Preparation, characterization, and anticancer effects of an inclusion complex of coixol with ß-cyclodextrin polymers.

CONTEXT: Coix [Coix lacryma-jobi L. var. mayuen (Roman.) Stapf (Poaceae)], a crop of medicinal and edible significance, contains coixol, which has demonstrated anticancer properties. However, the limited solubility of coixol restricts its potential therapeutic applications. OBJECTIVE: This study prepared a water-soluble coixol-ß-cyclodextrin polymer (CDP) inclusion compound and evaluated its anticancer effect. MATERIALS AND METHODS: The coixol-CDP compound was synthesized through a solvent-stirring and freeze-drying technique. Its coixol content was quantified using HPLC, and its stability was tested under various conditions. The anticancer effects of the coixol-CDP compound (4.129, 8.259, 16.518, and 33.035 mg/L for 24, 48, and 72 h) on the proliferation of non-small cell lung cancer (NSCLC) A549 cells were evaluated using an MTT assay; cell morphology was examined by Hoechst nuclear staining; apoptosis and cell cycle was detected by flow cytometry; and the expression of apoptosis-related proteins was assessed by Western blots. RESULTS: The water-soluble coixol-CDP inclusion compound was successfully prepared with an inclusion ratio of 86.6% and an inclusion yield rate of 84.1%. The coixol content of the compound was 5.63% and the compound remained stable under various conditions. Compared to coixol alone, all 24, 48, and 72 h administrations with the coixol-CDP compound exhibited lower IC50 values (33.93 ± 2.28, 16.80 ± 1.46, and 6.93 ± 0.83 mg/L) in A549 cells; the compound also showed stronger regulatory effects on apoptosis-related proteins. DISCUSSION AND CONCLUSIONS: These findings offer a new perspective for the potential clinical application of Coix in NSCLC therapy and its future research.

Coixol ameliorates Toxoplasma gondii infection-induced lung injury by interfering with T. gondii HSP70/TLR4/NF-κB signaling pathway.

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that causes pulmonary toxoplasmosis, although its pathogenesis is incompletely understood. There is no cure for toxoplasmosis. Coixol, a plant polyphenol extracted from coix seeds, has a variety of biological activities. However, the effects of coixol on T. gondii infection have not been clarified. In this study, we infected a mouse macrophage cell line (RAW 264.7) and BALB/c mice with the T. gondii RH strain to establish infection models in vitro and in vivo, respectively, to explore protective effects and potential mechanisms of coixol on lung injury caused by T. gondii infection. Anti-T. gondii effects and underlying anti-inflammatory mechanisms of coixol were investigated by real-time quantitative PCR, molecular docking, localized surface plasmon resonance, co-immunoprecipitation, enzyme-linked immunosorbent assay, western blotting, and immunofluorescence microscopy. The results show that coixol inhibits T. gondii loads and T. gondii-derived heat shock protein 70 (T.g.HSP70) expression. Moreover, coixol reduced inflammatory cell recruitment and infiltration, and ameliorated pathological lung injury induced by T. gondii infection. Coixol can directly bind T.g.HSP70 or Toll-like receptor 4 (TLR4) to disrupt their interaction. Coixol prevented overexpression of inducible nitric oxide synthase, tumor necrosis factor-α, and high mobility group box 1 by inhibiting activation of the TLR4/nuclear factor (NF)-κB signaling pathway, consistent with effects of the TLR4 inhibitor CLI-095. These results indicate that coixol improves T. gondii infection-induced lung injury by interfering with T.g.HSP70-mediated TLR4/NF-κB signaling. Altogether, these findings suggest that coixol is a promising effective lead compound for the treatment of toxoplasmosis.

Coixol Suppresses NF-κB, MAPK Pathways and NLRP3 Inflammasome Activation in Lipopolysaccharide-Induced RAW 264.7 Cells.

