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The methanol extract from the leaves of Ilex paraguariensis A. St.-Hil. (Aquifoliaceae), popularly known as mate, maté, or yerba maté, inhibits the intracellular triglyceride accumulation in HepG2 cells and suppresses the plasma triglyceride elevation in olive oil-treated mice. Three new triterpene saponins, termed mateosides I (1), II (2), and III (3), were isolated from the extract along with 29 known compounds. The structures of 1-3 were elucidated based on chemical and spectroscopic evidence. Among the isolates, principal saponin constituents, 2 and matesaponins 1 (7) and 2 (9), potently inhibited the triglyceride accumulation in HepG2 cells simultaneously treated with oleic acid and high glucose. In vivo assay of the methanol extract of I. paraguariensis revealed that 7 and 9 showed anti-hyperlipidemic activities in olive oil-treated mice. These results suggested that the saponin constituents of I. paraguariensis could be valuable bioactive marker for the anti-obesogenic activity.

Abstract During our studies characterizing functional substances from food resources for the prevention and treatment of lifestyle-related diseases, we isolated the active constituents, salacinol (1) and neokotalanol (4), and related thiosugar sulfoniums, from the roots and stems of the genus Salacia plants [Celastraceae (Hippocrateaceae)] such as Salacia reticulata Wight, S. oblonga Wall., and S. chinensis L., and observed their antidiabetic effects. These plant materials have been used traditionally in Ayurvedic medicine as a specific remedy at the early stage of diabetes, and have been extensively consumed in Japan, the United States, and other countries as a food supplement for the prevention of obesity and diabetes. Here, we review our studies on the antidiabetic effects of plants from the genus Salacia, from basic chemical and pharmacological research to their application and development as new functional food ingredients. Graphic abstract

The antidiabetic effects of a hot water extract of the stems of Salacia chinensis (SCE) were evaluated in vivo in ob/ob mice (genetically obese hyperglycemic mice). Administration of dietary feed containing 0.20 and 0.50% of SCE for 23 days to ob/ob mice significantly suppressed the elevation of both blood glucose and HbA1c levels, without significantly changing body weight and food intake. To characterize the antidiabetic effects of the thiosugar sulfonium constituent neokotalanol (1), which has potent α-glucosidase inhibitory activity, we performed a similar in vivo study. HbA1c levels were significantly suppressed in ob/ob mice after the administration of dietary feed containing 0.0003% of neokotalanol (1) for 20 days. These results indicate that SCE and neokotalanol (1) are potential leads for the development of novel antidiabetic agents.

Four chain-extended analogs (12a–12d) and two related de-O-sulfonated analogs (13a and 13c) by introducing alkyl groups (a: R = C3H7, b R = C6H13, c: R = C8H17, d: R = C10H21) to the side chains of salacinol (1), a natural α-glucosidase inhibitor from Ayurvedic traditional medicine “Salacia”, were synthesized. The α-glucosidase inhibitory activities of all the synthesized analogs were evaluated in vitro. Against human intestinal maltase, the inhibitory activities of 12a and 13a with seven-carbon side chain were equal to that of 1. In contrast, analogs (12b–12d, and 13c) exhibited higher level of inhibitory activity against the same enzyme than 1 and had equal or higher potency than those of the clinically used anti-diabetics, voglibose, acarbose, and miglitol. Thus, elongation of the side chains of 1 was effective for specifically increasing the inhibitory activity against human intestinal maltase.

Two new gallic acid glycosides, potentillanosides G (1) and H (2), were newly isolated from the methanol extract of the tuberous roots of Potentilla anserina (Rosaceae), together with a known compound, ellagic acid 3-O-α-l-rhamnopyranoside (3). Their structures were elucidated on the basis of chemical and physicochemical evidence. Among the constituents, potentillanoside H (2, IC50 = 99.5 μM) was found to show hepatoprotective activity.

An Orobanchaceae plant Cistanche tubulosa (SCHENK) WIGHT (Kanka-nikujuyou in Japanese), which is one of the authorized plant resources as Cistanches Herba in both Japanese and Chinese Pharmacopoeias, is a perennial parasitic plant growing on roots of sand-fixing plants. The stems of C. tubulosa have traditionally been used for treatment of impotence, sterility, lumbago, and body weakness as well as a promoting agent of blood circulation. In recent years, Cistanches Herba has also been widely used as a health food supplement in Japan, China, and Southeast Asian countries. Here we review our recent studies on chemical constituents from the stems of C. tubulosa as well as their bioactivities such as vasorelaxtant, hepatoprotective, and glucose tolerance improving effects.

An aqueous acetone extract from the fruit of Alpinia galanga (Zingiberaceae) demonstrated inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells (IC50 = 7.3 μg/mL). Through bioassay-guided separation of the extract, a new 7-O-9′-linked neolignan, named galanganol D diacetate (1), was isolated along with 16 known compounds including 14 phenylpropanoids (2–15). The structure of 1, including its absolute stereochemistry in the C-7 position, was elucidated by means of extensive NMR analysis and total synthesis. Among the isolates, 1 (IC50 = 2.5 μM), 1′S-1′-acetoxychavicol acetate (2, 5.0 μM), and 1′S-1′-acetoxyeugenol acetate (3, 5.6 μM) exhibited a relatively potent inhibitory effect without notable cytotoxicity at effective concentrations. The following structural requirements were suggested to enhance the inhibitory activity of phenylpropanoids on melanogenesis: (i) compounds with 4-acetoxy group exhibit higher activity than those with 4-hydroxy group; (ii) 3-methoxy group dose not affect the activity; (iii) acetylation of the 1′-hydroxy moiety enhances the activity; and (iv) phenylpropanoid dimers with the 7-O-9′-linked neolignan skeleton exhibited higher activity than those with the corresponding monomer. Their respective enantiomers [1′ (IC50 = 1.9 μM) and 2′ (4.5 μM)] and racemic mixtures [(±)-1 (2.2 μM) and (±)-2 (4.4 μM)] were found to exhibit melanogenesis inhibitory activities equivalent to those of the naturally occurring optical active compounds (1 and 2). Furthermore, the active compounds 1–3 inhibited tyrosinase, tyrosine-related protein (TRP)-1, and TRP-2 mRNA expressions, which could be the mechanism of melanogenesis inhibitory activity.

