Syntheses and Glycosidase Inhibitory Activities, and in Silico Docking Studies of Pericosine E Analogs Methoxy-Substituted at C6.

Inspired by the significant -glucosidase inhibitory activities of (+)- and (-)-pericosine E, we herein designed and synthesized 16 analogs of these marine natural products bearing a methoxy group instead of a chlorine atom at C6. Four of these compounds exhibited moderate -glucosidase inhibitory activities, which were weaker than those of the corresponding chlorine-containing species. The four compounds could be prepared by coupling reactions utilizing the (-)-pericosine B moiety. An additional in silico docking simulation suggested that the reason of reduced activity of the C6-methoxylated analogs might be an absence of hydrogen bonding between a methoxy group with the surrounding amino acid residues in the active site in -glucosidase.

Synthesis of Natural O-Linked Carba-Disaccharides, (+)- and (-)-Pericosine E, and Their Analogues as α-Glucosidase Inhibitors.

Pericosine E (6), a metabolite of Periconia byssoides OUPS-N133 was originally isolated from the sea hare Aplysia kurodai, which exists as an enantiomeric mixture in nature. The enantiospecific syntheses of both enantiomers of Periconia byssoides OUPS-N133 has been achieved, along with six stereoisomers, using a common simple synthetic strategy. For these efficient syntheses, highly regio- and steroselective processes for the preparation of bromohydrin and anti-epoxide intermediates were applied. In order to access the unique O-linked carbadisaccharide structure, coupling of chlorohydrin as a donor and anti-epoxide as an acceptor was achieved using catalytic BF3·Et2O. Most of the synthesized compounds exhibited selectively significant inhibitory activity against α-glycosidase derived from yeast. The strongest analog showed almost 50 times the activity of the positive control, deoxynojirimycin.

Simultaneous Addition of Shikonin and Its Derivatives with Lipopolysaccharide Induces Rapid Macrophage Death.

Macrophages play pivotal roles in inflammatory responses. Previous studies showed that various natural products exert antiinflammatory effects by regulating macrophage activation. Recent studies have shown that shikonin (SHK) and its derivatives (ß-hydroxyisovalerylshikonin, acetylshikonin, and isobutylshikonin), which are 1,4-naphthoquinone pigments extracted from the roots of Lithospermum erythrorhizon, have various pharmacological, including antiinflammatory and antitumor, effects. Even though there have been many studies on the antiinflammatory activities of SHK derivatives, only a few have described their direct effects on macrophages. We investigated the effects of SHK derivatives on lipopolysaccharide (LPS)-treated macrophages. Low doses of SHK derivatives induced significant macrophage cytotoxicity (mouse macrophage-like J774.1/JA-4 cells and mouse peritoneal macrophages) in the presence of LPS. SHK activated caspases-3 and -7, which led to DNA fragmentation, but this cytotoxicity was prevented through a pan-caspase inhibitor in LPS-treated JA-4 cells. Maximal cytotoxic effects were achieved when SHK was added immediately before LPS addition. These results indicate that SHK derivatives induce caspase-dependent apoptotic cell death of LPS-treated macrophages and suggest that SHK acts during an early stage of LPS signaling.

Synergistic antitumor effect of a combination of paclitaxel and carboplatin with nobiletin from Citrus depressa on non-small-cell lung cancer cell lines.

Non-small-cell lung carcinomas do not sufficiently respond to cancer chemotherapeutic drugs. Combination effects of cancer chemotherapy drugs (paclitaxel and carboplatin) with nobiletin or powdered Shiikuwasha extract from Citrus depressa were examined by isobologram and combination index analyses. It was demonstrated that the combination generated a synergistic inhibitory effect against the proliferation of the human non-small-cell lung carcinoma cell lines A549 and H460 and that of the two chemotherapy drugs, paclitaxel was responsible for this synergistic effect. Furthermore, the percentage of apoptotic cells was decreased with increasing rates of nobiletin to paclitaxel and carboplatin. These findings were considered to be attributed to the ability of nobiletin to regulate cells in the G1 phase, which escaped cell death initiated by paclitaxel and carboplatin. An antitumor activity assay showed that this combination significantly suppressed the growth of subcutaneous A549 tumor xenografts in nude mice.

