Junji Akaki

Quantitative determination of potent α-glucosidase inhibitors, salacinol and kotalanol, in Salacia species using liquid chromatography–mass spectrometry

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.

Structural analysis of monocyte activation constituents in cultured mycelia of Cordyceps sinensis.

It has been reported that mycelia of the Cordyceps sinensis (CS) can function as an immunostimulant. However, the active constituents of the mycelia are not well known. In this study, we investigated which components of the mycelia of CS induce monocyte activation and then structurally analyzed the active components. Assay of the effect of crude-(CS-P), soluble-(CS-Ps) and insoluble-(CS-Pp), polysaccharides extracted from the mycelia of CS, on macrophage production of TNF-alpha, indicated that CS-Pp enhanced TNF-alpha production to the highest extent. Furthermore, Structural analyses demonstrated that CS-Pp is a 1,3-beta-D-glucan contained some 1,6-branched chains and the mean particle diameter is 1.5 mum.

Structural analysis of glycogen-like polysaccharides having macrophage-activating activity in extracts of Lentinula edodes mycelia

An extract of Lentinula edodes mycelia (L.E.M.) was obtained with hot water after culturing in medium comprising bagasse and rice bran. It has been reported that L.E.M. exhibits physiological effects such as anticancer activity and immunoregulatory activity, and that such effects involve macrophage activation. We examined the active components in L.E.M. by fractionation using an anion exchange carrier and gel chromatography, and determined whether they stimulate production of the cytokine tumor necrosis factor-α (TNF-α) in RAW264.7 cells. We obtained 2 polysaccharide fractions, IA-a and IA-b, which potently stimulated cytokine production. We also confirmed that IA-a and IA-b significantly stimulate phagocytosis in RAW264.7 cells. To determine the structures of these fractions, we performed sugar component analysis, methylation analysis, FT-IR, 13C-NMR analysis, and molecular weight analysis. The results showed that the major component of IA-a is a 1,4-α-glucan exhibiting 1,6-branching and having a molecular weight of approximately 250,000. This branching structure suggests that IA-a has a glycogen-like structure. In addition, the major component of IA-b appears to be an arabinoxylan-like polysaccharide, mainly consisting of arabinose and xylose, with a molecular weight of approximately 8,500.

Salacinol and Related Analogs: New Leads for Type 2 Diabetes Therapeutic Candidates from the Thai Traditional Natural Medicine Salacia chinensis

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.

Effects of Salacia chinensis extract on reproductive outcome in rats

Salaciachinensis is a traditional South and Southeast Asian herb medicine and has been reported to have an antidiabetic function via α-glucosidases inhibitory activity. In this study, the effects of S. chinensis extract (SCE) on reproductive functions of F0 males and females and the effects on survival and growth of F1 offspring were examined using Sprague-Dawley rats. SCE was administered at dose levels of 0, 500, 1000 and 2000 mg/kg/day orally to groups consisting of 25 males and 25 females. Males were dosed once a day in the morning from 8 weeks before mating, throughout the mating period and until the day before necropsy and females were dosed once a day in the morning for 2 weeks before mating and through the mating, gestation and lactation periods (until day 20 of lactation). In all SCE treatment groups, no toxic signs were noted on reproductive outcome such as estrous cycle of F0 females or any parameters for reproductive function or survival, growth, sensory reflex or function development of F1 pups. Therefore, we concluded that SCE has no effects on the reproductive outcome even at a remarkably high dosage level, 2000 mg/kg/day, in Sprague-Dawley rats.

Evaluation of Salacia Species as Anti‐diabetic Natural Resources Based on Quantitative Analysis of Eight Sulphonium Constituents: A New Class of α‐Glucosidase Inhibitors

INTRODUCTION Stems and roots of Salacia genus plants have been used in Ayurveda as a specific remedy for early stage diabetes. Previous investigations identified four sulphonium sulphates, that is, salacinol (1), kotalanol (3), ponkoranol (5) and salaprinol (7), as the compounds responsible for the anti-diabetic activity. Their desulphonates (2, 4, 6 and 8) were also isolated as active constituents. Two separate quantitative analytical protocols, that is, for 1 and 3 and for 2 and 4, have been developed recently. OBJECTIVE To: validate the two analytical protocols with respect to all eight sulphoniums; evaluate the quality of a variety of Salacia samples collected in different geographical regions, that is, Thailand, Sri Lanka and India; and determine their distribution in each part of the plant, that is, stems/roots, leaves and fruits. METHODS Analyses of four sulphonium sulphates in 32 Salacia extracts were carried out on an Asahipak NH2P-50 column, and those of the corresponding desulphonates were conducted on an Inertsil ODS-3 column. RESULTS Neokotalanol (4) was the major constituent in Salacia samples from Thailand, whereas 1 was the primary constituent in extracts of the stems/roots of plants from Sri Lanka and India. These sulphoniums were only present in trace amounts in leaves and fruits of the plants. CONCLUSION Two analytical protocols were successfully applied to analyse 32 Salacia samples, and revealed that sulphoniums (1-8) had characteristic distributions due to the plant part and/or due to geographical region.

Beneficial Effects of the Genus Aloe on Wound Healing, Cell Proliferation, and Differentiation of Epidermal Keratinocytes

Aloe has been used as a folk medicine because it has several important therapeutic properties. These include wound and burn healing, and Aloe is now used in a variety of commercially available topical medications for wound healing and skin care. However, its effects on epidermal keratinocytes remain largely unclear. Our data indicated that both Aloe vera gel (AVG) and Cape aloe extract (CAE) significantly improved wound healing in human primary epidermal keratinocytes (HPEKs) and a human skin equivalent model. In addition, flow cytometry analysis revealed that cell surface expressions of β1-, α6-, β4-integrin, and E-cadherin increased in HPEKs treated with AVG and CAE. These increases may contribute to cell migration and wound healing. Treatment with Aloe also resulted in significant changes in cell-cycle progression and in increases in cell number. Aloe increased gene expression of differentiation markers in HPEKs, suggesting roles for AVG and CAE in the improvement of keratinocyte function. Furthermore, human skin epidermal equivalents developed from HPEKs with medium containing Aloe were thicker than control equivalents, indicating the effectiveness of Aloe on enhancing epidermal development. Based on these results, both AVG and CAE have benefits in wound healing and in treatment of rough skin.