Antiglycation Effects of Adlay Seed and Its Active Polyphenol Compounds: An In Vitro Study.

This study aimed to evaluate the antiglycation effects of adlay on protein glycation using in vitro glycation assays. Adlay seed was divided into the following four parts: the hull (AH), testa (AT), bran (AB), and polished adlay (PA). A solvent extraction technique and column chromatography were utilized to investigate the active fractions and components of adlay. Based on a BSA-glucose assay, the ethanolic extracts of AT (ATE) and AB (ABE) revealed a greater capacity to inhibit protein glycation. ATE was further consecutively partitioned into four solvent fractions with n-hexane, ethyl acetate (ATE-Ea), 1-butanol (ATE-BuOH), and water. ATE-BuOH and -Ea show marked inhibition of glucose-mediated glycation. Medium-high polarity subfractions eluted from ATE-BuOH below 50% methanol with Diaion HP-20, ATE-BuOH-c to -f, exhibited superior antiglycation activity, with a maximum inhibitory percentage of 88%. Two phenolic compounds, chlorogenic acid and ferulic acid, identified in ATE-BuOH with HPLC, exhibited potent inhibition of the individual stage of protein glycation and its subsequent crosslinking, as evaluated by the BSA-glucose assay, BS-methylglyoxal (MGO) assay, and G.K. peptide-ribose assay. In conclusion, this study demonstrated the antiglycation properties of ATE in vitro that suggest a beneficial effect in targeting hyperglycemia-mediated protein modification.

Adlay Seed (Coix lacryma-jobi L. var. Ma-yuen Stapf.) Ethanolic Extract Fractions and Subfractions Induce Cell Cycle Arrest and Apoptosis in Human Breast and Cervical Cancer Cell Lines.

The antitumor effects of Coix lacryma-jobi L. var. ma-yuen Stapf. (adlay seed) ethanolic extract have been increasingly shown. This study aimed to investigate the beneficial effects of both the fractions and subfractions of adlay seed ethanolic extract on the human breast (MCF-7) and cervical (HeLa) cancer cell lines, as well as exploring their possible mechanisms of action. The ethanolic extracts were obtained from different parts of adlay seed, including AHE (adlay hull extract), ATE (adlay testa extract), ABE (adlay bran extract) and PAE (polished adlay extract). The results of a 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl- tetrazolium bromide (MTT) assay showed that AHE-Ea and ATE-Ea showed significant growth inhibitory effects in a dose-dependent manner. The results also showed that the AHE-Ea-K, AHE-Ea-L, ATE-Ea-E and ATE-Ea-F subfractions inhibited cell proliferation, induced cell cycle arrest in the G0/G1 phase and decreased CDK4/Cyclin D1 protein expression. Finally, the extract activated caspase-3 activity and PARP protein expression, which induced MCF-7 and HeLa cell apoptosis. We then used liquid chromatography-mass spectrometry (LC/MS) to identify the potential active components., Quercetin showed an anticancer capacity. In conclusion, the AHE-Ea-K, AHE-Ea-L, ATE-Ea-E and ATE-Ea-F subfractions showed antitumor effects through the inhibition of MCF-7 and HeLa cell line viability, as well as inducing apoptosis and cell cycle arrest.

Adlay Testa (Coix lachryma-jobi L. var. Ma-yuen Stapf.) Ethanolic Extract and Its Active Components Exert Anti-Proliferative Effects on Endometrial Cancer Cells via Cell Cycle Arrest.

Endometrial cancer is the most common malignant tumors of gynecologic neoplasms in Western society. In recent years, the incidence of endometrial cancer has increased, and it has become the third most common female gynecological cancer (after ovarian and cervical cancer) in Taiwan. Adlay (Coix lachryma-jobi L. var. Ma-yuen Stapf.) has been demonstrated to have bioactive polyphenols, flavonoids, phytosterols, and essential nutrients for health benefits, including anticancer effects in humans. However, little is known about the effect of adlay seeds on endometrial cancer. Our study aimed to investigate the potential growth inhibitory effects of several adlay seed fractions, including ethyl acetate (ATE-EA) and its bioactive constituents, separately on endometrial cancer cells-HEC-1A (phosphatase and tensin homolog-positive) and RL95-2 (phosphatase and tensin homolog-negative)-and identify related active ingredients. In addition, the potential active fractions and the phytochemical compounds were elucidated. The results demonstrate superior activity of ATE-EA with significant in vitro cell proliferation inhibitory capacity, particularly its C.D.E.F-subfraction. Moreover, HPLC- and GC/FID-based quantification of ATE-EA subfractions showed that phenolic compounds (caffeic acid, protocatechuic acid, and p-hydroxybenzaldehyde), flavonoids, steroids, and fatty acid compounds exert anti-proliferative effects in the cell model. Finally, it was shown that cell growth and cell cycle arrest most significantly occurred in the in G1 or G2/M phase under ATE-EA treatment. Collectively, our results demonstrate an antiproliferative effect of ATE-EA on endometrial cancer cells that suggest a positive health outcome for women from consumption of these compounds.

