New biofunctional effects of oleanane-type triterpene saponins.

In the current review, we describe the novel biofunctional effects of oleanane-type triterpene saponins, including elatosides, momordins, senegasaponins, camelliasaponins, and escins, obtained from Aralia elata (bark, root cortex, young shoot), Kochia scoparia (fruit), Polygala senega var. latifolia (roots), Camellia japonica (seeds), and Aesculus hippocastanum (seeds), considering the following biofunctional activities: (1) inhibitory effects on elevated levels of blood alcohol and glucose in alcohol and glucose-loaded rats, respectively, (2) inhibitory effects on gastric emptying in rats and mice, (3) accelerative effects on gastrointestinal transit in mice, and (4) protective effects against gastric mucosal lesions in rats. In addition, we describe (5) suppressive effects of the extract and chakasaponins from Camellia sinensis (flower buds) on obesity based on inhibition of food intake in mice. The active saponins were classified into the following three types: (1) olean-12-en-28-oic acid 3-O-monodesmoside, (2) olean-12-ene 3,28-O-acylated bisdesmoside, and (3) acylated polyhydroxyolean-12-ene 3-O-monodesmoside. Furthermore, common modes of action, such as involvements of capsaicin-sensitive nerves, endogenous NO and PGs, and possibly sympathetic nerves, as well as common structural requirements, were observed. Based on our findings, a common mechanism of action might mediate the pharmacological effects of active saponins. It should be noted that the gastrointestinal tract is an important action site of saponins, and the role of the saponins in the gastrointestinal tract should be carefully considered.

Hepatoprotective Principles from the Rhizomes of Picrorhiza kurroa.

A methanol extract of rhizomes of Picrorhiza kurroa Royle ex Benth. (Plantaginaceae) showed hepatoprotective effects against D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. We had previously isolated 46 compounds, including several types of iridoid glycosides, phenylethanoid glycosides, and aromatics, etc., from the extract. Among them, picroside II, androsin, and 4-hydroxy-3-methoxyacetophenone exhibited active hepatoprotective effects at doses of 50-100 mg/kg, per os (p.o.) To characterize the mechanisms of action of these isolates and to clarify the structural requirements of phenylethanoid glycosides for their hepatoprotective effects, their effects were assessed in in vitro studies on (i) D-GalN-induced cytotoxicity in mouse primary 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. These isolates decreased the cytotoxicity caused by D-GalN without inhibiting LPS-induced macrophage activation and also reduced the sensitivity of hepatocytes to TNF-α. In addition, the structural requirements of phenylethanoids for the protective effects of D-GalN-induced cytotoxicity in mouse primary hepatocytes were evaluated.

Ursane-type triterpene oligoglycosides with anti-hepatosteatosis and anti-hyperlipidemic activity from the leaves of Ilex paraguariensis A. St.-Hil.

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.

Evaluation of in vitro transdermal permeation, mass spectrometric imaging, and in vivo analgesic effects of pregabalin using a pluronic lecithin organogel formulation in mice.

In clinical practice, pregabalin is orally administered for neuropathic pain, but causes severe central nervous system side effects, such as dizziness, which results in dose limitation or discontinuation. To reduce the central side effects of pregabalin, we developed four pregabalin preparations for transdermal application: 0.4% aqueous solution, pluronic lecithin organogel (PLO gel), hydrophilic cream, and lipophilic cream. Transdermal permeabilities of pregabalin among the four formulations were compared in vitro using hairless mouse skin. The longitudinal distribution of pregabalin within the skin was analyzed using mass spectrometric (MS) imaging. Furthermore, the in vivo analgesic effects of the formulations were evaluated using the von Frey filament test in a mouse model of diabetic neuropathy (DN). The PLO gel showed the highest permeability of pregabalin, followed by the aqueous solution, and no permeation was observed in the two cream formulations. The MS imaging analysis showed that pregabalin was distributed up to the dermis in the PLO gel 1 h after application, while the aqueous solution was distributed near the epidermis. A significant analgesic effect (p < .05) was observed 1.5 h after PLO gel application in the DN model mice, but the aqueous solution had no effect. This study indicated for the first time that pregabalin penetrated beyond the skin epidermis up to the dermis, from the PLO gel formulation, and that the application of this formulation exhibited an in vivo analgesic effect in the mouse model of DN.