Chemical quality evaluation and characteristics of interspecific hybrid (G. uralensis × G. glabra) C-18 strain as a new Licorice cultivar.

The development of new cultivars is essential for establishing a method of producing licorice in Japan. A suitable new cultivar for domestic licorice production, known as the interspecific hybrid strain C-18, was developed by crossbreeding Glycyrrhiza uralensis Fisch. (as the seed parent, possessing a high glycyrrhizin (GL) content, strain OMP-28) and Glycyrrhiza glabra L. (as the pollen parent, known for vigorous growth, strain OMP-10). Both G. uralensis and G. glabra are specified in the Pharmacopoeia of Japan (18th edition) as the source plants for Glycyrrhizae Radix. After 2 years of cultivation, strain C-18 exhibited robust growth, with a fresh weight of 148.8 g and a stem diameter of 0.89 mm. The GL content in the dry weight was measured at 3.61%. Seedlings cultivated from rhizomes in the field for 2 years showed a tap root fresh weight per plant of 120 ± 21 g, with an average GL content relative to the dry weight of 2.68% ± 0.38%. Although glabridin, a characteristic compound of G. glabra, was not detected, glycycoumarin, a characteristic compound of G. uralensis, was detected via HPLC analysis. Strain C-18 contained glycycoumarin as a characteristic compound of G. uralensis but lacked glabridin, a compound characteristic of G. glabra. Additionally, 2,3-dehydrokievitone (1) and parvisoflavone A (2) were identified as distinctive components of the interspecific hybrid (G. uralensis × G. glabra) C-18.

Characterization of a glycyrrhizin-deficient strain of Glycyrrhiza uralensis.

A triterpene saponin, glucoglycyrrhizin, was isolated from a glycyrrhizin-deficient strain 83-555 of Glycyrrhiza uralensis (Leguminosae), and the structure was determined by chemical and spectral data to be 3-O-[ß-D-glucopyranosyl-(1→2)-ß-D-glucuronopyranosyl]-glycyrrhetinic acid. Since this saponin has a 2'-O-ß-D-glucopyranosyl moiety instead of the 2'-O-ß-D-glucuronopyranosyl moiety of glycyrrhizin, the glucuronidation of 3-O-ß-D-glucuronopyranosyl-glycyrrhetinic acid leading to glycyrrhizin is inhibited in this strain. All 4 offspring of the 83-555 strain produced glucoglycyrrhizin. Interestingly, 2 of the offspring produced both glycyrrhizin and glucoglycyrrhizin, and sequence analysis of the pkr gene suggested that these 2 offspring were hybrids of 83-555 strain and glycyrrhizin-producing strains.

Seasonal variation in the total saponin content of platycodon roots cultivated in Japan.

Roots of Platycodon grandiflorus A. De Candolle (Campanulaceae), with the bark removed, have been used as food and frequently employed as herbal medicines for inflammatory diseases such as tonsillitis, dermatitis, and cough. Platycodins are the bioactive saponin components of these crude medicines. Recently, P. grandiflorus have been cultivated in Japan and are harvested from October to December according to conventional practices. Seasonal fluctuations in the total saponin content of these roots were determined using LC/MS methods to recommend harvesting times when the saponin content is high. Platycodins A and C are monoacetylated forms of platycodin D; however, the acetyl form is unstable and deacetylates easily. Here, the contents of platycodin D, platycodin D2, and platyconic acid A were measured as the total saponin content using alkaline hydrolysis for monoacetylated platycodins D, D2, and platyconic acid A. The results demonstrated that the saponin content in the roots decreased in summer, increased in autumn, but decreased again in late autumn.