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) Hull Extract and Active Compounds Inhibit Proliferation of Primary Human Leiomyoma Cells and Protect against Sexual Hormone-Induced Mice Smooth Muscle Hyperproliferation.

Uterine leiomyomas, also known as fibroids, are benign neoplasms of the uterus and have a high incidence rate in women of reproductive age. Hysterectomy or myomectomy is the initial treatment, but fibroids will recur if the patient is still exposed to similar risk factors. Therefore, developing new therapeutic strategies are urgently necessary. In this study, the anti-proliferation effects of each fraction of adlay seeds were evaluated in uterine leiomyomas, and we identified the potential phytochemical compounds. We found that the ethyl acetate fraction of adlay hull (AHE-ea) appeared to be highly efficient in the anti-proliferation of rat uterine leiomyoma ELT3 cells and primary human uterine leiomyoma (hUL) cells. The proliferation of primary human normal uterine smooth muscle (UtSMC) and normal uterine myometrial (hUM) cells were also suppressed by AHE-ea. Two phytosterols, stigmasterol and ß-sitosterol, were identified from AHE-ea fraction. Mice treated with AHE-ea and stigmasterol alone demonstrated reduced diethylstilbestrol/medroxyprogesterone 17-acetate (DES/MPA)-induced uterine myometrial hyperplasia, which is the critical step for the development of leiomyoma. Taken together, our results suggest that the AHE-ea fraction could be considered as a natural plant-based medicine in the prevention or treatment of uterine leiomyoma growth.

Dehulled Adlay (Coix lachryma-jobi L.) ameliorates hepatic gluconeogenesis and steatosis in streptozotocin/high-fat diet-induced diabetic rats.

Adlay (Coix lachryma-jobi L.) is a traditional Chinese herbal medicine with various biological activities. We investigated the anti-diabetic effects of different parts of adlay seeds, including polished adlay (PA), adlay bran (AB) and dehulled adlay (DA) in a streptozotocin (STZ)/high fat diet (HFD) diabetic rat model (DM). DM rats supplemented with or without PA (43%), AB (3%), or DA (46%) diet for 8 weeks. The plasma glucose and insulin levels and the insulin resistance index (HOMA-IR) were increased in DM group; among the three adlay diets, DA has the best effects attenuating all of these alterations in DM rats. Both AB and DA alleviated diabetes-impaired glucose tolerance. The increased hepatic phosphoenolpyruvate carboxykinase protein expression in DM group was improved by all of the three adlay diets. The increased ratio of glucose-6-phosphatase to glucokinase in DM group was suppressed by DA supplementation, further suggesting DA diet is most effective among the three diets. Both AB and DA diets had beneficial effects against hepatic steatosis, with better effects observed in DA group. These results suggest that the DA diet, composed of both polished adlay and adlay bran, possesses the best potential to improve glucose homeostasis, at least in part, by alleviating hepatic glucose metabolism and steatosis.

Investigating the Impact of Extruded Dehulled Adlay with Specific In Vitro Digestion Properties on Blood Lipids in Subjects with Mild to Moderate Dyslipidemia.