A review of antidiabetic active thiosugar sulfoniums, salacinol and neokotalanol, from plants of the genus Salacia.

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.

Dose-Dependent Suppression of Postprandial Hyperglycemia and Improvement of Blood Glucose Parameters by Salacia chinensis Extract: Two Randomized, Double-Blind, Placebo-Controlled Studies.

The number of diabetes mellitus and borderline diabetes cases is increasing and poses a serious problem worldwide. Plants of the genus Salacia are known to have α-glucosidase inhibitory activity and to lower postprandial hyperglycemia. Two randomized, double-blind, placebo-controlled clinical trials were conducted to evaluate the efficacy of Salacia chinensis extract. Study 1 was a single-dose crossover study of 150, 300, or 600 mg of Salacia extract or placebo to determine the dose dependency of the effect on postprandial hyperglycemia. The duration of the washout period between each experimental day was a minimum of 6 days. Study 2 was a 12-week, multiple-dose, parallel-group study to evaluate the effects of 600 mg/day of Salacia extract on blood glucose parameters. In Study 1, Salacia induced significant dose-dependent suppression of postprandial blood glucose, insulin, and their incremental area under the curve values. The dose of 600 mg appeared to have the most significant effect. In Study 2, Salacia significantly improved several blood glucose-related parameters, such as hemoglobin A1c, and glucose tolerance after glucose challenge. These results suggest that S. chinensis extract may have beneficial effects in patients with diabetes.

Correction to: Inhibition of melanin production by anthracenone dimer glycosides isolated from Cassia auriculate seeds.

The article Inhibition of melanin production by anthracenone dimer glycosides isolated from Cassia auriculata seeds, written by Weicheng Wang, Yi Zhang, Souichi Nakashima, Seikou Nakamura, Tao Wang, Masayuki Yoshikawa and Hisashi Matsuda.

Collagen synthesis-promoting and collagenase inhibitory activities of constituents isolated from the rhizomes of Picrorhiza kurroa Royle ex Benth.

Three new acylated phenylethanoid glycosides, kurroaosides A (14), B (15), and C (16), and a new acylated cucurbitane-type triterpene glycoside, kurroaoside D (17), were isolated from a methanol extract of the rhizomes of Picrorhiza kurroa Royle ex Benth. (Plantaginaceae) along with 29 known isolates including 10 acylated phenylethanoid glycosides (18-27), three cucurbitane-type triterpene glycosides (32-34), and a nortriterpene glycoside (35). The structures of these new compounds (14-17), including their stereochemistry, were determined based on chemical and physicochemical evidence derived from NMR and MS analysis. Among the isolates, acylated iridoid glycosides, picrosides I (8), II (9), III (10), and IV (11) and 6-feruloylcatalpol (12), phenylethanoid glycosides (14-16), triterpene glycosides, cucurbitacin B 2-O-ß-D-glucopyranoside (32) and 25-acetoxy-2-ß-D-glucopyranosyloxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5-en-22-one (35), and an acetophenone glycoside, picein (36), significantly promoted collagen synthesis at 10-30 µM, with no cytotoxicity being observed at the effective concentrations. Furthermore, acylated phenylethanoid glycosides, calceolarioside A (19, IC50 = 69.2 µM), plantamajoside (20, 51.8 µM), isoplantamajoside (21, 76.8 µM), and scroside E (23, 65.5 µM), exhibited collagenase inhibitory activity equivalent to that of positive agents caffeic acid (75.6 µM) and epigallocatechin 3-O-gallate (75.4 µM).

Acylated iridoid glycosides with hyaluronidase inhibitory activity from the rhizomes of Picrorhiza kurroa Royle ex Benth.

