulmoides Oliver (E. ulmoides) is a popular medicinal herb and health supplement in China, Japan, and Korea, and has a variety of pharmaceutical properties. The neuroendocrine–immune (NEI) network is crucial in maintaining homeostasis and physical or psychological functions at a holistic level, consistent with the regulatory theory of natural medicine. This review aims to systematically summarize the chemical compositions, biological roles, and pharmacological properties of E. ulmoides to build a bridge between it and NEI-associated diseases and to provide a perspective for the development of its new clinical applications. After a review of the literature, we found that E. ulmoides has effects on NEI-related diseases including cancer, neurodegenerative disease, hyperlipidemia, osteoporosis, insomnia, hypertension, diabetes mellitus, and obesity. However, clinical studies on E. ulmoides were scarce. In addition, E. ulmoides derivatives are diverse in China, and they are mainly used to enhance immunity, improve hepatic damage, strengthen bones, and lower blood pressure. Through network pharmacological analysis, we uncovered the possibility that E. ulmoides is involved in functional interactions with cancer development, insulin resistance, NAFLD, and various inflammatory pathways associated with NEI diseases. Overall, this review suggests that E. ulmoides has a wide range of applications for NEI-related diseases and provides a direction for its future research and development.

Ban I., Sugawa H., Nagai R.

Advanced glycation end products (AGEs) are associated with diabetes and its complications. AGEs are formed by the non-enzymatic reactions of proteins and reducing sugars, such as glucose and ribose. Ribose is widely used in glycation research as it generates AGEs more rapidly than glucose. This study analyzed the AGE structures generated from ribose-modified protein by liquid chromatography–quadrupole time-of-flight mass spectrometry. Among these AGEs, Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1) was the most abundant in ribose-glycated bovine serum albumin (ribated-BSA) among others, such as Nε-(carboxymethyl) lysine, Nε-(carboxyethyl) lysine, and Nω-(carboxymethyl) arginine. Surprisingly, MG-H1 was produced by ribated-BSA in a time-dependent manner, whereas methylglyoxal levels (MG) were under the detectable level. In addition, Trapa bispinosa Roxb. hot water extract (TBE) possesses several anti-oxidative compounds, such as ellagic acid, and has been reported to inhibit the formation of MG-H1 in vivo. Thus, we evaluated the inhibitory effects of TBE on MG-H1 formation using ribose- or MG-modified proteins. TBE inhibited MG-H1 formation in gelatin incubated with ribose and ribated-BSA, but not in MG-modified gelatin. Furthermore, MG-H1 formation was inhibited by diethylenetriaminepentaacetic acid. These results demonstrated that ribose reacts with proteins to generate Amadori compounds and form MG-H1 via oxidation.

Qian C., Zhang R., Li J., Huang Z., Liu X., Yu L., Yan L., Fu Y.

To find out the genuine characteristics of Eucommia ulmoides produced in Guizhou.The habitat, functional characters and the content of medicinal components of Eucommia ulmoides in Guizhou were studied by using the method of sample survey combined with typical survey, related laboratory experiments and quantitative analysis. The results showed that the yield of Eucommia ulmoides plantation in Guizhou was divided into low altitude, low middle and high temperature rain slope latitude mixing, short sunshine hours type(A type), medium altitude, low longitude and latitude, high temperature rain, positive oblique steep slope, medium sunshine hours type (B type), middle altitude, low longitude and latitude, moderate high temperature rain, shady side and sunny side have gentle deflection steep slope, medium sunshine hours type (C type), High altitude, low longitude and latitude, low temperature moderate rain, positive gentle slope, long sunshine hours type (D type); Different types of Eucommia ulmoides plantation, Different habitat quality, B type is intensity karst rocky desertification habitat, A type is potential karst rocky desertification habitat, the C and D types are light and moderate rocky desertification areas, respectively, the species diversity of shrub layer in Eucommia ulmoides plantation was higher in D type and B type, A type and C type followed; There was no significant difference in root carbon content and leaf nitrogen content in 4 types of Eucommia ulmoides plantation, Among the four types of A, B, C, D, there were significant or extremely significant differences in other indexes of plant functional traits; Both genipinic acid and aucubin had the highest content of root bark, followed by trunk bark and lowest leaves, Chlorogenic acid is the opposite, The content of geniposide was higher in trunk bark and lower in root bark and leaves; Genipinic acid is higher in D type, Aucubin is higher in A and D type, Chlorogenic acid has higher leaves content in B type, Geniposide was the highest in trunk bark of D type; The element enrichment coefficient K and Mn leaves are the largest, the largest in trunk bark is Ca and Zn, Fe root bark is the largest; Effects of soil potassium, phosphorus, pH value and bulk weight on the functional traits of Eucommia ulmoides were significant. The contents of medicinal components in root bark, trunk bark, and leaves was influenced by species diversity of shrub layer, The contents of geniposide in root bark, aucubin in root bark and trunk bark, genipinic acid in bark and chlorogenic acid in leaves were particularly affected by soil physical and chemical indexes and metal element contents, The functional traits of Eucommia ulmoides can affect the content of medicinal components in root bark, trunk bark, and leaves, Especially on the root bark, trunk bark, and leaves in the content of aucubin content; The content of medicinal components of Eucommia ulmoides was high and stable. The above research results have important theoretical reference significance for the cultivation of Eucommia ulmoides and the cultivation of target medicinal components and the comprehensive exploitation and utilization of resources.