A hitherto unreported member of γ-alkylidenebutenolides in Melodorum fruticosum (Annonaceae), (4E)-6-benzoyloxy-7-hydroxy-2,4-heptadiene-4-olide, named as isofruticosinol (4) was isolated from the methanol extract of flowers, along with the known related butenolides, namely, the (4Z)-isomer (3) of 4, melodrinol (1), and its (4E)-isomer (2). To unambiguously determine the absolute configuration at the C-6 position in these butenolides, the first total syntheses of both enantiomers of 2–4 were achieved over 6–7 steps from commercially available D- or L-ribose (D- and L-5). Using the same protocol, both enantiomers of 1 were also synthesized. Based on chiral HPLC analysis of all synthetic compounds (S- and R-1–4), all naturally occurring butenolides were assigned as partial racemic mixtures with respect to the chiral center at C-6 (enantiomeric ratio, 6S/6R = ∼83/17). Furthermore, the melanogenesis inhibitory activities of S- and R-1–4 were evaluated, with all shown to be potent inhibitors with IC50 values i...

A methanol extract of everlasting flowers of Helichrysum arenarium L. Moench (Asteraceae) was found to inhibit the increase in blood glucose elevation in sucrose-loaded mice at 500 mg/kg p.o. The methanol extract also inhibited the enzymatic activity against dipeptidyl peptidase-IV (DPP-IV, IC50 = 41.2 μg/ml), but did not show intestinal α-glucosidase inhibitory activities. From the extract, three new dimeric dihydrochalcone glycosides, arenariumosides V–VII (2–4), were isolated, and the stereostructures were elucidated based on their spectroscopic properties and chemical evidence. Of the constituents, several flavonoid constituents, including 2–4, were isolated, and these isolated constituents were investigated for their DPP-IV inhibitory effects. Among them, chalconaringenin 2′-O-β-D-glucopyranoside (16, IC50 = 23.1 μM) and aureusidin 6-O-β-D-glucopyranoside (35, 24.3 μM) showed relatively strong inhibitory activities.

The antidiabetic effect of a hot water extract of stems of Salacia chinensis (SCE) was evaluated in vivo in KK-Ay mice, a typical type 2 diabetes mellitus mice model. Administration of CE-2 dietary feed containing 0.25 and/or 0.50% of SCE for three weeks to KK-Ay mice significantly suppressed the elevation of both blood glucose and HbA1c levels without significant changes in body weight or food intake. Glucose tolerance was improved by administration to KK-Ay mice for 27 days of AIN93M purified dietary feed containing 0.12% of SCE. No suppressive effect with respect to HbA1c level was observed when AIN93M/Glc dietary feed in which all digestible glucides were replaced with glucose was administered with SCE. Thus, α-glucosidase inhibitory activity approved as the mechanism of action of the antidiabetic effect of SCE by in vitro investigation was reconfirmed also in in vivo studies. Evaluation of the α-glucosidase inhibitory activity of the active constituents, salacinol (1), kotalanol (3), and neokotalanol (4), by employing human α-glucosidases revealed that these compounds inhibited them as potently (IC50 = 3.9–4.9 μM for maltase) as they inhibited rat small intestinal α-glucosidase. The principal sulfonium constituents (1–4) were highly stable in an artificial gastric juice. In addition, 1–4 were hardly absorbed from the intestine in an experiment using the in situ rat ligated intestinal loop model. The results indicate that these sulfoniums are promising leads for a new type of anti-diabetic agents.

Three new limonoids, andirolides W-Y (1-3), were isolated from the flower oil of Carapa guianasis AUBLET (Meliaceae). Their structures were elucidated on the basis of spectroscopic analyses using 1D and 2D NMR spectra and FABMS. Seven known limonoids: 7-deacetoxy-7-oxogedunin (4), 6α-acetoxygedunin (5), methylangolensate (6), 6α-hydroxygedunin (7), 6α-acetoxy-7α-deacetoxy-7α-hydroxygedunin (8), gedunin (9), and 7-deacetoxy-7-hydroxygedunin (10) from this flower oil were evaluated for the effects on the production of NO in LPS-activated mouse peritoneal macrophages.

In our continuing study of biologically active natural products from the fruit of Alpinia galanga (Zingiberaceae), we newly isolated three new labdane-type diterpenes, termed galangalditerpenes A–C (1–3), along with four known sesquiterpenes (4–7) and two diterpenes (8 and 9). The stereostructures of 1–3 were elucidated on the basis of their spectroscopic properties. The melanogenesis inhibitory activities in theophylline-stimulated murine B16 melanoma 4A5 cells of these isolates, including the new diterpenes (1–3, IC50 = 4.4, 8.6, and 4.6 μM, respectively), were found to be more than 6–87-fold higher than that of arbutin (174 μM), a commercially available positive control.