Seven new acylated iridoid glycosides, picrorhizaosides A-G (1-7), were isolated from the methanol extract of the rhizomes of Picrorhiza kurroa Royle ex Benth. (Plantaginaceae), in addition to six known iridoid glycosides (8-13). The structures of these new iridoids, including their stereochemistry, were determined based on chemical and physicochemical evidence derived from NMR and MS analysis. Of the isolates, picrorhizaosides D (4, IC50 = 43.4 µM) and E (5, 35.8 µM); picrosides I (8, 60.7 µM), II (9, 22.3 µM), and IV (11, 59.2 µM); and minecoside (13, 57.2 µM), exhibited a similar or stronger hyaluronidase inhibitory activity than those of the antiallergic medicines disodium cromoglycate (64.8 µM), ketotifen fumarate (76.5 µM), and tranilast (227 µM).

Salacia chinensis stem extract and its thiosugar sulfonium constituent, neokotalanol, improves HbA1c levels in ob/ob mice.

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.

Inhibition of melanin production by anthracenone dimer glycosides isolated from Cassia auriculata seeds.

The methanol extract of Cassia auriculata seeds was found to inhibit melanogenesis in B16 melanoma 4A5 cells under conditions of theophylline stimulation. Two new phlegmacin-type anthracenone dimer glycosides, auriculataosides A and B, were isolated from the active methanol fraction, and their inhibitory effects were observed in the concentration range of 0.03 to 0.3 µM. Inhibition of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 protein expression was observed, suggesting that the inhibition of these factors is part of the mechanism of action underlying melanogenesis inhibition.

Cytotoxicity of sesquiterpene alkaloids from Nuphar plants toward sensitive and drug-resistant cell lines.

Multi-drug resistance (MDR) is a critical problem in cancer chemotherapy. MDR causes the overexpression of ATP-binding cassette (ABC) transporters and mutations in tumor suppressor genes and oncogenes. To tackle this issue, in this study, we focused on Nuphar plants, which have been traditionally used as food. Sesquiterpene alkaloids (1-3) were isolated from N. japonicum and dimeric sesquiterpene thioalkaloids (4-10) were isolated from N. pumilum. P-glycoprotein-overexpressing CEM/ADR5000 cells were cross-resistant to 6,6'-dihydroxythiobinupharidine (10). Using in silico molecular docking, we calculated the binding energies and simulated the interactions of these compounds with the corresponding amino acid residues at the binding site of P-gp. In addition, we investigated the cytotoxicity of these compounds towards cell lines overexpressing other ABC transporters (BCRP, ABCB5), cell lines with a knocked out tumor suppressor gene TP53 or cell lines overexpressing a deletion-activated EGFR oncogene. These cell lines were sensitive or only minimally cross-resistant to these compounds compared with their corresponding wild-type cell lines.

Triterpenes with Anti-invasive Activity from Sclerotia of Inonotus obliquus.

The methanolic extract [inhibition (%): 61.2±3.8 (p<0.01) at 100 µg/mL] and its EtOAc-soluble fraction [inhibition (%): 82.5±1.7 (p<0.01) at 100 µg/mL1 from the sclerotia of Inonotus obliquus collected in Japan significantly inhibited invasion of human fibrosarcoma HT1080 cells through matrigel-coated filters. In addition, the methanolic extract significantly inhibited lung tumor formation fifteen days after injection of BI6F10 melanoma cells in mice [inhibition (%) 66.1 ± 12:8 (p < 0.05) at 500 mg/kg/d, p.o.]. Lanostane-type triterpenes were isolated as the common principal constituents from Japanese and Russian . obliquus. Furtheremore, we examine the inhibitory effects of the constituents on the invasion of HT 1080 cells. Interestingly, 3ß-hydroxylanosta-8,24-dien- 21-al [inhibition (%) 37.9 ± 3.0 (p < 0.05) at 30 µM] significantly inhibited the invasion, and no cytotoxic effect at 30 µM was observed.

The Antiproliferative Effect of Chakasaponins I and II, Floratheasaponin A, and Epigallocatechin 3-O-Gallate Isolated from Camellia sinensis on Human Digestive Tract Carcinoma Cell Lines.

