Cytotoxic and antitumor activities of thiosulfinates from Allium tuberosum L

In this study we isolated crude thiosulfinates from Allium tuberosum L. using CH 2Cl 2 and then with silica gel column chromatography purified S-methyl methanthiosulfinate and S-methyl 2-propene-1-thiosulfinate from the crude thiosulfinates. Subsequently, in vitro cytotoxicities against human cancer cells and in vivo antitumor activities of the thiosulfinates were investigated. Their cytotoxicities were strong in human cancer cells, in the order of S-methyl 2-propene-1-thiosulfinate, crude thiosulfinates, and S-methyl methanthiosulfinate. When thiosulfinates were administered consecutively for 7 days at 10, 30, and 50 mg/kg ip, in mice, we found significant increases in the life spans of mice that had been inoculated with Sacorma-180 tumor cells. The crude thiosulfinates also induced apoptosis in MCF-7 cancer cells. These results suggest that thiosulfinates from Allium tuberosum L. inhibit the proliferation of cancer cells via apoptosis and have antitumor activities.     

Induction of apoptosis in cancer cells by Bilberry (Vaccinium myrtillus) and the anthocyanins

Among ethanol extracts of 10 edible berries, bilberry extract was found to be the most effective at inhibiting the growth of HL60 human leukemia cells and HCT116 human colon carcinoma cells in vitro. Bilberry extract induced apoptotic cell bodies and nucleosomal DNA fragmentation in HL60 cells. The proportion of apoptotic cells induced by bilberry extract in HCT116 was much lower than that in HL60 cells, and DNA fragmentation was not induced in the former. Of the extracts tested, that from bilberry contained the largest amounts of phenolic compounds, including anthocyanins, and showed the greatest 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. Pure delphinidin and malvidin, like the glycosides isolated from the bilberry extract, induced apoptosis in HL60 cells. These results indicate that the bilberry extract and the anthocyanins, bearing delphinidin or malvidin as the aglycon, inhibit the growth of HL60 cells through the induction of apoptosis. Only pure delphinidin and the glycoside isolated from the bilberry extract, but not malvidin and the glycoside, inhibited the growth of HCT116 cells.     

Identification of bitterness-masking compounds from cheese

Bitterness-masking compounds were identified in a natural white mold cheese. The oily fraction of the cheese was extracted and further fractionated by using silica gel column chromatography. The four fractions obtained were characterized by thin-layer chromatography and nuclear magnetic resonance spectroscopy. The fatty acid-containing fraction was found to have the highest bitterness-masking activity against quinine hydrochloride. Bitterness-masking activity was quantitated using a method based on subjective equivalents. At 0.5 mM, the fatty acid mixture, which had a composition similar to that of cheese, suppressed the bitterness of 0.008% quinine hydrochloride to be equivalent to that of 0.0049-0.0060% and 0.5 mM oleic acid to that of 0.0032-0.0038% solution. The binding potential between oleic acid and the bitter compounds was estimated by isothermal titration calorimetry. These results suggest that oleic acid masked bitterness by forming a complex with the bitter compounds.     

Determination of dioxins and furans in foods and biological tissues: review and update

Determination of trace residues of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs and PCDFs) in various matrixes is carried out by a limited number of laboratories in the United States, Canada, and other countries. Current methods for analysis of foods and biological tissues include a combination of preparation, extraction, cleanup, isolation, determination, and identity confirmation procedures. Soxhlet, liquid/liquid, solid-phase, and column extraction procedures are used as well as treatment with acid or base before solvent extraction. Cleanup and isolation steps include sulfuric acid partitioning; adsorption chromatography on Florisil, silica gel, or alumina; gel permeation chromatography; multi-stage column chromatography on sulfuric acid silica and alkali silica; carbon column chromatography; and liquid chromatography fractionation with size exclusion, normal-phase, and reverse-phase columns. Activated carbon and multistage chromatographic columns are widely used in cleanup schemes. Isomer-specific identification and quantitation of PCDD and PCDF congeners at parts-per-trillion levels or lower are carried out by high resolution (capillary) gas chromatography (HRGC) and multiple ion detection mass spectrometry. In addition to chemical methods, bioassay procedures have been recommended (e.g., use of monoclonal antibodies, for immunoassay determination of PCDDs and PCDFs).     

Bitter gourd suppresses lipopolysaccharide-induced inflammatory responses

Bitter gourd ( Momordica charantia L.) is a popular tropical vegetable in Asian countries. Previously it was shown that bitter gourd placenta extract suppressed lipopolysaccharide (LPS)-induced TNFalpha production in RAW 264.7 macrophage-like cells. Here it is shown that the butanol-soluble fraction of bitter gourd placenta extract strongly suppresses LPS-induced TNFalpha production in RAW 264.7 cells. Gene expression analysis using a fibrous DNA microarray showed that the bitter gourd butanol fraction suppressed expression of various LPS-induced inflammatory genes, such as those for TNF, IL1alpha, IL1beta, G1p2, and Ccl5. The butanol fraction significantly suppressed NFkappaB DNA binding activity and phosphorylation of p38, JNK, and ERK MAPKs. Components in the active fraction from bitter gourd were identified as 1-alpha-linolenoyl-lysophosphatidylcholine (LPC), 2-alpha-linolenoyl-LPC, 1-lynoleoyl-LPC, and 2-linoleoyl-LPC. Purified 1-alpha-linolenoyl-LPC and 1-linoleoyl-LPC suppressed the LPS-induced TNFalpha production of RAW 264.7 cells at a concentration of 10 microg/mL.     