A methanol extract of mace, the aril of Myristica fragrans (Myristicaceae), was found to inhibit the release of β-hexosaminidase, a marker of antigen-IgE-stimulated degranulation in rat basophilic leukemia cells (RBL-2H3, IC50 = 45.7 μg/ml). From the extract, three new 8-O-4′ type neolignans, maceneolignans I–K (1–3), were isolated, and the stereostructures of 1–3 were elucidated based on spectroscopic and chemical evidence. Among the isolates, maceneolignans A (5), D (6), and H (8), (−)-(8R)-∆8′-4-hydroxy-3,3′,5′-trimethoxy-8-O-4′-neolignan (13), (−)-(8R)-∆8′-3,4,5,3′,5′-pentamethoxy-8-O-4′-neolignan (14), (−)-erythro-(7R,8S)-∆8′-7-acetoxy-3,4-methylenedioxy-3′,5′-dimethoxy-8-O-4′-neolignan (17), (+)-licarin A (20), nectandrin B (24), verrucosin (25), and malabaricone C (29) were investigated as possible degranulation inhibitors (IC50 = 20.7–63.7 μM). These inhibitory activities were more potent than those of the antiallergic agents tranilast (282 μM) and ketotifen fumalate (158 μM). Compounds 5, 25, and 29 also inhibited antigen-stimulated tumor necrosis factor-α production (IC50 = 39.5–51.2 μM), an important process in the late phase of type I allergic reactions.

Acylated phenylethanoid glycosides, echinacoside (1) and acteoside (2), principal constituents in stems of Cistanche tubulosa (Orobanchaceae), inhibited the increase in postprandial blood glucose levels in starch-loaded mice at doses of 250-500 mg/kg p.o. These compounds (1 and 2) also significantly improved glucose tolerance in starch-loaded mice after 2 weeks of continuous administration at doses of 125 and/or 250 mg/kg/day p.o. without producing significant changes in body weight or food intake. In addition, several constituents from C. tubulosa, including 1 (IC50 = 3.1 μM), 2 (1.2 μM), isoacteoside (3, 4.6 μM), 2'-acetylacteoside (4, 0.071 μM), tubulosides A (5, 8.8 μM) and B (9, 4.0 μM), syringalide A 3-O-α-L-rhamnopyranoside (10, 1.1 μM), campneoside I (13, 0.53 μM), and kankanoside J1 (14, 9.3 μM), demonstrated potent rat lens aldose reductase inhibitory activity. In particular, the potency of compound 4 was similar to that of epalrestat (0.072 μM), a clinical aldose reductase inhibitor.

A methanol extract of the flower buds of Chimonanthus praecox (L.) Link (Calycanthaceae) demonstrated inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells. From the extract, five dimeric pyrrolidinoindoline alkaloids and four sesquiterpenes were isolated, together with 16 known compounds. Among them, (-)-chimonanthine (1, IC50 = 0.93 μM), (-)-folicanthine (2, 1.4 μM), and (-)-calycanthidine (3, 1.8 μM) showed potent inhibitory effects without notable cytotoxicity at the effective concentrations. The most potent alkaloid (1) inhibited both tyrosinase and tyrosine-related protein-1 mRNA expressions, to which the melanogenesis inhibitory activity would be ascribable.

An acylated flavonol glycoside, helichrysoside, at a dose of 10 mg/kg/day per os for 14 days, improved the glucose tolerance in mice without affecting the food intake, visceral fat weight, liver weight, and other plasma parameters. In this study, using hepatoblastoma-derived HepG2 cells, helichrysoside, trans-tiliroside, and kaempferol 3-O-&beta, D-glucopyranoside enhanced glucose consumption from the medium, but their aglycones and p-coumaric acid did not show this activity. In addition, several acylated flavonol glycosides were synthesized to clarify the structural requirements for lipid metabolism using HepG2 cells. The results showed that helichrysoside and related analogs significantly inhibited triglyceride (TG) accumulation in these cells. The inhibition by helichrysoside was more potent than that by other acylated flavonol glycosides, related flavonol glycosides, and organic acids. As for the TG metabolism-promoting activity in high glucose-pretreated HepG2 cells, helichrysoside, related analogs, and their aglycones were found to significantly reduce the TG contents in HepG2 cells. However, the desacyl flavonol glycosides and organic acids derived from the acyl groups did not exhibit an inhibitory impact on the TG contents in HepG2 cells. These results suggest that the existence of the acyl moiety at the 6'' position in the D-glucopyranosyl part is essential for glucose and lipid metabolism-promoting activities.

A quantitative analytical method for five aporphine alkaloids, nuciferine (1), nornuciferine (2), N-methylasimilobine (3), asimilobine (4), and pronuciferine (5), and five benzylisoquinoline alkaloids, armepavine (6), norarmepavine (7), N-methylcoclaurine (8), coclaurine (9), and norjuziphine (10), identified as the constituents responsible for the melanogenesis inhibitory activity of the extracts of lotus flowers (the flower buds of Nelumbo nucifera), has been developed using liquid chromatography-mass spectrometry. The optimum conditions for separation and detection of these 10 alkaloids were achieved on a πNAP column, a reversed-phase column with naphthylethyl group-bonded silica packing material, with CH3CN–0.2% aqueous acetic acid as the mobile phase and using mass spectrometry equipped with a positive-mode electrospray ionization source. According to the protocol established, distributions of these 10 alkaloids in the petal, receptacle, and stamen parts, which were separated from the whole flower, were examined. As expected, excellent correlations were observed between the total alkaloid content and melanogenesis inhibitory activity. Among the active alkaloids, nornuciferine (2) was found to give a carbamate salt (2′′) via formation of an unstable carbamic acid (2′) by absorption of carbon dioxide from the air.

