Kensaku Takara

Evaluation of an Oral Carrier System in Rats: Bioavailability and Antioxidant Properties of Liposome-Encapsulated Curcumin

To enhance the curcumin absorption by oral administration, liposome-encapsulated curcumin (LEC) was prepared from commercially available lecithins (SLP-WHITE and SLP-PC70) and examined for its interfacial and biochemical properties. A LEC prepared from 5 wt % of SLP-PC70 and 2.5 wt % of curcumin gave a good dispersibility with 68.0% encapsulation efficiency for curcumin, while those from SLP-WHITE did not. Moreover, the resulting LEC using SLP-PC70 was confirmed to be composed of small unilamellar vesicles with a diameter of approximately 263 nm. The resulting LEC was then examined for its effect on bioavailability in Sprague-Dawley (SD) rats. Three forms of curcumin [curcumin, a mixture of curcumin and SLP-PC70 (lecithin), and LEC] were then administered orally to SD rats at a dose of 100 mg curcumin/kg body weight. The pharmacokinetic parameters following curcumin administration were determined in each form. Pharmacokinetic parameters after oral administration of LEC were compared to those of curcumin and a mixture of curcumin and lecithin. High bioavailability of curcumin was evident in the case of oral LEC; a faster rate and better absorption of curcumin were observed as compared to the other forms. Oral LEC gave higher C(max) and shorter T(max) values, as well as a higher value for the area under the blood concentration-time curve, at all time points. These results indicated that curcumin enhanced the gastrointestinal absorption by liposomes encapsulation. Interestingly, the plasma antioxidant activity following oral LEC was significantly higher than that of the other treatments. In addition, the plasma curcumin concentration was significantly correlated to plasma antioxidant activities, and enhanced curcumin plasma concentrations might exert a stronger influence on food functionality of curcumin. The available information strongly suggests that liposome encapsulation of ingredients such as curcumin may be used as a novel nutrient delivery system.

New antioxidative phenolic glycosides isolated from Kokuto non-centrifuged cane sugar.

Nine compounds, 3-hydroxy-4,5-dimethoxyphenyl-β-D-glucopyranoside (1),β-D-fructfuranosyl-α-D-(6-vanilloyl)-glucopyranoside (2), β-D-fructfuranosyl-α-D-(6-syringyl)-glucopyranoside (3), 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxy-1-(E)-propenyl)- 2-methoxyphenoxy]propyl-β-D-glucopyranoside(4), 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3- hydroxy-1-(E)-propenyl)-2,6-dimethoxyphenoxy] propyl-β-D-glucopyranoside (5), dehydrodiconiferyl alcohol-9′-β-D-glucopyranoside (6), 4-[ethane-2-[3-(4-hydroxy-3-methoxyphenyl)-2-propen]oxy]-2,6-dimethoxyphenyl-β-D-glucopyranoside (7), 4-[ethane-2-[3-(4-hydroxy-3-methoxyphenyl)-2-propen]oxy]-2-methoxyphenyl-β-D-glucopyranoside (8), and 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-2-[4-(3-hydroxy-1-(E)-propenyl)-2,6-dimethoxyphenoxy]propyl-β-D-glucopyranoside (9), were isolated from Kokuto non- centrifuged cane sugar. Their structures were elucidated by spectroscopic evidence, mainly based on the NMR technique. Among them, seven new glycosides were identified. The 2-deoxyribose oxidation method was used to measure their antioxidative activity. All of these compounds showed antioxidative activities.

Coral tumors store reduced level of lipids

The reef building coral Montipora informis (Scleractinia, Acroporidae) found on the colony surface of the fringing reef of Sesoko Island, Okinawa, Japan frequently carries hemispherical protuberances (tumors). We compared the total lipid content and the compositions of lipid and fatty acid between normal and tumorous tissues. The lipid content of tumor was 10.6% of the dry tissue weight, and was much lower than that for normal tissues (32.2%). The decrease in the total lipid content of tumorous tissue accompanied a reduced proportion of wax and triacylglycerol (TG). The major fatty acid component was 16:0 (palmitic) acid, and comprised comparable proportion of polar lipids and free fatty acid from both tumorous and normal tissues. An increasing tendency of polyunsaturated fatty acid (PUFA; C>20) was noted in the free fatty acid and polar lipid fractions from tumorous tissues compared with the normal tissue. Thus, the present study first demonstrated the reduced lipid storage level in the tumorous coral, and discussed the rationale for these observations.