Anti-tumor activity of new orally bioavailable 2-amino-5-(thiophen-2-yl)benzamide-series histone deacetylase inhibitors, possessing an aqueous soluble functional group as a surface recognition domain.

New orally bioavailable 5-(thiophen-2-yl)-substituted 2-aminobenzamide-series histone deacetylase inhibitors were synthesized. These compounds possess a morpholine or piperadine-derived moiety as an aqueous soluble functional group. Among them, 8b, having a 4-ethyl-2,3-dioxopiperazine-1-carboxamide group as a surface recognition domain, showed promising inhibitory activities against HCT116 cell growth and HDAC1/2. Notably, unlike MS-275, this compound did not induce apoptosis in the cell cycle tests. We therefore conducted antitumor tests of 8b and MS-275 against HCT116 cell xenografts in nude mice. Compound 8b reduced the volume of tumor mass to T/C: 60% and 47% at 45 and 80mg/kg over 16days, respectively. These values were comparable to the rate (T/C: 51% at 45mg/kg) for MS-275. Furthermore, 8b, at neither 45 nor 80mg/kg, induced the weight loss which was observed in the mice given MS-275 at 45mg/kg.

Divergent synthesis and evaluation of inhibitory activities against cyclooxygenases-1 and -2 of natural withasomnines and analogues.

The divergent synthesis of natural withasomnines and analogues was achieved from 4-hydroxypyrazoles, which was prepared via alkaline hydrolysis of the Baeyer-Villiger oxidation products from 4-formylpyrazoles. Key steps of this synthesis are regioselective Claisen rearrangement of 4-allyloxypyrazoles and the Suzuki-Miyaura coupling of 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl trifluoromethanesulfonate and commercially available arylboronic acids. The Suzuki-Miyaura coupling at the final step of this strategy enabled facile access to natural withasomnines and their analogues. The biological activities of the twelve synthesized compounds against cyclooxygenases-1 and -2 (COX-1 and COX-2) were evaluated.

New microtubule polymerization inhibitors comprising a nitrooxymethylphenyl group.

We have designed cancer antiproliferative compounds, starting from aniline or phenol derivative, which comprise one or two nitrooxymethylphenyl groups as do the hybrid drugs NCX4040 and NCX530. Compound 2a with p-nitrooxymethylbenzoyl-oxy and -amino groups as well as 8a with a p-nitrooxymethylbenzoylamino group showed more promising effects than NCX4040 against human colon and breast cancer cells. Since 2a and 8a, but not NCX4040, arrested human colon carcinoma HCT116 cells in the M phase, the former two compounds may inhibit cell growth differently from NCX4040. Merged images of immunofluorescence-stained α-tubulin and Hoechst-stained nuclei in human fibrosarcoma HT1080 cells showed that 2a and 8a disrupted microtubule formation just as did vincristine, the tubulin polymerization inhibitor. In experiments in vivo, the intraperitoneal administration of 8a at 80 mg/kg/day reduced the growth of HCT116 xenografts in nude mice to T/C 55%.

Inhibitory effects of furanocoumarin derivatives in Kampo extract medicines on P-glycoprotein at the blood-brain barrier.

Furanocoumarin derivatives, known as components of grapefruit juice, showing inhibitory effects against P-glycoprotein (P-gp) in the intestine are also contained in the plants of rutaceae and umbelliferae families, which are used as components of Kampo extract medicines. In this study, we investigated the inhibitory effects of byakangelicol and rivulobirin A, known as furanocoumarins showing P-gp inhibitory effect using Caco-2 monolayer, against P-gp at the blood-brain barrier (BBB) under both in vitro and in vivo conditions. First we studied the membrane permeability of furanocoumarins to clarify whether they can be absorbed from the intestine. Both furanocoumarins showed high permeability through the Caco-2 monolayer, suggesting that they can easily reach the systemic circulation after oral administration. Then, we evaluated the effect of these furanocoumarins on the uptake of calcein acetoxymethyl ester (calcein-AM), a P-gp substrate, into bovine brain microvascular endothelial cells (BBMEC). Both furanocoumarins significantly increased the uptake amount of calcein-AM into BBMEC by the inhibition of P-gp at the BBB in vitro. Next we also investigated the P-gp inhibitory effect of these furanocoumarins at the rat BBB in vivo using verapamil as a P-gp substrate. Both furanocoumarins increased the B/P ratio of verapamil compared to the control, even under in vivo conditions; however, the extent of the inhibitory effect was much lower than in vitro condition. In conclusion, byakangelicol and rivulobirin A may inhibit P-gp expressed at the BBB even under in vivo conditions. Further studies using Kampo extract medicines under in vivo condition are necessary for safe drug therapy.