Using an in silico method, seven analogs bearing hydrophobic substituents (8a: Me, 8b: Et, 8c: n-Pent, 8d: n-Hept, 8e: n-Tridec, 8f: isoBu and 8g: neoPent) at the 3'-O-position in salacinol (1), a highly potent natural α-glucosidase inhibitor from Ayurvedic traditional medicine 'Salacia', were designed and synthesized. In order to verify the computational SAR assessments, their α-glucosidase inhibitory activities were evaluated in vitro. All analogs (8a-8g) exhibited an equal or considerably higher level of inhibitory activity against rat small intestinal α-glucosidases compared with the original sulfonate (1), and were as potent as or higher in potency than the clinically used anti-diabetics, voglibose, acarbose or miglitol. Their activities against human maltase exhibited good relationships to the results obtained with enzymes of rat origin. Among the designed compounds, the one with a 3'-O-neopentyl moiety (8g) was most potent, with an approximately ten fold increase in activity against human maltase compared to 1.

Three gedunin-type limonoids, gedunin (1), 6α-acetoxygedunin (2), and 7-deacetoxy-7-oxogedunin (3), which were isolated from the seed and flower oils of andiroba (Carapa guianensis Aublet, Meliaceae), exhibited hepatoprotective effects at doses of 25 mg/kg, p.o. against d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. To characterize the mechanisms of action of 1–3 and clarify the structural requirements for their hepatoprotective effects, 17 related limonoids (1–17) isolated from the seed and/or flower oils of C. guianensis were examined in in vitro studies assessing their effects on (i) d-GalN-induced cytotoxicity in primary cultured mouse hepatocytes, (ii) LPS-induced nitric oxide (NO) production in mouse peritoneal macrophages, and (iii) tumor necrosis factor-α (TNF-α)-induced cytotoxicity in L929 cells. The mechanisms of action of 1–3 are likely to involve the inhibition of LPS-induced macrophage activation and reduced sensitivity of hepatocytes to TNF-α; however, these compounds did not decrease the cytotoxicity caused by d-GalN. In addition, the structural requirements of limonoids (1–17) for inhibition of LPS-induced NO production in mouse peritoneal macrophages and TNF-α-induced cytotoxicity in L929 cells were evaluated.

A methanol extract of the bark of Shorea roxburghii (Dipterocarpaceae) was found to inhibit plasma glucose elevation in sucrose-loaded mice. From the extract, three new 3-ethyl-4-phenyl-3,4-dihydroisocoumarins, 1′ S -dihydrophayomphenol A 2 ( 1 ) and phayomphenols B 1 ( 2 ) and B 2 ( 3 ), were isolated together with 24 known compounds including 20 stilbenoids and oligostilbenoids. The structures of 1 – 3 were determined on the basis of their spectroscopic properties as well as of chemical evidences. Among the isolates, (−)-hopeaphenol ( 6 ), hemsleyanol D ( 8 ), (+)-α-viniferin ( 15 ), and (−)-balanocarpol ( 18 ) showed inhibitory activity against plasma glucose elevation in sucrose-loaded rats at doses of 100–200 mg/kg, p.o. To clarify the mode of action of the antihyperglycemic property, effects of these oligostilbenoids on gastric emptying in mice, those on glucose uptake in isolated intestinal tissues as well as inhibitory activities against rat intestinal α-glucosidase and rat lens aldose reductase were examined.

A novel sugar ester poacynose (1) was isolated from the flowers of Poacynum hendersonii together with 31 known compounds. The structure of 1 was established mainly on the basis of 1D and 2D NMR spectral data. Among the isolates, several constituents, such as kaempferol 3-O-sophoroside (5), picein (16), and 4-hydroxybenzoic acid 4-O-β-d-glucopyranoside (17) moderately promoted adipogenesis of 3T3-L1 cells. In addition, simultaneous quantitative analysis of eight flavonoid constituents from the flower and leaf parts of P. hendersonii was developed.

The methanolic extract from the leaves of Salacia chinensis collected in Thailand was found to show a protective effect on D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes. From the methanolic extract, eight new glycosides, named foliachinenosides E, F, G, H, and I, and foliasalaciosides J, K and L, were isolated together with 26 known constituents. The structures of new glycosides were determined on the basis of physicochemical and chemical evidence. In addition, the hepatoprotective effects of the isolated compounds on D-galactosamine-induced cytotoxicity were examined. Among them, lignans, eleutheroside E₂ and 7R,8S-dihydrodehydrodiconiferyl alcohol 4-O-β-D-glucopyranoside, were found to show the protective effects [inhibition (%) 41.4 ± 3.6 (p < 0.01), 45.5 ± 2.7 (p < 0.01) at 100 µM, respectively].

Four new acylated phenylethanoid oligoglycosides, kankanosides J(1) (1), J(2) (2), K(1) (3), and K(2) (4), were isolated from stems of Cistanche tubulosa (Orobanchaceae) together with isocampneoside I (5). Their structures were elucidated on the basis of chemical and physicochemical evidence. Among them, 3-5 were found to inhibit D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes.

Three iridoid glycosides, kankanosides L, M, and N, and two acyclic monoterpene glycosides, kankanosides O and P, were isolated from fresh stems of Cistanche tubulosa (Orobanchaceae) together with eight iridoid glycosides, five acyclic monoterpene glycosides, three phenylpropanoid glycosides, and four lignan glycosides. Their structures were elucidated on the basis of chemical and physicochemical evidence.

The 80% aqueous acetone extract from the flowers of Hedychium coronarium was found to show a protective effect on D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes. On the other hand, two new labdane-type diterpene glycosides, coronalactosides I (1) and II (2), and a new labdane-type trinorditerpene, coronadiene (3), were isolated together with 8 known compounds from the extracts, which were obtained with chloroform and 80% aqueous acetone from the flowers of H. coronarium. The structures of new constituents were elucidated on the basis of chemical and physicochemical evidence. In addition, the principal constituents, coronaririn C and 15-hydroxylabda-8(17),11,13-trien-16,15-olide, displayed hepatoprotective effects, which were stronger than that of the hepatoprotective agent, silybin.