Acylated oleanane-type triterpene saponins, namely chakasaponins I (1) and II (2), floratheasaponin A (3), and their analogs, together with catechins-including (-)-epigallocatechin 3-O-gallate (4), flavonoids, and caffeine-have been isolated as characteristic functional constituents from the extracts of "tea flower", the flower buds of Camellia sinensis (Theaceae), which have common components with that of the leaf part. These isolates exhibited antiproliferative activities against human digestive tract carcinoma HSC-2, HSC-4, MKN-45, and Caco-2 cells. The antiproliferative activities of the saponins (1-3, IC50 = 4.4-14.1, 6.2-18.2, 4.5-17.3, and 19.3-40.6 µM, respectively) were more potent than those of catechins, flavonoids, and caffeine. To characterize the mechanisms of action of principal saponin constituents 1-3, a flow cytometric analysis using annexin-V/7-aminoactinomycin D (7-AAD) double staining in HSC-2 cells was performed. The percentage of apoptotic cells increased in a concentration-dependent manner. DNA fragmentation and caspase-3/7 activation were also detected after 48 h. These results suggested that antiproliferative activities of 1-3 induce apoptotic cell death via activation of caspase-3/7.

New biofunctional effects of the flower buds of Camellia sinensis and its bioactive acylated oleanane-type triterpene oligoglycosides.

We review the biofunctional effects of the flower buds of Camellia sinensis and C. sinensis var. assamica, such as antihyperlipidemic, antihyperglycemic, antiobesity, and gastroprotective effects in vivo, and antiallergic, pancreatic lipase inhibitory, and amyloid ß (Aß) aggregation inhibitory activities in vitro. Although the biofunctional effects of tea leaves have been extensively studied, less attention has been given to those of the flowers and seeds of the tea plant. Our studies focused on the saponin constituents of the extracts of the flower buds of C. sinensis cultivated in Japan and China, and C. sinensis var. assamica cultivated in India, and we review their beneficial biofunctions for health promotion.

Quantitative Determination of Alkaloids in Lotus Flower (Flower Buds of Nelumbo nucifera) and Their Melanogenesis Inhibitory Activity.

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.

Acylated oleanane-type triterpene saponins from the flowers of Bellis perennis show anti-proliferative activities against human digestive tract carcinoma cell lines.

Seven oleanane-type triterpene saponin bisdesmosides, perennisaponins N-T (1-7), were newly isolated from a methanol extract of daisy, the flowers of Bellis perennis L. (Asteraceae). The structures were determined based on chemical and physicochemical data and confirmed using previously isolated related compounds as references. The isolates, including 13 previously reported perennisaponins A-M (8-20), exhibited anti-proliferative activities against human digestive tract carcinoma HSC-2, HSC-4, and MKN-45 cells. Among them, perennisaponin O (2, IC50 = 11.2, 14.3, and 6.9 µM, respectively) showed relatively strong activities. The mechanism of action of 2 against HSC-2 was found to involve apoptotic cell death.

Chemical structures of constituents from the whole plant of Bacopa monniera.

Two new dammarane-type triterpene oligoglycosides, bacomosaponins A and B, and three new phenylethanoid glycosides, bacomosides A, B1, and B2, were isolated from the whole plant of Bacopa monniera Wettst. The chemical structures of the new constituents were characterized on the basis of chemical and physicochemical evidence. In the present study, bacomosaponins A and B with acyl groups were obtained from the whole plant of B. monniera. This is the first report of acylated dammarane-type triterpene oligoglycosides isolated from B. monniera. In addition, dammarane-type triterpene saponins significantly inhibited the aggregation of 42-mer amyloid ß-protein.

Degranulation Inhibitors from Medicinal Plants in Antigen-Stimulated Rat Basophilic Leukemia (RBL-2H3) Cells.

Mast cells and basophils play important roles in both immediate- and late-phase reactions of type 1 allergy. Histamine, which is released from mast cells and basophils stimulated by an antigen or degranulation inducers, is usually determined as a degranulation marker in experiments on immediate allergic reactions in vitro. ß-Hexosaminidase is also stored in secretory granules of the cells and is released concomitantly with histamine when the cells are immunologically activated, and recently this enzyme activity in the medium has been used as a marker of the degranulation. In this paper, we review our studies on the search for degranulation inhibitors, such as flavonoids, stilbenes, and curcuminoids, from medicinal plants using rat basophilic leukemia (RBL-2H3) cells.