Coixol, a plant polyphenol extracted from coix (Coix lachryma-jobi L.var.ma-yuen Stapf), has not been investigated for its anti-inflammatory effect. In this study, using a lipopolysaccharide (LPS)-induced macrophage cell model, we observed that coixol can effectively reduce the expression of interleukin (IL)-1ß, IL-6, IL-18, tumor necrosis factor (TNF)-α, nitric oxide (NO), inducible nitric oxide synthases (iNOS), and cyclooxygenase (COX)-2, but had no effect on the expression of the anti-inflammatory mediator IL-10. Furthermore, we found that coixol inhibits mitogen-activated protein kinases (MAPKs), nuclear transcription factor κ B (NF-κB) pathways, and NOD-like receptor protein (NLRP) 3 inflammasome activation. In conclusion, the present study demonstrates that coixol exerts certain anti-inflammatory effects by inhibiting the expression of pro-inflammatory mediators in vitro. The mechanism of this effect was in part related to its ability to inhibit the activation of NF-κB, MAPKs pathways, and NLRP3 inflammasome.

Analysis of Phenolic Compounds in Coix-seed Reactive Derivatives(CRD) by Using Liquid Chromatography/Mass Spectrometry(LC-MS) / 日本補完代替医療学会誌

To clarify the functions of Coix-seed Reactive Derivatives(CRD), we analyzed 13 kinds of phenolic compounds and coixol(an alkaloid) in CRD powder by using Liquid Chromatography/Mass Spectrometry(LC-MS).The following are the results: p -Coumaric acid 275.0 (μg/g), p -Hydroxybenzaldehyde 50.2 (μg/g), Caffeic acid 36.9 (μg/g), Protocatechuic acid 31.9 (μg/g), Ferulic acid 27.1 (μg/g), Quercetin 16.2 (μg/g), Syringaldehyde 12.1 (μg/g), Chlorogenic acid 11.3 (μg/g), Naringenin 11.1 (μg/g), Vanillin 9.2 (μg/g), trans -Coniferylaldehyde 9.1 (μg/g), Tricin 0.7 (μg/g), Luteolin 0.5 (μg/g) and Coixol(6-Methoxy-2-benzoxazolinone) 5.5 (μg/g). CRD is composed of many substances other than these phenoic compounds. Therefore, further studies are needed to understand the various clinical features associated with CRD intake.

Coixol amplifies glucose-stimulated insulin secretion via cAMP mediated signaling pathway.

Recently, we reported the role of coixol (6-methoxy-2(3H)-benzoxazolone), an alkaloid from Scoparia dulcis, in glucose-dependent insulin secretion; however, its insulin secretory mechanism(s) remained unknown. Here, we explored the insulinotropic mechanism(s) of coixol in vitro and in vivo. Mice islets were batch incubated, perifused with coixol in the presence of agonists/antagonists, and insulin secretion was measured by ELISA. Intracellular cAMP levels were measured using enzyme immunoassay. K+- and Ca2+-currents were recorded in MIN6 cells using whole-cell patch-clamp technique. The in vivo glucose tolerance and the insulinogenic index were evaluated in diabetic rats treated with coixol at 25 and 50 mg/kg, respectively. Coixol, unlike sulfonylurea, enhanced insulin secretion in batch incubated and perifused islets at high glucose, with no effect at basal glucose concentrations. Coixol showed no pronounced effect on the inward rectifying K+- and Ca2+-currents in whole-cell patch recordings. Moreover, coixol-induced insulin secretion was further amplified in the depolarized islets. Coixol showed an additive effect with forskolin (10 µM)-induced cAMP level, and in insulin secretion; however, no additive effect was observed with isobutylmethylxanthine (IBMX, 100 µM)-induced cAMP level, nor in insulin secretion. The PKA inhibitor H-89 (50 µM), and Epac2 inhibitor MAY0132 (50 µM) significantly inhibited the coixol-induced insulin secretion (P < 0.01). Furthermore, insulin secretory kinetics revealed that coixol potentiates insulin secretion in both early and late phases of insulin secretion. In diabetic animals, coixol showed significant improvement in glucose tolerance and on fasting blood glucose levels. These data suggest that coixol amplifies glucose-stimulated insulin secretion by cAMP-mediated signaling pathways.

