Chemical constituents of the Ajuga multiflora bunge and their protective effects on dexamethasone-induced muscle atrophy in C2C12 myotubes.

Ajuga multiflora Bunge is a perennial ornamental herb and has been used for the treatment of fever in Korean folk medicine. In the course of searching for protective agents associated with the potential of A. multiflora against dexamethsone (DEX)-induced muscle atrophy, a new phytoecdysteroid, 29-hydroxyprecyasterone (1), together with four known compounds (2-5), were isolated from A. multiflora. The structures of the compounds were determined by spectroscopic analyses, including 1D-, 2D-NMR and HR-MS interpretation. To elucidate the effects of obtained compounds on DEX-induced muscle atrophy, the myotubes diameter, myosin heavy chain (MyHC) positive area, and fusion index were evaluated by immunofluorescence staining. Overall, each compound treatment effectively prevented the atrophic myotubes through an increase of MyHC-positive myotubes and the number of nuclei. Particularly, the measurement of myotube diameter showed that compounds 1 and 5 treatment significantly alleviated the myotube thickness.

Bioassay-Guided Isolation of Two Eudesmane Sesquiterpenes from Lindera strychnifolia Using Centrifugal Partition Chromatography.

In this study, a centrifugal partition chromatography (CPC) separation was applied to identify antioxidant-responsive element (ARE) induction molecules from the crude extract of Lindera strychnifolia roots. CPC was operated with a two-phase solvent system composed of n-hexane-methanol-water (10:8.5:1.5, v/v/v) in dual mode (descending to ascending), which provided a high recovery rate (>95.5%) with high resolution. Then, ARE induction activity of obtained CPC fractions was examined in ARE-transfected HepG2 cells according to the weight ratios of the obtained fractions. The fraction exhibiting ARE-inducing activity was further purified by preparative HPLC that led to isolation of two eudesmane type sesquiterpenes as active compounds. The chemical structures were elucidated as linderolide U (1) and a new sesquiterpene named as linderolide V (2) by spectroscopic data. Further bioactivity test demonstrated that compounds 1 and 2 enhanced ARE activity by 22.4-fold and 7.6-fold, respectively, at 100 µM concentration while 5 µM of sulforaphane induced ARE activity 24.8-fold compared to the control.

Bioassay-Guided Separation of Centipeda minima Using Comprehensive Linear Gradient Centrifugal Partition Chromatography.

A comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) was developed for the bioassay-guided isolation of natural compounds. The gradient solvent system consisted of three different ternary biphasic solvents types: n-hexane-acetonitrile-water (10:2:8, v/v), ethyl acetate-acetonitrile-water (10:2:8, v/v), and water-saturated n-butanol-acetonitrile-water (10:2:8, v/v). The lower phase of the n-hexane-acetonitrile-water (10:2:8, v/v) was used as the stationary phase, while its upper phase, as well as ethyl acetate-acetonitrile-water (10:2:8), and water-saturated n-butanol-acetonitrile-water (10:2:8, v/v) were pumped to generate a linear gradient elution, increasing the mobile phase polarity. We used the gradient CPC to identify antioxidant response elements (AREs), inducing compounds from Centipeda minima, using an ARE-luciferase assay in HepG2 cells, which led to the purification of the active molecules 3-methoxyquercetin and brevilin A. The developed CPC solvent systems allow the separation and isolation of compounds with a wide polarity range, allowing active molecule identification in the complex crude extract of natural products.

Silymarin-Laden PVP-Nanocontainers Prepared Via the Electrospraying Technique for Improved Aqueous Solubility and Dissolution Rate

Abstract The aim of the present research was to develop a silymarin-laden PVP-nanocontainer providing ameliorated aqueous solubility and dissolution of the drug. Several silymarin-laden formulations were formed with varying quantities of PVP and SDS via the solvent evaporation method using the electrospraying technique. The influence of the hydrophilic carriers on solubility and dissolution was explored. The solid-state characterization was carried out by particle-size analysis, PXRD, DSC, FTIR and SEM. All of the formulations demonstrated better solubility and dissolution than did silymarin plain powder. Both the SDS and PVP had positive effects on solubility and dissolution of silymarin in the aqueous media. An increased solubility was attained as the drug/PVP ratio was 1/4; however, further increase in PVP did not provide significant improvement. In particular, a nanocontainer formulation prepared with silymarin, PVP and SDS (1/4/0.5, w/w/w) exhibited the best solubility (26432.76 ± 1749.00 μg/mL) and an excellent dissolution (~92 % in 20 min) than did silymarin plain powder. Also, it demonstrated similar dissolution profiles compared to a commercial product; therefore, might be bioequivalent to the commercial product (f 1 = 3 and f 2 = 69). Moreover, cumulative undersize distribution values as represented by X10, X50 and X90 were 201 ± 21.01 nm, 488 ± 36.05 nm and 392 ± 48.10 nm, respectively. The drug existed in the amorphous state in the PVP-nanocontainers with no strong chemical bonding with other excipients. Thus, this formulation might be used for more effective administration of silymarin via the oral route.