Formononetin attenuates H2O2-induced cell death through decreasing ROS level by PI3K/Akt-Nrf2-activated antioxidant gene expression and suppressing MAPK-regulated apoptosis in neuronal SH-SY5Y cells.

Formononetin is an isoflavone, found in herbs like Trifolium pratense, which executes a variety of physiological activities including anti-neurodegenerative effect. However, the molecular mechanism of formononetin-mediated neuroprotection remains unclear. In this study, we investigated the protective effect of formononetin on hydrogen peroxide (H2O2)-induced death of human neuroblastoma SH-SY5Y cells and its underlying molecular mechanism. Formononetin suppressed H2O2-induced cytotoxicity. H2O2-induced increase in the intracellular reactive oxygen species (ROS) levels was decreased by formononetin, together with the enhanced expression of the antioxidant genes. H2O2-induced elevation of the Bax/Bcl-2 ratio and cleaved caspase-3 and caspase-7 levels were lowered by formononetin treatment. Moreover, formononetin repressed H2O2-induced phosphorylation of mitogen-activated protein kinases (MAPKs). Nuclear factor erythroid 2-related factor 2 (Nrf2) siRNA decreased antioxidant gene expression and elevated the H2O2-induced ROS level in the formononetin-treated cells. Furthermore, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling is involved in the activation of the nuclear translocation of Nrf2. These results indicate that the neuroprotective effect of formononetin against H2O2-induced cell death is due to a decrease in the ROS level with the enhanced expression of the antioxidant genes through activation of the PI3K/Akt-Nrf2 signaling. In addition, formononetin suppressed apoptosis through inhibition of phosphorylation of MAPKs in SH-SY5Y cells. Thus, formononetin is a potential therapeutic agent for the treatment of neurodegenerative diseases.

Furanocoumarin derivatives in Kampo extract medicines inhibit cytochrome P450 3A4 and P-glycoprotein.

Furanocoumarins in grapefruit are known to show inhibitory effects against P-glycoprotein (P-gp) and CYP3A4 in intestinal epithelial cells; however, furanocoumarin derivatives are widely contained in the plants of Rutaceae and Umbelliferae families, which are used as components of Kampo extract medicines. In this study, we investigated the inhibitory effects of 12 furanocoumarins extracted from plants in the Umbelliferae family against P-gp and CYP3A4 activity. Furthermore, we studied their inhibitory effect on P-gp when furanocoumarins are used as Kampo extract medicine rather than as an isolated single compound. From screening of the CYP3A4 inhibitory effect, notopterol and rivulobirin A, the only dimer types of furanocoumarin, were found to be potent inhibitors of CYP3A4. On the other hand, byakangelicol and rivulobirin A showed strong P-gp inhibition from the screening of P-gp inhibitor evaluated by quinidine permeation through the Caco-2 monolayer; however, the chemical structural relationship of furanocoumarins between P-gp and CYP3A4 inhibitory effects could not be obtained. We also investigated the effect of these furanocoumarins on the transport of digoxin through the Caco-2 monolayer. The inhibitory effect of rivulobirin A was more potent than that of byakangelicol. Application of either Senkyu-cha-cho-san or Sokei-kakketsu-to, which are composed of herbal remedies in the Umbelliferae group, significantly decreased the efflux ratio of digoxin. In conclusion, it was found that some furanocoumarins extracted from the plants in the Umbelliferae family strongly inhibited P-gp and CYP3A4. Kampo extract medicines containing herbal remedies belonging to the Umbelliferae family may cause a drug-drug interaction with P-gp or a CYP3A4 substrate drug.