Dyslipidemia, a major risk factor for cardiovascular diseases (CVDs), is modifiable by diet and lifestyle changes. A large population with mild to moderate dyslipidemia is at risk of developing CVDs, and early initiation of preventive measures can avert advancing into severe medical conditions. Studies suggest increasing slowly digestible starch (SDS) in diets can help lower blood lipids. We processed dehulled adlay, a cereal rich in bioactive compounds, such as polyphenols and phytosterols, into an instant meal by extrusion and milling and then assessed its starch composition and in vitro digestibility. The dehulled adlay was found to consist of 32% SDS and resistant starch combined. Then, eligible subjects with dyslipidemia were recruited to explore the adlay's hypolipidemic potential, safety, and acceptability. Subjects consumed the dehulled adlay as the sole carbohydrate source in their breakfast, without changing other components in the diet or lifestyle, for 12 weeks. After intervention, serum total cholesterol (TC) decreased significantly in subjects with hypercholesterolemia. In addition, both TC and triglyceride levels decreased significantly in those above 50 years old. In conclusion, the extruded dehulled adlay displays potential for favorably modulating blood lipids, and the effect is more pronounced in the middle-aged population.

Anti-inflammatory effects and chemical study of a flavonoid-enriched fraction from adlay bran.

Anti-inflammation-guided fractionation and purification were used to evaluate the bioactivity and components of adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) bran. Results showed that the fraction with high phenolic and flavonoid contents from the ethanol extracts of adlay bran suppressed LPS-stimulated IL-6 and TNF-α secretions in a concentration-dependent manner in RAW 264.7 cells and murine peritoneal macrophages. Fifteen compounds, including a novel aurone derivative, two chromones, one dihydrochalcone, one chalcone, four flavanones, five flavones and one isoflavone, were isolated from the active fraction. The structure of the new compound was elucidated by spectroscopic methods, including 1D and 2D NMR and MS. All of the isolates are reported for the first time from adlay except naringenin. LC/MS was also provided as an analytical platform. Our results suggest that flavonoids in adlay bran, partially at least, contribute to its anti-inflammatory effect. Thus, adlay bran may be beneficial to the health of consumers.

Effects of adlay bran and its ethanolic extract and residue on preneoplastic lesions of the colon in rats.

BACKGROUND: Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) is a cereal crop used in traditional Chinese medicine and as a nutritious food. Epidemiologists have suspected that the low cancer rates in southeastern China might be related to adlay. Previous studies have shown that adlay has anti-tumour and anti-inflammatory activity. This study investigated the effect of adlay bran and its fractions on chemically induced colon carcinogenesis in rats. RESULTS: Adlay bran and its ethanolic extract and residue significantly reduced the number of preneoplastic aberrant crypt foci (ACF) and modified their mucin composition. The inhibitory effect of adlay bran ethanolic extract on ACF showed a dose dependence. Adlay bran and its ethanolic extract suppressed small ACF (one, two or three crypts) and ACF in the distal colon, while the residue suppressed large ACF (four or more crypts). CONCLUSION: These findings suggest the possibility that adlay bran and its ethanolic extract and residue inhibit colonic preneoplastic lesions in an early stage. Adlay and its fractions may have the potential to be developed as chemopreventive cereal products.

Suppression on allergic airway inflammation of dehulled adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) in mice and anti-degranulation phytosterols from adlay bran.

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) seeds have been used in Asia for thousands years to treat warts, chapped skin, rheumatism, and neuralgia. The anti-allergic activity of dehulled adlay (DA) seeds was identified, and the bran (AB) is regarded as the main functional constituent in the edible part. However, no study has focused on in vivo acute anti-allergic airway inflammation. In the present report, we investigated DA methanolic extract (DAM) reversed ovalbumin (OVA)/methacholine (Mch)-induced airway hypersensitivity, decreased interleukin (IL)-4, IL-5, and IL-13 levels from splenocytes, suppressed tumor necrosis factor (TNF)-α, IL-1ß, and IL-13 levels and reduced eosinophil counts and eotaxin in bronchoalveolar lavage fluid (BALF), which imply that the modulatory effects of DA should involve allergic degranulation. Further, seven phytosterols were isolated from AB ethanolic extract (ABE); among them, 3-O-caffeoyl-5ß-sitostan-3-ol, ß-sitosterol 3-O-glucopyranoside and ß-sitosterol inhibited ß-hexosaminidase release from A23187-stimulated RBL-2H3 cells with percentages of 54.1%, 52.0% and 48.5%, respectively, at 50 µM. In addition, ß-sitosterol reduced immunoglobulin (Ig)E-stimulated degranulation on RBL-2H3 cells in a dose-dependent manner. The phytosterols were the predominant components based on gas chromatography (GC) analysis. This is the first study to demonstrate that DA suppressed OVA/Mch-induced acute airway inflammation. The phytosterols in AB showed significant anti-degranulation activities, and may be regarded as the indicative components of AB for anti-allergy effects.