A methanol extract from the rhizomes of Kaempferia parviflora Wall. ex Baker (Zingiberaceae) has shown inhibitory effects against melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells (IC50 = 9.6 μg/mL). Among 25 flavonoids and three acetophenones isolated previously (1-28), several constituents including 5-hydroxy-7,3',4'-trimethoxyflavone (6, IC50 = 8.8 μM), 5,7,3',4'-tetramethoxyflavone (7, 8.6 μM), 5,3'-dihydroxy-3,7,4'-trimethoxyflavone (12, 2.9 μM), and 5-hydroxy-3,7,3',4'-tetramethoxyflavone (13, 3.5 μM) showed inhibitory effects without notable cytotoxicity at the effective concentrations. Compounds 6, 7, 12, and 13 inhibited the expression of tyrosinase, tyrosine-related protein (TRP)-1, and TRP-2 mRNA, which could be the mechanism of their melanogenesis inhibitory activity. In addition, a quantitative analytical method for 12 methoxyflavones (1, 2, 4-11, 13, and 14) in the extract was developed using HPLC. The optimal condition for separation and detection of these constituents were achieved on an ODS column (3 μm particle size, 2.1 mm i.d. × 100 mm) with MeOH-0.1 % aqueous acetic acid solvent systems as the mobile phase, and the detection and quantitation limits of the method were estimated to be 0.08-0.66 ng and 0.22-2.00ng, respectively. The relative standard deviation values of intra- and interday precision were lower than 0.95 and 1.08 %, respectively, overall mean recoveries of all flavonoids were 97.9-102.9 %, and the correlation coefficients of all the calibration curves showed good linearity within the test ranges. For validation of the protocol, extracts of three kinds of the plant's rhizomes collected from different regions in Thailand (Leoi, Phetchabun, and Chiang Mai provinces) were evaluated. The results indicated that the assay was reproducible, precise, and could be readily utilized for the quality evaluation of the plant materials.

[structure: see text] Four new dolabellane-type diterpene alkaloids, nigellamines A(1) (1), A(2) (2), B(1) (3), and B(2) (4), were isolated from the seeds of Nigella sativa. Their absolute stereostructures were determined on the basis of chemical and physicochemical evidence. Nigellamines A(1) (1), B(1) (3), and B(2) (4) were found to show potent lipid metabolism promoting activity in primary cultured mouse hepatocytes, and their activities were equivalent to that of a PPAR-alpha agonist, clofibrate.

Six new sesquiterpenes, cyperusols A(1) (1), A(2) (2), B(1) (3), B(2) (4), C (5), and D (6), together with two monoterpenes and 13 sesquiterpenes were isolated from an Egyptian herbal medicine, the whole plants of Cyperus longus. The stereostructures of the new sesquiterpenes were determined on the basis of chemical and physicochemical evidence. In addition, the principal constituents were found to exhibit inhibitory activity on D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes.

New dolabellane-type diterpene alkaloids, nigellamines A(3), A(4), A(5), and C, were isolated from the methanolic extract of an Egyptian medicinal food, black cumin (the seeds of Nigella sativa). Their absolute configurations were determined on the basis of chemical and physicochemical evidence. Nigellamines were found to lower triglyceride levels in primary cultured mouse hepatocytes, and in particular, the activity of nigellamine A(5) was equivalent to that of the hypolipidemic agent, clofibrate.

The saponin fraction from the flower buds of Panax notoginseng exhibited protective effect on liver injury induced by d-galactosamine and lipopolysaccharide. From the saponin fraction with hepatoprotective effect, five new dammarane-type triterpene saponins, notoginsenosides-O (1), -P (2), -Q (3), -S (4), and -T (5), were isolated together with nine known protopanaxadiol oligoglycosides. The structures of the new saponins were elucidated on the basis of chemical and physicochemical evidence. The principal dammarane-type triterpene saponins from the roots and flower buds of Panax notoginseng were found to show potent hepatoprotective effects.

The methanolic extract of the roots of Saussurea lappa C larke , a Chinese medicinal herb Saussureae Radix, was found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-activated mouse peritoneal macrophages. Among the constituents from the methanolic extract, two sesquiterpene lactones (costunolide and dehydrocostus lactone) and two amino acid-sesquiterpene conjugates (saussureamines A and B) potently inhibited LPS-induced NO production (IC 50 =1.2–2.8 μM). Saussureamines A and B in addition to costunolide and dehydrocostus lactone did not inhibit iNOS enzyme activity, but they inhibited both induction of inducible NO synthase and activation of nuclear factor-κB in accordance with induction of heat shock protein 72.

The methanolic extract from the flowers of Tilia argentea (linden) was found to show a hepatoprotective effect against d -galactosamine ( d -GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. By bioassay-guided separation using in vitro d -GalN-induced damage to hepatocytes, five flavonol glycosides were isolated as the hepatoprotective constituents of the methanolic extract. Tiliroside, the principal flavonol glycoside, strongly inhibited serum GPT and GOT elevations at doses of 25–100 mg/kg (p.o.) in d -GalN/LPS-treated mice. By comparing the inhibitory effects of tiliroside with those of its components alone, the kaempferol 3-O-β- d -glucopyranoside moiety was found to be essential for the activity, and its effect was suggested to depend on the inhibition of tumor necrosis factor-α (TNF-α) production, decreased sensitivity of hepatocytes to TNF-α, and on the protection of hepatocytes against d -GalN.