New orally bioavailable 2-aminobenzamide-type histone deacetylase inhibitor possessing a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group.

New 2-aminobenzamide-type histone deacetylase (HDAC) inhibitors were synthesized. They feature a sulfur-containing bicyclic arylmethyl moiety-a surface recognition domain introduced to increase in cellular uptake-and a substituted tert-amino group which affects physicochemical properties such as aqueous solubility. Compound 22 with a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group reduced the volume of human colon cancer HCT116 xenografts in nude mice to T/C 67% by oral administration at 45mg/kg, which was comparable to the rate (T/C 62%) for a positive control, MS-275. Western blot analyses as well as cell cycle and TUNEL assays by flow cytometry suggested that the two compounds inhibited the growth of cancer cells via similar mechanisms.

Determination of flavonoids in licorice using acid hydrolysis and reversed-phase HPLC and evaluation of the chemical quality of cultivated licorice.

Licorice contains flavonoids and triterpenoids as the major bioactive components. Most of the flavonoids are the glycosidic form of liquiritigenin (LIQ), isoliquiritigenin (ISO) and formononetin (FOR). A reversed-phase HPLC method for the quantification of LIQ, ISO and FOR in licorice was developed. This method does not measure each glycoside but measures the aglycones using acid hydrolysis. All calibration curves showed good linear regression (r > 0.9998). The method showed good precision for intraday (RSD < 2.14 %) and interday (RSD < 0.51 %) assays. The limit of detection was 0.031 microg for LIQ, 0.011 microg for ISO and 0.006 microg for FOR. The limit of quantification was 0.31 microg for LIQ, 0.11 microg for ISO and 0.06 microg for FOR. The flavonoid contents along with the glycyrrhizin content of cultivated licorice from seedling plants in Japan and commercial wild licorice were investigated. This new method could be extremely useful for evaluating the quality of licorice.

Phytochemical characterization of Rosa multiflora Thunb. (Rosaceae) in Japan and South Korea, with a focus on the bioactive flavonol glycoside 'multiflorin A'.

Dried achene or anthocarpous accessory fruits of Rosa multiflora Thunb., Rosae fructus ("Eijitsu" in Japanese), have been used in clinical practice to improve constipation within traditional Japanese medicine. Recently, it has been claimed that the efficacy of this crude drug is decreasing, and multiflorin A, the purgative component, was not detected within the tested samples. In order to clarify the causes of this issue, we investigated Rosa section Synstylae (Rosaceae), including R. multiflora, growing in Japan and South Korea with a focus on the secondary metabolite, multiflorin A. We recognize that there are two chemotypes based on the presence (Type I) or absence (Type II) of multiflorin A. Type I contains quercitrin, multinoside A, multiflorin B, and multinoside A acetate as major index compounds. Type II contains hyperin, isoquercitrin, quercetin 3-O-glucuronide, and 3'-methoxy-isoquercitrin as the major index compounds. The chemotype of Rosa section Synstylae (Rosaceae) plants collected in Japan (excluding Tsushima Island) were all classified as Type I with exception of two species, R. luciae and R. sambucina. On the other hand, both Type I and Type II were detected within Rosae fructus obtained from R. multiflora collected in South Korea and Tsushima Island, Japan. The results indicate that Rosae fructus from R. multiflora (Type I) from Japan, excluding Tsushima Island, should be employed clinically, which we describe as purgative.

Suppression of Fatty Acid and Triglyceride Synthesis by the Flavonoid Orientin through Decrease of C/EBPδ Expression and Inhibition of PI3K/Akt-FOXO1 Signaling in Adipocytes.