1,1-Diphenyl-2-picrylhydrazyl Radical Scavenging Activity and Tyrosinase Inhibitory Effects of Constituents of Sugarcane Molasses

In the course of our work into the use of cane by-products, we have studied the isolation and structural determination of bioactive compounds in sugarcane molasses. In this study, three stereo isomers of syringyl glycerol 3′-O-β-D-glucopyranoside, three stereo isomers of guaiacyl glycerol 3′-O-β-D-glucopyranoside, a syringyl glycerol 2′-O-β-D-glucopyranoside, tachioside and a 2,3-dihydro-3,5-dihydroxy-6-methyl-4-(H)-pyran-4-one (DDMP) were isolated from the 25% methanol eluate by Amberlite XAD-2 column chromatography of sugarcane molasses. The structures of these compounds were determined on the basis of spectroscopic evidence. These isolated compounds were examined for their scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical species, and for their inhibitory activity against mushroom tyrosinase. All of the isolated compounds showed DPPH radical scavenging activity, while DDMP and tachioside showed mushroom tyrosinase inhibitory activity.

Triterpene synthases from the Okinawan mangrove tribe, Rhizophoraceae

Oleanane‐type triterpene is one of the most widespread triterpenes found in plants, together with the lupane type, and these two types often occur together in the same plant. Bruguiera gymnorrhiza (L.) Lamk. and Rhizophora stylosa Griff. (Rhizophoraceae) are known to produce both types of triterpenes. Four oxidosqualene cyclase cDNAs were cloned from the leaves of B. gymnorrhiza and R. stylosa by a homology‐based PCR method. The ORFs of full‐length clones termed BgbAS (2280 bp, coding for 759 amino acids), BgLUS (2286 bp, coding for 761 amino acids), RsM1 (2280 bp, coding for 759 amino acids) and RsM2 (2316 bp coding for 771 amino acids) were ligated into yeast expression plasmid pYES2 under the control of the GAL1 promoter. Expression of BgbAS and BgLUS in GIL77 resulted in the production of β‐amyrin and lupeol, suggesting that these genes encode β‐amyrin and lupeol synthase (LUS), respectively. Furthermore, RsM1 produced germanicol, β‐amyrin, and lupeol in the ratio of 63 : 33 : 4, whereas RsM2 produced taraxerol, β‐amyrin, and lupeol in the proportions 70 : 17 : 13. This result indicates that these are multifunctional triterpene synthases. Phylogenetic analysis and sequence comparisons revealed that BgbAS and RsM1 demonstrated high similarities (78–93%) to β‐amyrin synthases, and were located in the same branch as β‐amyrin synthase. BgLUS formed a new branch for lupeol synthase that was closely related to the β‐amyrin synthase cluster, whereas RsM2 was found in the first branch of the multifunctional triterpene synthase evolved from lupeol to β‐amyrin synthase. Based on these sequence comparisons and product profiles, we discuss the molecular evolution of triterpene synthases and the involvement of these genes in the formation of terpenoids in mangrove leaves.

Lipid composition of mangrove and its relevance to salt tolerance

Lipid compositions of mangrove trees were studied in relation to the salt-tolerance mechanism. Leaves and roots were obtained from seven mature mangrove trees on Iriomote Island, Okinawa: Bruguiera gymnorrhiza, Rhizophora stylosa, Kandelia candel, Lumnitzera racemosa, Avicennia marina, Pemphis acidula and Sonneratia alba. Lipids of mangrove leaves mainly consisted of 11 lipid classes: polar lipids, unknown (UK) 1–6, sterols, triacyl glycerols, wax ester and sterol ester (UK 3 and 4 were found to be tri-terpenoid alcohol in this study). Of these lipid classes, sterol ester was the main lipid in all species comprising 17.6–33.7% of total lipids. Analysis of the chemical structure found that the sterol esters mainly consisted of fatty acid esters of tri-terpenoid alcohols. One major tri-terpenoid alcohol was identified to be lupeol by interpretation of infrared resonance, nuclear magnetic resonance and mass spectrometry. Because of the unique anatomy of the mangrove root, lipid analyses were made separately for epidermis, cortex and innermost stele, respectively. The concentration of free tri-terpenoid alcohols showed a higher tendency in the outside part than in the inside portion of the roots, suggesting their protective roles. Relevance of lipid composition to salt tolerance was studied with propagules of K. candel and B. gymnorrhiza planted with varied salt concentrations. The proportions of free tri-terpenoids increased with salinity in both leaves and roots of K. candel, and only in roots of B. gymnorrhiza. No salt-dependent changes were noted in the phospholipid and fatty acid compositions in both species. These findings suggested that salt stress specifically modulated the terpenoid concentrations in mangroves.

Tumor-selective cytotoxicity of benzo[c]phenanthridine derivatives from Toddalia asiatica Lam.