Basic inhibitory mechanisms of costunolide and its active component, alpha-methylene-gamma-butyrolactone (alpha-MGBL), on blood-ethanol elevation were investigated in rats. In normal rats, blood-ethanol elevation (30 min later) induced by 20% (v/v) ethanol [5 ml/kg, per os (p.o.)] was strongly inhibited by pretreatment (30 min earlier) with costunolide and alpha-MGBL (50 mg/kg, p.o.). In pylorus-ligated rats given ethanol, blood-ethanol level (30 min) was barely elevated compared with that of normal rats. Neither costunolide nor alpha-MGBL affected the blood-ethanol elevation in pylorus-ligated rats or that induced by intraperitoneal and intraduodenal ethanol administration. Moreover, these compounds given orally induced no irreversible changes in alcohol dehydrogenase activity in rat liver. We continuously investigated the rate of gastric emptying in rats given various test meals. Costunolide and alpha-MGBL suppressed gastric emptying in rats given 20% ethanol and 1% sodium carboxymethyl cellulose. alpha-MGBL (50 mg/kg), but not costunolide, suppressed gastric emptying in 20% glucose-loaded rats. In an in vitro experiment, alpha-MGBL contracted the pylorus strip at a high concentration (20 mM), which was the estimated concentration in the stomach when the substance was given orally in vivo. These findings suggested that alpha-MGBL constricted the pylorus and caused delay of gastric emptying. Moreover, both compounds increased gastric fluid secretion with pepsin and mucus. In conclusion, the inhibitory effects of costunolide and alpha-MGBL on blood-ethanol elevation were based on inhibition of gastric emptying and dilution of the ethanol concentration by the increased gastric fluid.

The hepatoprotective effects of the hot water (SRHW) and methanolic (SRM) extracts from the roots and stems of Salacia reticulata were examined using an oxidative stress-induced liver injury model. Both SRHW and SRM extracts (400 mg/kg, p.o.) significantly suppressed the increase in glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities in carbon tetrachloride (CCl4)-treated mice. These extracts also inhibited CCl4-induced thiobarbituric acid-reactive substance (TBA-RS) formation, which indicates increased lipid peroxidation in the liver. A good correlation (r=0.945, p0.01) was observed between the amount of phenolic compounds in the extracts and their inhibitions of TBA-RS formation. The IC50 values of the extracts on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging were less than 10 microg/ml and the antioxidative activities of six phenolic compounds from the roots of S. reticulata were examined. Mangiferin, (-)-4'-O-methylepigallocatechin, and (-)-epicatechin-(4beta--8)-(-)-4'-O-methylepigallocatechin, which a principal phenolic compounds, showed potent scavenging activity on DPPH radicals and their concentrations required for 50% reduction of 40 microM DPPH radicals were 5.9, 10, and 3.2 microM, respectively. On the other hand, against the CCl4-induced serum GOT and GPT elevations and TBA-RS formation in mice, mangiferin and (-)-4'-O-methylepigallocatechin showed potent activity at a dose of 100 mg/kg, but (-)-epicatechin-(4beta--8)-(-)-4'-O-methylepigallocatechin did not. These results suggest that the antioxidative activity of the principal phenolic compounds is involved in the hepatoprotective activity of S. reticulata.

The 80% aqueous acetone extract of Zedoariae Rhizoma was found to show a protective effect against D-galactosamine (D-GalN)/lipopolysaccharide-induced acute liver injury in mice. To clarify the active compounds, the principal constituents were examined and 11 sesquiterpenes (furanodiene, curdione, neocurdrione, dehydrocurdione, germacrone, 13-hydroxygermacrone, curcumenol, isocurcumenol, aerugidiol, zedoarondiol, and curcumenone) and a diarylheptanoid (curcumin) were found to inhibit the increase in serum aspartate aminotransaminase and alanine aminotransaminase at a dose of 50 mg/kg p.o. in agreement with the previous in vitro studies, except for dehydrocurdione, aerugidiol, and zedoarondiol. In particular, curdione, neocurdione, curcumenol, and isocurcumenol potently inhibited the increase at a dose of 12.5 mg/kg p.o. Furthermore, the eight sesquiterpenes, furanodiene, curdione, neocurdione, dehydrocurdione, germacrone, 13-hydroxygermacrone, curcumenol, and curcumenone, also showed a protective effect against D-GalN/tumor necrosis factor-alpha-induced liver injury in mice at a dose of 50 mg/kg p.o.

A simultaneous quantitative analytical method for 13 stilbenoids including (−)-hopeaphenol (1), (+)-isohopeaphenol (2), hemsleyanol D (3), (−)-ampelopsin H (4), vaticanols A (5), E (6), and G (7), (+)-α-viniferin (8), pauciflorol A (9), hopeafuran (10), (−)-balanocarpol (11), (−)-ampelopsin A (12), and trans-resveratrol 10-C-β-d-glucopyranoside (13), and two dihydroisocoumarins, phayomphenols A1 (14) and A2 (15) in the extract of Shorea roxburghii (dipterocarpaceae) was developed. According to the established protocol, distributions of these 15 polyphenols (1–15) in the bark and wood parts of S. roxburghii and a related plant Cotylelobium melanoxylon were evaluated. In addition, the principal polyphenols (1, 2, 8, 13–15) exhibited hepatoprotective effects against d-galactosamine (d-galN)/lipopolysaccharide (LPS)-induced liver injury in mice at a dose of 100 or 200 mg/kg, p.o. To characterize the mechanisms of action, the isolates were examined in in vitro studies assessing their effects on (i) d-GalN-induced cytotoxicity in primary cultured mouse hepatocytes; (ii) LPS-induced nitric oxide (NO) production in mouse peritoneal macrophages; and (iii) tumor necrosis factor-α (TNF-α)-induced cytotoxicity in L929 cells. The mechanisms of action of these polyphenols (1, 2, and 8) were suggested to be dependent on the inhibition of LPS-induced macrophage activation and reduction of sensitivity of hepatocytes to TNF-α. However, none of the isolates reduced the cytotoxicity caused by d-GalN.