Application of Centrifugal Partition Chromatography for Bioactivity-Guided Purification of Antioxidant-Response-Element-Inducing Constituents from Atractylodis Rhizoma Alba.

Activity-guided separation of antioxidant response element (ARE)-inducing constituents from the rhizomes of Atractylodis Rhizoma Alba was performed by the combination of centrifugal partition chromatography (CPC) and an ARE luciferase reporter assay. From 3 g of the active n-hexane fraction, one polyacetylene, (6E,12E)-tetradeca-6,12-dien-8,10-diyne-1,3-diyl diacetate (47.3 mg), and two sesquiterpenes, atractylenolide I (40.9 mg), and selina-4(14),7(11)-dien-8-one (6.0 mg) were successfully isolated by CPC with n-hexane⁻ethyl acetate⁻methanol⁻water (8:2:8:2, v/v). The chemical structures of the isolated compounds were determined by ¹H- and 13C-NMR and ESI-MS. Among the isolated compounds, (6E,12E)-tetradeca-6,12-diene-8,10-diyne-1,3-diol diacetate and selina-4(14),7(11)-dien-8-one increased ARE activity 32.9-fold and 16.6-fold, respectively, without significant cytotoxicity, when 5 µM sulforaphane enhanced ARE activity 27.1-fold. However, atractylenolide I did not increase ARE activity at 100 µM, and showed cytotoxicity at concentrations over 10 µM.

A new diacetylated flavonol triglycoside from the aerial parts of Actinidia polygama.

Phytochemical investigation of a methanolic extract of aerial parts Actinidia polygama Miq. led to the isolation of one new diacetylated flavonol triglycoside, kaempferol 3-O-[α-L-rhamnopyranosyl(1→3)-(4-O-acetyl)-O-α-L-rhamnopyranosyl(1→6)-(2-Oacetyl)-O-ß-D-galactopyranoside] (1) along with 12 known compounds (2-13). The chemical structures were determined using their spectroscopic data including 1D and 2D NMR. To the best of our knowledge, this is the first time that compounds 2, 4, 6, 7, 8, 9, 12 and 13 are isolated from this plant. All purified compounds were tested for free radical scavenging effect using DPPH and ABTS assays. Our results showed that compounds 4, 6, 7 and 13 have potential antioxidative effect for scavenging both DPPH· and ABTS·+ radicals that are comparable with those of ascorbic acid used as positive control, whereas compounds 1 and 2, which are di- and mono- acetylated flavonol triglycoside respectively, were not found to be potent scavengers of free radicals.

Cyclocurcumin, an Antivasoconstrictive Constituent of Curcuma longa (Turmeric).

Despite the increasing attention on the therapeutic potential of Curcuma longa (turmeric), the biological activities of curcuminoids other than curcumin are not well understood. Here, we investigated antivasoconstrictive activities of C. longa extract and its ingredients using freshly isolated rat aortic rings. C. longa extract significantly suppressed agonist-stimulated vasoconstriction, and cyclocurcumin was found to be the most potent (IC50 against phenylephrine-induced vasoconstriction: 14.9 ± 1.0 µM) among the 10 tested ingredients including four curcuminoids. Cyclocurcumin significantly inhibited contraction of vascular smooth muscle, which was mediated by the suppression of myosin-light-chain phosphorylation and calcium influx via the L-type calcium channel. The inhibitory effect of cyclocurcumin was observed to be reversible and without cytotoxicity. Taken together, we demonstrated that cyclocurcumin, a bioactive ingredient in C. longa, may have a therapeutic potential as a novel antivasoconstrictive natural product.

Potent Anti-inflammatory and Analgesic Actions of the Chloroform Extract of Dendropanax morbifera Mediated by the Nrf2/HO-1 Pathway.