PurposeTo develop a novel anti-cancer drug of low side effect against lung adenocarcinoma, the authors screened the bioresources of Okinawa Island, Japan. The medicinal plant Toddalia asiatica Lam. contained three benzo[c]phenanthridine derivatives: dihydronitidine (DHN), nitidine (NTD) and demethylnitidine (DMN). Of the three derivatives, DHN had been shown to selectively inhibit the growth of cancer cells in our previous study. Because of similar molecular topology of NTD or DMN to DHN, it can be expected that NTD and DMN also show selective cytotoxicity. The aim of the present study was therefore to examine the selective cytotoxicity of these two compounds in vitro and in vivo.MethodsBenzo[c]phenanthridine derivatives were isolated from T. asiatica Lam., and their chemical structures were identified by interpretation of NMR and MS spectrum. Of the isolated compounds, NTD and DMN were evaluated for cytotoxicity in vitro or in vivo.ResultsNTD as well as DHN selectively reduced the growth of murine and human lung adenocarcinoma in vitro with selective intracellular accumulation. NTD has also been proven to be highly effective in vivo to inhibit the growth of both murine and human lung adenocarcinoma in a subcutaneous xenograft model without any deteriorating side effect. In contrast, DMN had no selective cytotoxicity suggesting that 8-methoxy group of NTD is the critical structural feature for the selective cytotoxicity.ConclusionsThis study thus proves the effectiveness of benzo[c]phenanthridine derivatives as anti-cancer agent in vivo for the first time, and discusses the mechanisms responsible for the selective cytotoxicity.

New Phenolic Compounds from Kokuto, Non-centrifuged Cane Sugar

Five new phenolic compounds, 4-(β-D-glucopyranosyloxy)-3,5-dimethoxyphenyl-propanone (8), 3-[5-[(threo) 2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxybenzofurany]]-propanoic acid (12), 2-[4-(3-hydroxy-1-propenyl)-2,6-dimethoxyphenoxy]-3-hydroxy-3-(4-hydroxy-3,5-dimethoxyphenyl)propyl-β-D-glucopyranoside (13), 4-[(erythro) 2,3-dihydro-3(hydroxymethyl)-5-(3-hydropropyl)-7-methoxy-2-benzofuranyl]-2,6-dimethoxyphenyl-β-D-glucopyranoside (14), 9-O-β-D-xylopyranoside of icariol A2 (15), and known phenolic compounds were isolated from Kokuto, non-centrifuged cane sugar (Saccharum officinarum L.). Their structures were determined by a spectral investigation.

Characterization and Bioavailability of Liposomes Containing a Ukon Extract

In order to use liposomes as an efficient carrier of functional food materials, liposomes encapsulating a ukon extract (LUE) were prepared by the mechanochemical method under different conditions, and were physico-chemically and biochemically characterized. After a homogenization treatment, the size of LUE decreased with decreasing concentration of the extract from 10 to 2.5 wt %, but did not decrease below 570 nm. LUE were thus subjected to microfluidization. The LUE solutions obtained from less than 5 wt % of the extract remained well dispersed for at least 14 d, whereas those from 10 wt % showed phase separation. With 5 wt % of the extract, the size of LUE obtained at an inlet pressure of 100 MPa was smaller than that obtained at 20 MPa, and reached below 180 nm. Under optimal conditions, resulting LUE was confirmed to be small unilamellar vesicles (SUV) with a diameter of approximately 100 nm by freeze-fracture electron microscopy (FFEM). When used for treating simulated gastric and intestinal fluids, LUE obtained by microfluidization showed a 2-fold higher residual rate of curcumin than the uncapsuled extract itself. The bioactivity of LUE was further examined for its suppressive effect on carbon tetrachloride (CCl4)-induced liver injury by using mice. Orally administrated LUE at a dose of 10 mg/kg as the extract had a much higher suppressive effect on the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, compared to the uncapsuled extract at a dose of 33 mg/kg.

The tumor specific cytotoxicity of dihydronitidine from Toddalia asiatica Lam

Purpose: In recent years, a number of reports have shown the anticancer activity of plant extracts and phytoalkaloid. Methods: We have evaluated the cytotoxicity profiles of 157 extracts prepared from dietary or medical plants growing in the Okinawa island, using 10 different cell lines. In vitro cytotoxicity screening indicated the presence of a highly selective cytotoxic compound in the extract of Toddalia asiaticaLam. The known alkaloid (1,3)benzodioxolo(5,6-c)phenanthridine, 12,13-dihydro-2,3-dimethoxy-12-methyl-(dihydronitidine) was identified as an active material from this plant. This alkaloid had highly specific cytotoxicity to human lung adenocarcinoma (A549) cells. Results: The results of the fluorescence activated cell sorter (FACS) analysis and the measurement of caspase-3 activity showed that dihydronitidine induced specific apoptotic cell death in A549 cells. Gene expression analysis in the apoptotic cells found that dihydronitidine variously regulated the cell cycle related genes (CDK2 and CCNE), and up-regulated the cell death related genes specifically in tumor cells. Thus dihydronitidine manifested its characteristics in the tumor selective cytotoxicity, contrasting with the case of a known anticancer agent camptothecin (CPT). Microscopic observation further revealed the specific accumulation of dihydronitidine within the cytosolic organelle, but not in the nuclei of adenocarcinoma. No accumulation was observed with CPT in all cell lines. Conclusion: The data suggested that dihydronitidine toxicity targeted a particular intracellular organelle in the tumor cells.