An 80% aqueous acetone extract of Cassia auriculata leaves was found to show a protective effect on D-galactosamine-induced cytotoxicity in primary cultured mouse hepatocytes. From the 80% aqueous acetone extract, we isolated a new benzocoumarin glycoside, avaraoside I (1), and a new flavanol dimer, avaraol I (2), together with 29 known constituents. The structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. In addition, three isolated compounds, pseudosemiglabrin (15, 0.0011%), (2S)-7,4'-dihydroxyflavan(4β→8)-catechin (22, 0.00075%), and (2S)-7,4'-dihydroxyflavan(4β→8)-gallocatechin (23, 0.092%), displayed hepatoprotective effects equivalent to that of the hepatoprotective agent, silybin.

New carabrane-type sesquiterpene lactones, curcumenolactones A, B, and C, were isolated from the 80% aqueous acetone extract of Zedoariae Rhizoma (Zingiberaceae), together with 41 sesquiterpenes and two diarylheptanoids. The absolute stereostructures of curcumenolactones A, B, and C were determined on the basis of physicochemical evidence, which included nuclear Overhauser effect (NOE) and circular dichroic (CD) spectroscopic analyses. Curcumenone, a principal carabrane-type sesquiterpene from Zedoariae Rhizoma, was found to show potent protective effect on D-galactosamine/lipopolysaccharide-induced acute liver injury in mice. In addition, curcumenolactones A and B and the other constituents showed protective effect on D-galactosamine-induced cytotoxicity in primary cultured rat hepatocytes.

A new ionone glucoside, pisumionoside, a phenethyl glycoside, sayaendoside, and two acylated flavonol oligoglycosides, pisumflavonosides I and II, were isolated from the young seedpods of garden peas, Pisum sativum L., together with quercetin and kaempferol 3-O-(6-O-trans-p-coumaroyl)-beta-D-glucopyranosyl (1--2)-beta-D-glucopyranosyl (1--2)-beta-D-glucopyranosides and quercetin and kaempferol 3-sophorotriosides. The structures of pisumionoside, sayaendoside, and pisumflavonosides I and II were determined on the basis of chemical and physicochemical evidence, respectively. Quercetin 3-sophorotrioside, a principle component, was found to show protective effects on liver injury induced by D-galactosamine and lipopolysaccharide and by carbon tetrachloride in mice.

A methanol extract from the tuberous roots of Potentilla anserina (Rosaceae) exhibited hepatoprotective effects against d-galactosamine (d-GalN)/lipopolysaccharide-induced liver injuries in mice. Six triterpene 28-O-monoglucopyranosyl esters, potentillanosides A-F, were isolated from the extract along with 32 known compounds, including 15 triterpenes. The structures of potentillanosides A-F were determined on the basis of spectroscopic properties and chemical evidence. Four ursane-type triterpene 28-O-monoglycosyl esters, potentillanoside A (IC50=46.7μM), 28-O-β-d-glucopyranosyl pomolic acid (IC50=9.5μM), rosamutin (IC50=35.5μM), and kaji-ichigoside F1 (IC50=14.1μM), inhibited d-GalN-induced cytotoxicity in primary cultured mouse hepatocytes. Among these four triterpenes, potentillanoside A, rosamutin, and kaji-ichigoside F1 exhibited in vivo hepatoprotective effects at doses of 50-100mg/kg, p.o. The mode of action was ascribable to the reduction in cytotoxicity caused by d-GalN.

With the aid of an in silico method, α-glucosidase inhibitors with far more potent activities than salacinol (1), a potent natural α-glucosidase inhibitor isolated from an Ayurvedic traditional medicine Salacia reticulata, have been developed.

A methanol extract of the flowers of Mammea siamensis (Calophyllaceae) was found to inhibit nitric oxide (NO) production in lipopolysaccharide-activated RAW264.7 cells. From the extract, two new geranylated coumarins, mammeasins A (1) and B (2), were isolated together with 17 known compounds including 15 coumarins. The structures of 1 and 2 were determined on the basis of their spectroscopic properties as well as of their chemical evidence. Among the isolates, 1 (IC(50) = 1.8 μM), 2 (6.4 μM), surangins B (3, 5.0 μM), C (4, 6.8 μM), and D (5, 6.2 μM), kayeassamins E (7, 6.1 μM), F (8, 6.0 μM), and G (9, 0.8 μM), mammea A/AD (11, 1.3 μM), and mammea E/BB (16, 7.9 μM) showed NO production inhibitory activity. Compounds 1, 9, and 11 were found to inhibit induction of inducible nitric oxide synthase (iNOS). With regard to mechanism of action of these active constituents (1, 9, and 11), suppression of STAT1 activation is suggested to be mainly involved in their suppression of iNOS induction.

Two new phenolic glycosides, kaempferiaosides A and B were isolated from the rhizomes of Kaempferia parviflora (Zingiberaceae) together with 24 known compounds. Their structures including absolute stereochemistry were elucidated on the basis of chemical and spectroscopic evidence. Among the isolates, 5,3'-dihydroxy-3,7,4'-trimethoxyflavone showed higher activity than silybin, a commercial hepatoprotective agent.