Sugawa H., Yachi A., Fujimoto Y., Nagai R.

Abstract Advanced glycation end-products (AGEs) are formed when proteins react with carbonyl compounds, and they gradually accumulate with age. However, AGE accumulation with ageing is not fully understood because longevity studies in mammals are time-consuming. Therefore, we used Caenorhabditis elegans to evaluate the correlation between ageing and AGE accumulation. Age-synchronized C.elegans nematodes were cultured for 3 and 12 days. The levels of Nε-(carboxymethyl) lysine, Nω-(carboxymethyl) arginine, Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl) ornithine and Nε-(carboxyethyl) lysine (CEL) were compared. Glucose, methylglyoxal and acetol were incubated with human serum albumin, and CEL formation was evaluated. The levels of methylglyoxal and ketone bodies in C.elegans were quantified. CEL accumulation increased significantly with culture duration. Methylglyoxal and ketone bodies—possible forerunners of AGE accumulation—were also quantified with respect to culture duration. The levels of ketone bodies increased significantly during culture, and correlated closely with CEL accumulation (R2 = 0.72, P = 0.0008), whereas the levels of methylglyoxal did not increase over time. CEL was formed in vitro in a time-dependent manner from methylglyoxal and acetol when incubated with human serum albumin (HSA) at the same temperature as C.elegans culture, suggesting that increased levels of CEL in C.elegans are attributable to ketone bodies.

Tominaga Y., Sugawa H., Hirabayashi K., Ikeda T., Hoshi Y., Nagai R.

The accumulation of advanced glycation end-products (AGEs) correlates with aging and accompanies the onset of age-related diseases, such as diabetes and arteriosclerosis. Therefore, a daily intake of natural compounds that inhibit the production of AGEs may be beneficial in preventing these diseases. In this study, we evaluated the inhibitory effects of 14 natural crude extracts, including those of Drosera species, which possess anti-inflammatory activity, on the formation of AGEs, such as Nω-(carboxymethyl)arginine (CMA) and Ne-(carboxymethyl)lysine (CML). Crude extracts of Drosera inhibited the formation of CMA and CML by incubation on gelatin with ribose more effectively than with other extracts, so active compounds that prevent AGE formation were purified from Drosera tokaiensis, which is endemic to Japan. Several compounds were purified from D. tokaiensis extracts using HPLC and identified by NMR analysis. These compounds included ellagic acid, 3,3'-di-O-methylellagic acid 4'-glucoside, myricitrine, and quercimelin. Furthermore, all compounds showed a significantly higher inhibitory effect on CMA and CML formations than aminoguanidine. Specifically, ellagic acid and myricitrine had the highest inhibitory effects of the compounds tested. However, not all compounds showed inhibition of CMA formation in a mixture of gelatin and glyoxal (GO). These results suggest that the compounds in D. tokaiensis inhibit CMA and CML formations via the antioxidative activity of phenolic compounds, rather than GO trapping action. This study provides the first evidence that D. tokaiensis inhibits CMA and CML formations and that phenolic compounds such as ellagic acid and myricitrine play an important role as active components of D. tokaiensis extracts.