Endoplasmic reticulum stress induced by an ethanol extract of Coicis semen in Chang liver cells.

BACKGROUND: It is well known that endoplasmic reticulum (ER) stress plays a huge role in development of metabolic diseases. Specially, ER stress-induced cellular dysfunction has a significant involvement in the pathogenesis of human chronic disorders. This study was designed to study to assess whether an ethanol extract of Coicis Semen (CSE) and coixol induces the ER stress in Chang liver cells. METHODS: Coicis Semen was mixed with 95% ethanol at a ratio of 1:10 (w/v) and freeze dried. Chang liver cells were seeded to 96-well plates and treated with or without CSE (100, 200, 300, 500, or 1000 µg/mL) or coixol (100, 200, 300, 500, 750, or 1000 µg/mL). cell viability was analyzed with MTT assay. Effects of CSE and coixol on expression of the genes for ER stress markers were determined with qRT-PCR and the expression of the protein levels of ER stress markers were determined with western blotting. RESULTS: The concentration causing 50% inhibition (IC50) for CSE and coixol was 250 and 350 µg/mL, respectively. The CSE and coixol increased the gene expression of BiP and CHOP in a dose-dependent manner. Furthermore, CSE and coixol dose-dependently increased the the expression of XBP1. CONCLUSIONS: CSE or coixol may have cytotoxic effect to Chang liver cells and, may induce ER stress and stimulate the UPR via activation of the PERK and IRE1 pathways in normal liver cells.

Impact of germination on nutritional and physicochemical properties of adlay seed (Coixlachryma-jobi L.).

Adlay has garnered a great deal of research attentions in recent years as a highly nutritious food material and herbal medicine. This study characterized the changes of nutritional and physicochemical properties of adlay seeds during a 60-h germination. The results showed that the 60-h germination brought about a 3.4-fold increase in γ-aminobutyric acid (GABA) and 3.6-fold increase in coixol compared to ungerminated adlay seeds, while the triolein content slightly decreased. Some high molecular proteins were hydrolyzed into smaller proteins, peptides and amino acids after germination. Scanning electron micrographs (SEM) showed that the germination process destroyed the continuous matrix structure of adlay flour and created pits and holes on the surface of some starch granules. Germination resulted to changes in the pasting and gelatinization properties of adlay flour. The results of present study suggest that germination efficiently enhances the nutritional compounds while altering the physicochemical characteristics of adlay seeds.

Melanogenesis inhibitors from Coix lacryma-jobi seeds in B16-F10 melanoma cells.

An adenine derivative, 9-ß-D-glucopyranosyl adenine, reported for the first time from a natural source, in addition to nine known compounds were isolated from the seeds of Coix lacryma-jobi. Their structures were elucidated based on extensive spectroscopic and chemical studies. The isolated compounds and the ethanol extract have been assayed for melanin inhibition using B16-F10 melanoma cell line. The results of our study suggested the potential use of Coix lacryma-jobi seeds as a skin whitening agent and reveal the seeds to be a rich source of important phytochemicals with melanogenesis inhibitory activity. Among the isolated compounds, coixol (2) and 2-O-ß-glucopyranosyl-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (8) exhibited potent melanogenesis inhibitory activity with no obvious melanocytotoxicity. The rest of the compounds showed weak to moderate activity.

Sensitive quantification of coixol, a potent insulin secretagogue, in Scoparia dulcis extract using high-performance liquid chromatography combined with tandem mass spectrometry and UV detection.