The methanol extract from the bark of Shorea roxburghii (Dipterocarpaceae, "Phayom" in Thai) was found to suppress plasma triglyceride elevation in olive oil-treated mice, and also to inhibit pancreatic lipase activity (IC(50) = 31.6 μg/ml). From the extract, two new 3-acetyl-4-phenyl-3,4-dihydroisocoumarins, phayomphenols A(1) (1) and A(2) (2) were isolated, together with 22 known compounds. The structures of 1 and 2 were elucidated on the basis of chemical and spectroscopic evidence, including X-ray crystallographic analysis. Among the isolates, several oligostilbenoids, including (-)-hopeaphenol (3) and (+)-isohopeaphenol (4), showed inhibitory effects on plasma triglyceride elevation at a dose of 200 mg/kg p.o. and pancreatic lipase inhibitory activity (IC(50) = 32.9 and 26.5 μM, respectively).

Five new triterpene saponins perennisosides VIII (1), IX (2), X (3), XI (4), and XII (5) were isolated from the MeOH-eluated fraction of the methanolic extract from the flowers of Bellis perennis. The MeOH-eluted fraction of the methanolic extract from the flowers of B. perennis was found to inhibit gastric emptying in olive oil-loaded mice at a dose of 200 mg/kg, per os (p.o.). The stereostructures of 1-5 were elucidated on the basis of chemical and spectroscopic evidence.

Two hitherto missing members of sulfonium salts family in Salacia genus plants as a new class of α-glucosidase inhibitors, neoponkoranol (7) and neosalaprinol (8), were isolated from the water extracts, and their structures were unambiguously identified. For further SAR studies on this series of sulfonium salts, several epimers of 7 and 8 were synthesized, and their inhibitory activities against rat small intestinal α-glucosidases were evaluated. Among them, 3′-epimer of 7 was found most potent in this class of molecules, and revealed as potent as currently used antidiabetics, voglibose and acarbose.

Four new acylated acyclic sesquiterpene oligoglycosides (1-4) were isolated from the pericarps of Sapindus rarak DC. together with four known acyclic sesquiterpene oligoglycosides, mukuroziosides Ia (5), Ib (6), IIa (7), and IIb (8). Their structures were elucidated on the basis of chemical and physicochemical evidence. These newly isolated compounds (1-4) were found to show inhibitory effects on tumor necrosis factor-alpha-induced cytotoxicity in L929 cells at concentrations of 30-100 microM.

The methanolic extract from fresh stems of Cistanche tubulosa (Orobanchaceae) was found to show hepatoprotective effects against D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. From the extract, three new phenylethanoid oligoglycosides, kankanosides H(1) (1), H(2) (2), and I (3), were isolated together with 16 phenylethanoid glycosides (4-19) and two acylated oligosugars (20, 21). The structures of 1-3 were determined on the basis of spectroscopic properties as well as of chemical evidence. Among the isolates, echinacoside (4, IC(50)=10.2 microM), acteoside (5, 4.6 microM), isoacteoside (6, 5.3 microM), 2'-acetylacteoside (8, 4.8 microM), and tubuloside A (10, 8.6 microM) inhibited D-GalN-induced death of hepatocytes. These five isolates, 4 (31.1 microM), 5 (17.8 microM), 6 (22.7 microM), 8 (25.7 microM), and 10 (23.2 microM), and cistantubuloside B(1) (11, 21.4 microM) also reduced TNF-alpha-induced cytotoxicity in L929 cells. Moreover, principal constituents (4-6) exhibited in vivo hepatoprotective effects at doses of 25-100mg/kg, po.

A practical HPLC-MS method for the quantitative determination of salacinol (1) and kotalanol (2), potent alpha-glucosidase inhibitors from Salacia species (Hippocrateaceae) as a specific remedy for diabetes in Ayurvedic system, was developed. The optimum conditions of separation and detection of these two constituents were achieved on a Asahipak NH2P-50 column (5 mcirom particle size, 2.0 mm i.d. x 150 mm) with a CH(3)CN-H(2)O mobile phase, associated with MS using electrospray ionization source. The overall recoveries of 1 (85.8-112.6%) and 2 (99.7-106.1%), and relative standard deviation values of intra- and inter-day precision were lower than 6.8 and 8.5%, respectively. The detection (S/N=3) and quantitation limits (S/N=10) were established to be 0.015 and 0.050 ng for 1, and 0.030 and 0.10 ng for 2, respectively. The correlation coefficients of all the calibration curves showed good linearity within test ranges. The extraction process was also optimized as 2 h immersion in water under reflux. The method was applied to evaluate extracts of three kinds of Salacia species, i.e. S. reticulata, S. oblonga, and S. chinensis, and those of four different parts, i.e. roots, stems, leaves and fruits of the same material, revealing that the extract from the roots of S. reticulata had the highest contents of these compounds. The results indicated that the assay was reproducible and precise and could be readily utilized for the evaluation of Salacia species.

The 80% aqueous acetone extract from the fruit of Piper chaba (Piperaceae) was found to have hepatoprotective effects on D-galactosamine (D-GalN)/lipopolysaccharide-induced liver injury in mice. From the ethyl acetate-soluble fraction, three new amides, piperchabamides E, G, and H, 33 amides, and four aromatic constituents were isolated. Among the isolates, several amide constituents inhibited D-GalN/tumor necrosis factor-alpha (TNF-alpha)-induced death of hepatocytes, and the following structural requirements were suggested: (i) the amide moiety is essential for potent activity; and (ii) the 1,9-decadiene structure between the benzene ring and the amide moiety tended to enhance the activity. Moreover, a principal constituent, piperine, exhibited strong in vivo hepatoprotective effects at doses of 5 and 10 mg/kg, po and its mode of action was suggested to depend on the reduced sensitivity of hepatocytes to TNF-alpha.