Zhou Q., Cheng K., Xiao J., Wang M.

Background Advanced glycation end products (AGEs) play a crucial role in the onset, progression, and propagation of both macroangiopathy and microangiopathy complications associated with diabetes mellitus, while some dietary flavonoids and their derivatives were proved to be potential in the controlling of AGEs during the past decades. Scope and approach In this review, the multifunctional effects of flavonoids were summarized in the aspect of inhibiting the formation of AGEs and attenuating AGEs-induced harmful effects. Key findings and conclusions A multitude of plant-derived flavonoids have been examined of their antiglycative capability in in vitro models, while glycosylation and methylation/methoxylation of flavonoids might decrease the ability of aglycones. Furthermore, the attenuation of AGEs-induced damages by flavonoids were mainly targeted on the receptor of AGEs (RAGE) and through the prevention of MAPKs/NF-κB or TGF-β1 signaling pathway. Besides, during the inhibition of AGEs formation, flavonoids can trap reactive di-carbonyl species (RCS) and form new adducts. Some of these flavonoid-RCS adducts were further proved to possess bioactivities in foods and biological systems.

Ma B., Zhu Z., Zhang J., Ren C., Zhang Q.

Oxidative stress and inflammation are two important pathological mechanisms in diabetic nephropathy (DN). Aucubin (AU), a natural iridoid glucoside, possessing remarkable anti-inflammatory and anti-oxidation activity, has been used to treat diabetic complications. However, underlying mechanism of protective action in DN remains unclear. In the present study, AU significantly alleviated albuminuria, glomerular extracellular matrix expansion, renal fibrosis, and inflammation caused by diabetes after ten weeks of treatment. Moreover, AU significantly inhibited oxidative stress markers in kidney tissue. In addition, we demonstrated that AU promoted endogenous antioxidant defenses by triggering Nrf2 and FOXO3a nuclear translocation, respectively, and up-regulation of antioxidant genes, including NQO1, HO-1, Mn-SOD, and CAT. These effects may be, in part, mediated by inducing SIRT1 and SIRT3 activities. Furthermore, AU also attenuated pro-inflammatory cytokines in renal tissue by inhibiting the NF-κB signaling pathway. Taken together, our results suggested that AU is a potential natural agent for the prevention of diabetes-induced DN.

Ohno R., Ichimaru K., Tanaka S., Sugawa H., Katsuta N., Sakake S., Tominaga Y., Ban I., Shirakawa J., Yamaguchi Y., Ito E., Taniguchi N., Nagai R.

Prolonged hyperglycemia generates advanced glycation end-products (AGEs), which are believed to be involved in the pathogenesis of diabetic complications. In the present study, we developed a polyclonal antibody against fructose-modified proteins (Fru-P antibody) and identified its epitope as glucoselysine (GL) by NMR and LC-electrospray ionization (ESI)- quadrupole TOF (QTOF) analyses and evaluated its potential role in diabetes sequelae. Although the molecular weight of GL was identical to that of fructoselysine (FL), GL was distinguishable from FL because GL was resistant to acid hydrolysis, which converted all of the FLs to furosine. We also detected GL in vitro when reduced BSA was incubated with fructose for 1 day. However, when we incubated reduced BSA with glucose, galactose, or mannose for 14 days, we did not detect GL, suggesting that GL is dominantly generated from fructose. LC-ESI-MS/MS experiments with synthesized [13C6]GL indicated that the GL levels in the rat eye lens time-dependently increase after streptozotocin-induced diabetes. We observed a 31.3-fold increase in GL 8 weeks after the induction compared with nondiabetic rats, and Nɛ-(carboxymethyl)lysine and furosine increased by 1.7- and 21.5-fold, respectively, under the same condition. In contrast, sorbitol in the lens levelled off at 2 weeks after diabetes induction. We conclude that GL may be a useful biological marker to monitor and elucidate the mechanism of protein degeneration during progression of diabetes.