Dendropanax morbifera LEVEILLE (DP) has been used in traditional Korean medicines to treat a variety of inflammatory diseases. Although the in vitro anti-inflammatory potential of this plant is understood, its in vivo efficacy and underlying molecular mechanism of anti-inflammatory effects are largely unknown. We elucidated the anti-inflammatory and analgesic activities and the underlying molecular mechanisms of DP using in vitro and in vivo models. Lipopolysaccharide (LPS)-stimulated murine macrophages were used to analyze the in vitro anti-inflammatory potential of DP extract and to elucidate the underlying mechanisms. In vivo animal models of phorbol 12-myristate 13-acetate (TPA)-induced ear edema and acetic acid-induced writhing response tests were used to analyze the in vivo anti-inflammatory effects and anti-nociceptive effects of DP extract, respectively. Methanolic extract of DP (DPME) significantly inhibited the release of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-activated macrophages. Among the five sub-fractions, the chloroform fraction (DP-C) showed the most potent suppressive effects against pro-inflammatory mediators and cytokines in LPS-stimulated macrophages. These effects were attributed to inhibition of nuclear factor-κB (NF-κB) nuclear translocation and c-Jun N terminal kinase (JNK) 1/2 phosphorylation and to activation of NF-E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling. DP-C exhibited strong protective in vivo effects in TPA-induced ear edema mouse model and acetic acid-induced writhing response test. Our data suggest that DP-C has potent anti-inflammatory and analgesic activities and may be a promising treatment against a variety of inflammatory diseases.

Preparative separation of sesamin and sesamolin from defatted sesame meal via centrifugal partition chromatography with consecutive sample injection.

A preparative separation method using consecutive sample injection centrifugal partition chromatography (CPC) was developed to obtain sesamin and sesamolin from defatted sesame meal extracts. A two-phase solvent system consisting of n-hexane-ethyl acetate-methanol-water (8:2:8:2, v/v) was applied in reversed-phase mode (descending mode). Preliminary experiments with an SCPC-100 (column volume: 100mL) were performed to select the appropriate two-phase solvent system and sample injection times; these parameters were then used with an SCPC-1000 (column volume: 1000mL) in a 10-fold scale-up preparative run. A sample containing 3g of crude extract was consecutively injected four times onto the SCPC-1000, which yielded 328mg of sesamin and 168mg of sesamolin. These compounds were analyzed by high-performance liquid chromatography and determined to have purities of 95.6% and 93.9%, respectively. Sesamin and sesamolin (30µM) increased antioxidant response element (ARE) luciferase activity 2.6-fold and 1.9-fold, respectively.

Heme oxygenase 1-mediated novel anti-inflammatory activities of Salvia plebeia and its active components.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia plebeia R. Br. (SP) has been widely used as a traditional folk medicine for the treatment of infectious diseases and pain. An anti-inflammatory potential of SP has remains largely unknown. AIM OF THE STUDY: We tried to elucidate the principle mechanism and the active ingredients underlying the anti-inflammatory activities of SP. MATERIALS AND METHODS: We investigated the protective activities of SP methanolic extract (SPME) and seven representative ingredients against inflammation. Quantitative analysis using HPLC-DAD-ESI/MS was conducted to determine the relative amounts of these seven active ingredients in SPME. Both in vitro murine macrophages and in vivo mouse models were employed to elucidate SP- and active ingredient-mediated anti-inflammatory effects. RESULTS: SPME significantly reduced inflammatory processes both in vivo in a TPA-induced ear edema model and in vitro in lipopolysaccharide (LPS)-activated macrophages. SPME decreased the release of nitric oxide (NO) and prostaglandin E2 (PGE2) and expression of inducible nitric oxide synthase (iNOS). Seven active components (luteoloside (C1), nepitrin (C2), homoplantagenin (C3), luteolin (C4), nepetin (C5), hispidulin (C6), and eupatorin (C7)) of SPME were analyzed and their relative concentrations were determined, demonstrating that C2, C3, C5 and C6 were present in higher amounts than were C1, C4, and C7. These major compounds inhibited NO and PGE2 production, and iNOS and COX-II protein expression through heme oxygenase-1 (HO-1) induction via activation of nuclear factor erythroid 2-related factor2 (Nrf2). CONCLUSION: Our data demonstrate that SPME possesses potent in vitro and in vivo anti-inflammatory activities. Nepetin and hispidulin, and their glycosides are the major active compounds in SPME, and their effects are mediated by Nrf2/HO-1 signaling. Taken together, we propose that SPME and its active ingredients may serve as novel therapeutic candidates for diseases associated with excessive inflammation.