Plant flavonoids have a variety of biological properties. In a previous study, we found that the tea of the Asian dayflower, Commelina communis L., decreased the body weight gain in high-fat diet-fed mice. In this study, we studied the anti-adipogenic ability of a flavonoid orientin that is found in abundance in C. communis. Orientin repressed the accumulation of intracellular triglyceride (TG) in mouse adipocyte 3T3-L1 cells. The treatment with orientin also decreased the mRNA levels of the genes involved in adipogenesis, lipogenesis, lipolysis, and TG synthesis, and reduced the release of glycerol. Orientin lowered the expression of CCAAT/enhancer binding protein (C/EBP) δ in the early stage of adipogenesis, leading to a decrease in the expression of the adipogenic master transcription factors such as peroxisome proliferator-activated receptor (PPAR) γ and C/EBPα. Moreover, the anti-adipogenic effect of orientin repressed the phosphorylation of Akt and subsequent phosphorylation of forkhead box protein O1 (FOXO1), which inhibits the transcription of the Ppar gene. These results indicate that a plant flavonoid orientin suppressed the expression of the Pparγ gene through repression of C/ebpδ expression and inhibition of the phosphoinositide 3-kinase /Akt-FOXO1 signaling in adipocytes.

Anti-inflammatory activities of Ophiopogonis Radix on hydrogen peroxide-induced cellular senescence of normal human dermal fibroblasts.

Ophiopogonis Radix (Ophiopogon root), which nourishes the yin, has been used in clinical practice to promote fluid secretion and to moisturize the lungs and skin in traditional Chinese and Japanese (Kampo) medicine. To evaluate this traditional medicinal effect, we investigated the anti-chronic inflammatory effect of Radix Ophiopogonis on senescent cells. Conversely, although several phenotypes of Ophiopogon japonicus Ker-Gawler (Liliaceae) are prevalent in Japan and China, we used these Ophiopogon roots by considering them as one crude drug, Ophiopogonis Radix. In this study, it was revealed that there are two chemotypes (Types A and B) in the root of the original plant, O. japonicus. Methylophiopogonanone A (compound 1) and methylophiopogonanone B (compound 2) were isolated as index compounds from Type A and compound 1 and ophiopogonanone A (compound 3) from Type B. In addition, ophiopogonin B (compound 4) was isolated as the main steroidal saponin from both Type A and B. The results indicated that two different methanol extracts (from Types A and B) and the main constituents of O. japonicus (compound 1-4), significantly downregulated the expression of interleukin (IL)-6 and IL-8, which were enhanced by senescent normal human dermal fibroblasts. Moreover, the two different methanol extracts and compounds 1-4 decreased IL-6 production in a strong and concentration-dependent manner by the ELISA method.

Evaluation of the chemical quality of Sekkoku () in current Japanese commercial crude drugs: constituents of Flickingeria xantholeuca (Rchb. f.) A.D. Hawkes.

In our investigation, most Shihu (; Japanese name, Sekkoku) in current Japanese commercial crude drugs were from Flickingeria xantholeuca (Orchidaceae). As the index compounds, three new ent-pimarane-type diterpenes, flickinxthanthosides A-C (1-3), one known analogue (7), and three new ent-kaurane-type diterpenes, flickinxanthosides D (4) and E (5) and flickinxanthol A (6) were isolated from the stem of F. xantholeuca. The structures of the new compounds were elucidated on the basis of spectroscopic analyses and chemical methods. We attempted to detect these index compounds from the MeOH extracts of other Dendrobium or Flickingeria plants using TLC and LC/MS.

Biosynthesis of 1-deoxynojirimycin in Commelina communis: a difference between the microorganisms and plants.

1-Deoxynojirimycin is a glycosidase-inhibitory alkaloid obtained from several plants and microorganisms. Administration experiments using [1-(13C)] glucose in the higher plant Commelina communis and 13C NMR spectroscopic analyses of products suggested that 1-deoxynojirimycin was biosynthesized through a different route compared with that in Streptomyces and Bacilli microorganisms.