Kinoshita S., Mera K., Ichikawa H., Shimasaki S., Nagai M., Taga Y., Iijima K., Hattori S., Fujiwara Y., Shirakawa J., Nagai R.

Advanced glycation end products (AGEs) accumulate in proteins during aging in humans. In particular, the AGE structureNω-(carboxymethyl)arginine (CMA) is produced by oxidation in glycated collagen, accounting for one of the major proteins detected in biological samples. In this study, we investigated the mechanism by which CMA is generated in collagen and detected CMA in collagen-rich tissues. When various protein samples were incubated with glucose, the CMA content, detected using a monoclonal antibody, increased in a time-dependent manner only in glycated collagen, whereas the formation ofNε-(carboxymethyl)lysine (CML), a major antigenic AGE, was detected in all glycated proteins. Dominant CMA formation in glycated collagen was also observed by electrospray ionization-liquid chromatography-tandem mass spectrometry (LC-MS/MS). During incubation of glucose with collagen, CMA formation was enhanced with increasing glucose concentration, whereas it was inhibited in the presence of dicarbonyl-trapping reagents and a metal chelator. CMA formation was also observed upon incubating collagen with glyoxal, and CMA was generated in a time-dependent manner when glyoxal was incubated with type I–IV collagens. To identify hotspots of CMA formation, tryptic digests of glycated collagen were applied to an affinity column conjugated with anti-CMA. Several CMA peptides that are important for recognition by integrins were detected by LC-MS/MS and amino acid sequence analyses. CMA formation on each sequence was confirmed by incubation of the synthesized peptides with glyoxal and ribose. LC-MS detected CMA in the mouse skin at a higher level than other AGEs. Furthermore, CMA accumulation was greater in the human aorta of older individuals. Overall, our study provides evidence that CMA is a representative AGE structure that serves as a useful index to reflect the oxidation and glycation of collagen.

Xing Y., He D., Wang Y., Zeng W., Zhang C., Lu Y., Su N., Kong Y., Xing X.

Eucommia ulmoides Oliver is a native plant and valuable tonic Chinese medicine in China with a long history, great economic value and comprehensive development potential. Traditionally, the comprehensive utilization rate of E. ulmoides Oliv. is still very low, only bark has been used as medicine and other parts of Eucommia ulmoides Oliv. cannot be fully utilized, even the leaves have been well utilized in food products in Japan in the past decades. In order to improve the comprehensive utilization efficiency of E. ulmoides Oliv., in this review, we summarized the varieties and contents of main active compounds, biological functions and pharmacological effects in different parts of E. ulmoides Oliv. The findings suggest that other parts of E. ulmoides Oliv. could replace the bark of E. ulmoides Oliv. to some extent besides of their respective applications. The unique and extensive physiological functions between different parts of E. ulmoides Oliv. indicate that the comprehensive utilization of E. ulmoides Oliv. has a wide space to develop, which is also an effective way to protect E. ulmoides Oliv. resources and improve its the utilization rate.

Sugawa H., Matsuda S., Shirakawa J., Kabata K., Nagai R.

Approximately 20% of diabetic patients develop diabetic cataracts. As lens proteins are known to be only slightly metabolized during the lifetime, cataracts are difficult to recover from once they have progressed. Therefore, the daily intake of natural compounds would be an important strategy for the prevention of diabetic cataracts. Aphanothece sacrum Okada (Asa) is a freshwater blue-green algae endemic to Japan. It has been eaten since the Edo period in Kyushu. In this study, the inhibitory effects of Asa on the pathogenesis of diabetic cataracts were evaluated. Furthermore, the inhibitory effects of Asa on the formation of Nε-(carboxymethyl) lysine (CML), an oxidation-dependent advanced glycation end-product, were also measured. After 3-month administration, the CML contents in the lens were measured by liquid chromatography tandem mass spectrometry using an internal standard of CML or lysine. Asa significantly inhibited the progression of cataractogenesis and accumulation of CML in diabetic lens compared with the normal diet group. These results suggested that daily intake of Asa reduces oxidative stress and prevents the pathogenesis of cataracts.