Ethanolic Extracts of Adlay Testa and Hull and Their Active Biomolecules Exert Relaxing Effect on Uterine Muscle Contraction through Blocking Extracellular Calcium Influx in Ex Vivo and In Vivo Studies.

Dysmenorrhea is one of the most prevalent disorders in gynecology. Historically, adlay (Coix lachryma-jobi L. var. Ma-yuen Stapf.) has been explored for its anti-tumor, pain relief, anti-inflammatory, and analgesic effects. The aim of this study was to evaluate the effects of adlay seeds on the inhibition of uterine contraction and thus dysmenorrhea relief, in vitro and in vivo. HPLC-MS and GC were used to elucidate the ethyl acetate fraction of adlay testa ethanolic extract (ATE-EA) and ethyl acetate fraction of adlay hull ethanolic extract (AHE-EA). Elucidation yielded flavonoids, phytosterols, and fatty acids. Uterine leiomyomas and normal adjacent myometrial tissue were evaluated by oxytocin- and PG-induced uterine contractility. ATE-EA and AHE-EA suppressed uterine contraction induced by prostaglandin F2 alpha (PGF2α), oxytocin, carbachol, and high-KCl solution ex vivo. In addition, the external calcium (Ca2+) influx induced contraction, and increased Ca2+ concentration was inhibited by ATE-EA and AHE-EA on the uterine smooth muscle of rats. Furthermore, ATE-EA and AHE-EA effectively attenuated the contraction of normal human myometrium tissues more than adjacent uterine leiomyoma in response to PGF2α. 3,5,6,7,8,3',4'-Heptamethoxyflavone and chrysoeriol produced a remarkable inhibition with values of IC50 = 24.91 and 25.59 µM, respectively. The experimental results showed that treatment with ATE-EA at 30 mg/day effectively decreased the writhing frequency both on the oxytocin-induced writhing test and acetic acid writhing test of the ICR mouse.

The gastroprotective effect of the foxtail millet and adlay processing product against stress-induced gastric mucosal lesions in rats.

Foxtail millet (Setaria italica (L.) P. Beauv.) and adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) seeds have substantial benefits possesses remarkable edible and nutritive values, and ease of processing and food manufacturing. They have nutraceutical properties in the form of antioxidants which prevent deterioration of human health and have long been used in traditional Chinese medicine as a remedy for many diseases. The present study is designed to investigate the gastroprotective effect of foxtail millet and adlay processing product (APP) diet on water immersion restraint stress (WIRS) induced ulceration in rats. We examined the effects of intake of AIN-93G diet containing either foxtail millet (10, 20 and 40%, 4 weeks) or APP (15 and 30%, 5 weeks) on macroscopic ulcer index (UI), plasma calcium level, lipid peroxidation products (estimated by the thiobarbituric acid reactive substances; TBARS), non-protein sulfhydryl (NPSH), digestive enzyme activities, and histopathology were determined. The results showed that pretreatment with millet and adlay diets significantly prevented the gastric mucosal lesion development. In addition, ulcerated rats showed depletion of NPSH levels whereas treatment with millet and adlay reverted this decline in stress-induced rats. Histological studies confirmed the results. The finding suggests that millet and adlay diets promote ulcer protection by the decrease in ulcer index, TBARS values and increase NPSH concentrations. Millet and adlay diets retain the advantage of being a natural product which may protect the gastric mucosa against ulceration.

Effect of adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) hull extracts on testosterone release from rat Leydig cells.

Adlay has been used as a traditional Chinese medicine for the treatment of many diseases. However, few studies have reported the effects of adlay seeds on the endocrine system. In the present study, the effects of methanol extracts of adlay hull (AHM) on testosterone synthesis were studied. Rat Leydig cells were incubated with different reagents including human chorionic gonadotropin, 8-bromo-adenosine-3',5'-cyclic monophosphate, forskolin, A23187, progesterone and androstenedione in the presence or absence of AHM. The rat anterior pituitary (AP) gland was treated with gonadotropin-releasing hormone (GnRH) in vitro in the presence or absence of AHM, and the concentrations of luteinizing hormone (LH) in the media were measured. AHM decreased testosterone release via the inhibition of (1) the PKA and PKC signal transduction pathways, (2) 17beta-HSD enzyme activity in rat Leydig cells, and (3) in vitro GnRH-induced LH secretion.