Diabetes is a major global health problem which requires new studies for its prevention and control. Scoparia dulcis, a herbal product, is widely used for treatment of diabetes. Recent studies demonstrate coixol as a potent and nontoxic insulin secretagog from S. dulcis. This study focuses on developing two quantitative methods of coixol in S. dulcis methanol-based extracts. Quantification of coixol was performed using high-performance liquid chromatography-tandem mass spectrometry (method 1) and high-performance liquid chromatography-ultraviolet detection (method 2) with limits of detection of 0.26 and 11.6 pg/µL, respectively, and limits of quantification of 0.78 and 35.5 pg/µL, respectively. S. dulcis is rich in coixol content with values of 255.5 ± 2.1 mg/kg (method 1) and 220.4 ± 2.9 mg/kg (method 2). Excellent linearity with determination coefficients >0.999 was achieved for calibration curves from 10 to 7500 ng/mL (method 1) and from 175 to 7500 ng/mL (method 2). Good accuracy (bias < -8.6%) and precision (RSD < 8.5%) were obtained for both methods. Thus, they can be employed to analyze coixol in plant extracts and herbal formulations.

Determination of effective contents triolein and coixol in Coix lacrymajobi var. mayuen from different origins / 中草药

Objective: To compare the differences of contents of effective components triolein and coixol in Coix lacryma-jobi var. mayuen from different origins. Methods: The Discover C18 column was used for the detection of triolein with mobile phase of acetonitrile-dichloromethanol (66:34). The flow rate of mobile phase was 0.7 mL/min. The column temperature was controlled at 30℃. The ELSD was used. The tube temperature was 45℃, and the flow rate of nitrogen was 1.5 L/min. The Agilent Zorbax SB-C18 column was used for the detection of the coixol with the mobile phase of acetonitrile-water (25:75), a flow rate of 1 mL/min, a temperature of 25℃ and a wavelength of 232 nm. Results: The triolein content of Zhejiang province was the highest, at 1.04%, and the lowest was in Hainan Province, which was 0.53%; The highest coixol content was in Shanghai, reaching 3.469 7 mg/g, but the lowest content was only 0.619 3 mg/g in Anhui province. Conclusion: The contents of triolein and coixol in adlay from eight Provinces are significantly different, the contents of triolein and coixol in adlay from Zhejiang and Fujian Provinces were relatively high, and medicinal material quality is better.

Potent Insulin Secretagogue from Scoparia dulcis Linn of Nepalese Origin.

Ethno-botanical inspired isolation from plant Scoparia dulcis Linn. (Sweet Broomweed) yielded six compounds, coixol (1), glutinol (2), glutinone (3), friedelin (4), betulinic acid (5), and tetratriacontan-1-ol (6). There structures were identified using mass and 1D- and 2D-NMR spectroscopy techniques. Compounds 1-6 were evaluated for their insulin secretory activity on isolated mice islets and MIN-6 pancreatic ß-cell line, and compounds 1 and 2 were found to be potent and mildly active, respectively. Compound 1 was further evaluated for insulin secretory activity on MIN-6 cells. Compound 1 was subjected to in vitro cytotoxicity assay against MIN-6, 3T3 cell lines, and islet cells, and in vivo acute toxicity test in mice that was found to be non-toxic. The insulin secretory activity of compounds 1 and 2 supported the ethno-botanic uses of S. dulcis as an anti-diabetic agent.

A Study on the Useful Components of Adlay (<i>Coix lachryma-jobi</i> L. <i>var.ma-yuen</i> Stapf) / 日本補完代替医療学会誌

Hot water extract of adlay (<i>Coix lachryma-jobi</i> L. <i>var. ma-yuen</i> Stapf) seed, commonly called Yokuinin, has been used as herbal medicine for treating verruca vulgaris, et al. Although there have been a number of studies on the usefulness of Yokuinin, the pharmacological assessment of its husk, pellicle, and astringent skin remains unclear. In this line, we evaluated the effect of methanol extract from all parts of adlay grain (seed, husk, pellicle, astringent skin) on cancer cells and identified its useful chemical components. Results revealed that a fraction of the extract have weak growth-suppressing activity on human cervical cancer cell line (HeLa cell). In particular, 5,7-dihydroxychromone and coixol were isolated and identified from the active fraction. This indicates the possible cancer chemopreventive efficacy of methanol extract from adlay. Moreover, further tests are needed to determine the role of 5,7-dihydroxychromone.<br>