Food-Drug Interaction between the Adlay Bran Oil and Drugs in Rats.

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) contains various phytonutrients for treating many diseases in Asia. To investigate whether orally administered adlay bran oil (ABO) can cause drug interactions, the effects of ABO on the pharmacokinetics of five cytochrome P450 (CYP) probe drugs were evaluated. Rats were given a single oral dose (2.5 mL/kg BW) of ABO 1 h before administration of a drug cocktail either orally or intravenously, and blood was collected at various time points. A single oral dose of ABO administration did not affect the pharmacokinetics of five probe drugs when given as a drug cocktail intravenously. However, ABO increased plasma theophylline (+28.4%), dextromethorphan (+48.7%), and diltiazem (+46.7%) when co-administered an oral drug cocktail. After 7 days of feeding with an ABO-containing diet, plasma concentrations of theophylline (+45.4%) and chlorzoxazone (+53.6%) were increased after the oral administration of the drug cocktail. The major CYP enzyme activities in the liver and intestinal tract were not affected by ABO treatment. Results from this study indicate that a single oral dose or short-term administration of ABO may increase plasma drug concentrations when ABO is given concomitantly with drugs. ABO is likely to enhance intestinal drug absorption. Therefore, caution is needed to avoid food-drug interactions between ABO and co-administered drugs.

Adlay Bran Oil Suppresses Hepatic Gluconeogenesis and Attenuates Hyperlipidemia in Type 2 Diabetes Rats.

This study aimed to examine the antidiabetic effects of various concentrations of adlay bran oil (ABO) in high fat diet and streptozotocin-induced diabetic rats. Dietary supplementation with 10% ABO for 4 weeks effectively decreased the blood triacylglycerol, glucose, and total cholesterol levels in diabetic rats, although body weight remained the same. The mRNA and protein expressions of hepatic glucose transporter 2 (GLUT-2) and phosphoenolpyruvate carboxykinase (PEPCK) were increased and that of glucokinase (GCK) were decreased in diabetic rats. However, 10% ABO treatment reduced the mRNA and protein expressions of GLUT-2 and PEPCK and elevated the expression of hepatic GCK in diabetic rats. Thus, ABO enhanced hepatic glucose metabolism to decrease blood glucose in diabetic rats. In addition, 10% ABO supplementation increased the expression of phosphorylated protein kinase B (Akt) relative to the total Akt levels in the muscles of diabetic rats, indicating enhanced insulin sensitivity. The results indicate that ABO displays a potential for improving hyperlipidemia and hyperglycemia in diabetes by enhancing insulin sensitivity and hepatic glucose metabolism.

Dietary Glycotoxins, Advanced Glycation End Products, Inhibit Cell Proliferation and Progesterone Secretion in Ovarian Granulosa Cells and Mimic PCOS-like Symptoms.

Women with polycystic ovary syndrome (PCOS) have been reported to have an elevated serum advanced glycation end product (AGE) level. However, the effect of AGEs on the pathophysiological ovarian granulosa cells of PCOS is still unclear. In this study, five indented BSA-derived AGE products were used to evaluate their effect on the function of human granulosa cells. We found that the proliferation of both primary human ovarian granulosa (hGC) cells and human granulosa-like tumor (KGN) cells were inhibited by treatment with these five AGE products. The progesterone secretion level was also reduced in both hGC and KGN cells by treatment with these AGE products through downregulation of LH receptor/cAMP regulatory activity. The granulosa cell layer and serum progesterone level were reduced in rats by treatment with MG-BSA; moreover, an increased number of follicle cysts and an irregular estrous cycle were observed. MG-BSA treatment had a similar effect on the phenotypes of the DHEA-induced PCOS model. Additionally, the insulin resistance and hepatic lesions seen in the DHEA-induced PCOS model were observed in the MG-BSA treatment group. Taken together, we found that AGEs exert a toxic effect on ovarian granulosa cells, ovarian morphology, and the estrous cycle that mimics the DHEA-induced PCOS phenotypes.

Lysophosphatidic acid-induced oxidized low-density lipoprotein uptake is class A scavenger receptor-dependent in macrophages.

Lysophosphatidic acid (LPA) is a low-molecular-weight lysophospholipid enriched in platelets and mildly oxidized low-density lipoprotein (OxLDL). It is suggested that LPA is involved in atherosclerosis, and our previous studies showed that LPA regulates inflammation in multiple cell types. The main aim of this study was to investigate the effects of LPA on the uptake of OxLDL by mouse J774A.1 macrophages. We observed that LPA upregulated fluorescence-labeled DiI-OxLDL uptake in J774A.1 cells. Meanwhile, expression of the class A scavenger receptor (SR-A), a receptor for modified LDL, was also enhanced. Furthermore, pertussis toxin (PTx) or Ki16425 significantly abolished LPA's effects, indicating that G(i) and LPA(3) are involved in OxLDL uptake and SR-A expression. Of most importance, the LPA-induced OxLDL uptake could be inhibited when cells were incubated with a functional blocking antibody of SR-A. Our results suggest that LPA-enhanced OxLDL uptake is mediated via LPA(3)-G(i) activation and subsequent SR-A expression.

Isolation and characterization of new lactam compounds that inhibit lung and colon cancer cells from adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) bran.

Five active compounds that inhibit cancer cells were isolated from adlay bran (Coix lachryma-jobi L. var. ma-yuen Stapf), and their structures and activities in vitro were characterized. The ethyl acetate-soluble fraction of methanol extracts of adlay bran (ABM-EtOAc) exhibited a stronger anti-proliferative effect on human lung cancer cell A549, human colorectal carcinoma cell HT-29, and COLO 205 than other fractions by MTT (3-4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide) assay. Assay-guided isolation gave five lactams including three that were previously undocumented; coixspirolactam A (1), coixspirolactam B (2), and coixspirolactam C (3); one isolated from the natural plant for the first time, coixlactam (4); and one known compound, methyl dioxindole-3-acetate (5). Pure active compounds were identified by spectral analysis including IR, 1H and 13C NMR, UV-vis, MS and 2D NMR techniques. All the compounds were tested for their anti-proliferative effect on A549, HT-29 and COLO 205 cells. These compounds showed anti-cancer activities with IC50 values between 28.6 and 72.6microg/mL.

Hexane fraction of adlay (Coix lachryma-jobi L.) testa ethanolic extract inhibits human uterine sarcoma cancer cells growth and chemosensitizes human uterine sarcoma cells to doxorubicin.

BACKGROUND: Cancer has remained among the top ten causes of death in Taiwan since 1982. Uterine sarcoma is a rare gynecologic cancer, and chemotherapy is one type of cancer treatment. Doxorubicin (Dox) is widely used for treating several cancers, including uterine sarcoma, however, multidrug resistance (MDR) is a major clinical problem and a critical cause of treatment failure. The ethanolic extracts of adlay testa (ATE) exhibited significant anticancer activities against many cancer types. PURPOSE: In this study we investigated the antitumor effects of the hexane fraction of the adlay testa ethanolic extracts (ATE-Hex) on the human uterine sarcoma cancer cell line MES-SA, as well as on the multidrug-resistant human uterine sarcoma cancer cell line MES-SA/Dx5. METHODS: The MTT assay was performed to assess the effects of the extracts of different parts of the adlay on the proliferation of human uterine sarcoma cells (MES-SA and MES-SA/Dx5) and human uterine smooth muscle cells (HUtSMCs). To determine whether ATE-Hex has a chemosensitizing effect on drug-resistant uterine sarcoma cells, the MTT assay was performed to examine the synergistic effects of ATE-Hex, the chemotherapeutic drug Dox alone, and in combination. Rhodamine accumulation was analyzed using fluorescence detection. Apoptotic cells were analyzed via flow cytometry. In addition, employing a flame ionization detector (GC/FID) gas chromatography was also developed as the analysis platform for ATE-Hex. RESULTS: The results demonstrated that ATE-Hex exhibited the best effects of inhibition on MES-SA and MES-SA/Dx5 cells. Co-treatment of ATE-Hex and Dox could synergistically inhibit the proliferation of cancer cells. ATE-Hex reduced the rhodamine efflux in MES-SA/Dx5 cells, indicating that ATE-Hex could reduce the expression of P-gp. In addition, our results showed that treatment with ATE-Hex alone or in combination with Dox significantly inhibited the growth of cancer cells and induced apoptosis by increasing the sub-G1 phase and poly(ADP-ribose) polymerase (PARP) being cleaved. Flow cytometry revealed that ATE-Hex induced apoptosis. CONCLUSION: These results suggest that ATE-Hex can inhibit human uterine sarcoma cancer cells by inducing apoptosis and increasing the chemosensitivity of the multidrug-resistant human uterine sarcoma cancer cell MES-SA/Dx5 to Dox. Furthermore, the combination of ATE-Hex and Dox could decrease MDR and increase the synergistic effect.

Effects of adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) hull extracts on the secretion of progesterone and estradiol in vivo and in vitro.

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) has been used as a traditional Chinese medicine for dysfunction of the endocrine system. However, there have been few studies on the effects of adlay seed on the endocrine system. In the present study, both the in vivo and in vitro effects of methanolic extracts of adlay hull (AHM) on progesterone synthesis were studied. AHM was partitioned with four different solvents: water, 1-butanol, ethyl acetate, and n-hexane. Four fractions, namely, AHM-Wa (water fraction), AHM-Bu (1-butanol fraction), AHM-EA (ethyl acetate fraction), and AHM-Hex (n-hexane fraction), were respectively obtained. Granulosa cells (GCs) were prepared from pregnant mare serum gonadotropin-primed immature female rats and were challenged with different reagents, including human chorionic gonadotropin (hCG; 0.5 IU/ml), 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP; 0.1 mM), forskolin (10 microM), 25-OH-cholesterol (10 microM), and pregnenolone (10 microM), in the presence or absence of AHM (100 microg/ml). The functions of steroidogenic enzymes, including protein expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage enzyme (P450scc), protein kinase A (PKA), and aromatase activity, were investigated. The expression of StAR mRNA was also explored by using real-time reverse transcription-polymerase chain reaction. In the in vivo study, AHM decreased plasma progesterone and estradiol levels after an intravenous injection of AHM (2 mg/ ml/kg). In the in vitro studies, AHM decreased progesterone and estradiol via inhibition of (i) the cAMP-PKA signal transduction pathway, (ii) cAMP accumulation, (iii) P450scc and 3beta-HSD enzyme activities, (iv) PKA, P450scc and StAR protein expressions and StAR mRNA expression, and (v) aromatase activity in rat GCs. These results suggest that AHM decreased the production of progesterone via mechanisms involving the inhibition of the cAMP pathway, enzyme activities, and the protein expressions of P450scc and StAR in rat GCs.

Effects of crude adlay hull acetone extract on corticosterone release from rat zona fasciculata-reticularis cells.

Adlay is a grass crop which has been used in traditional Chinese medicine and also as a nourishing food. It has been shown to posses anti-allergic, antimutagenic and hypolipemic effects. However, the effects and action mechanisms of crude adlay hull acetone extract (AHA) on adrenal zona fasciculata-reticularis (ZFR) cells are still unclear. This study explored the effects of AHA on corticosterone release. ZFR cells were incubated with AHA in the presence or absence of adrenocorticotropin (ACTH), 8-bromo-cyclic 3': 5'- adenosine monophosphate (8-Br-cAMP), forskolin (FSK), 25-hydroxy cholesterol (25-OH-cholesterol), pregnenolone, progesterone or deoxycorticosterone. The concentrations of corticosterone or pregnenolone in the media were measured by radioimmunoassay (RIA). The cells were used to measure the expression of steroidogenic acute regulatory (StAR) protein by Western blot. The present data demonstrated that: (1) AHA inhibited ACTH-, 8-Br-cAMP-, forskolin-, 25-OH-cholesterol-, pregnenolone-, progesterone- or deoxycorticosterone-stimulated corticosterone release; (2) AHA (800 microg/ml) caused more pregnenolone release in control group, but not in 25-OH-cholesterol, trilostane or 25-OH-cholesterol+trilostane group; (3) kinetic study showed an uncompetitive inhibition model of AHA to P450 side chain cleavage enzyme (P450scc); (4) kinetic study showed a noncompetitive inhibition model of AHA to 11beta-hydroxylase; and (5) AHA inhibited the expression of StAR protein. These results suggest that AHA acts directly upon rat ZFR cells to diminish corticosterone release. These results indicate the inhibitory mechanism of AHA mediates through an inhibition of the activities of the post-cAMP corticosterone synthesis enzymes, i.e. 3beta-HSD, 21-hydroxylase, 11beta-hydroxylase, and inhibition of StAR protein expression.