Improved separation method for highly purified lutein from Chlorella powder using jet mill and flash column chromatography on silica gel

We investigated an improved method for the separation of high-purified lutein from a commercially available spray-dried Chlorella powder (CP) using fine grinding by jet mill and flash column chromatography on a silica gel. Saponification and extraction of lutein were enhanced 2.3-2.9-fold in jet mill-treated CP (mean particle size, 20 microm) as compared to untreated CP (mean particle size, 67 microm). The carotenoid extract was dissolved in ether-hexane (1:1 v/v) and subjected to flash column chromatography on silica gel. A mixture of alpha- and beta-carotene was eluted with hexane, followed by elution with hexane-acetone-chloroform (7:2:1 v/v). Lutein (dark-orange band) was collected after the elution of an unknown colorless compound (detected based on UV absorbance). The purity of lutein in this fraction was over 99%, and the yield was 60%. The present study provides key information for obtaining highly purified lutein using flash column chromatography on a silica gel.     

Mechanisms of cancer chemoprevention by hop bitter acids (beer aroma) through induction of apoptosis mediated by Fas and caspase cascades

The bitter acids of hops (Humulus lupulus L.) mainly consist of alpha-acids, beta-acids, and their oxidation products that contribute the unique aroma of the beer beverage. Hop bitter acids displayed a strong growth inhibitory effect against human leukemia HL-60 cells, with an estimated IC(50) value of 8.67 microg/mL, but were less effective against human histolytic lymphoma U937 cells. Induction of apoptosis was confirmed in HL-60 cells by DNA fragmentation and the appearance of a sub-G1 DNA peak, which were preceded by dissipation of mitochondrial membrane potential, cytochrome c release, and subsequent induction of pro-caspase-9 and -3 processing. Cleavages of PARP and DFF-45 were accompanied with activation of caspase-9 and -3 triggered by hop bitter acids in HL-60 cells. The change in the expression of Bcl-2, Bcl-X(L), and Bax in response to hop bitter acids was studied, and the Bcl-2 protein level slightly decreased; however, the Bcl-X(L) protein level was obviously decreased, whereas the Bax protein level was dramatically increased, indicating that the control of Bcl-2 family proteins by hop bitter acids might participate in the disruption of mitochondrial integrity. In addition, the results showed that hop bitter acids promoted the up-regulation of Fas and FasL prior to the processing and activation of pro-caspase-8 and cleavage of Bid, suggesting the involvement of a Fas-mediated pathway in hop bitter acids-induced cells. Taken together, these findings suggest that a certain intimate link might exist between receptor- and mitochondria-mediated death signalings that committed to cell death induced by hop bitter acids. The induction of apoptosis by hop bitter acids may offer a pivotal mechanism for their chemopreventive action.     

Watermelon (Citrullus lanatus) hydroperoxide lyase greatly increases C6 aldehyde formation in transgenic leaves

Fatty acid hydroperoxide lyase (HL) is the key enzyme for the production of the "green note"compounds, leaf aldehyde [(2E)-hexenal] and leaf alcohol [(3Z)-hexenol], in plant tissues. A cDNA encoding HL was cloned from leaves of watermelon (Citrullus lanatus) and expressed in Nicotiana tabacum. The enzyme is 3 times more active with 13-hydroperoxylinolenic acid than with 13-hydroperoxylinoleic acid. The activity against 9-hydroperoxides of polyunsaturated fatty acids is minimal. Enzyme activity of the watermelon HL in the transgenic leaves was approximately 50 times higher than endogenous HL activity in the wild-type N. tabacum plants. When compared with Arabidopsis HL also expressed in N. tabacum, the highest HL activity is 10 times higher in watermelon HL overexpressing leaves than in Arabidopsis HL overexpressers.     

Silicon nutrition of tomato and bitter gourd with special emphasis on silicon distribution in root fractions

Two plant species, tomato (Lycopersicon esculentum Mill.) and bitter gourd (Momordica charantia), were used for in‐depth studies on the dynamics of silicon (Si) uptake and translocation to the shoots and compartmentation of Si in the roots. The experiments were conducted under controlled environmental conditions in nutrient solutions, which were partly amended with 1 mM Si in the form of silicic acid. At harvest, xylem exudates were collected, and Si concentrations and biomass of roots and shoots were determined. Mass flow of Si was calculated based on the Si concentration of the nutrient solution and transpiration determined in a parallel experiment. Plant roots were subjected to a fractionated Si analysis, allowing attributing Si to different root compartments. Silicon concentrations in the roots compared to the shoots were higher in tomato but lower in bitter gourd. A more ready translocation from the roots to the shoots in bitter gourd was in agreement with Si concentrations in the xylem exudates which were higher than in the external solution. In tomato, the xylem‐sap Si concentration was lower than in the nutrient solution. Calculated Si mass flow to the root exceeded Si uptake in tomato, which was consistent with the measured accumulation of Si in the root water‐free space (WFS). In contrast, Si concentration in the root WFS was lower than in the nutrient solution in bitter gourd, reflecting the calculated Si depletion at the root surface based on the comparison of Si mass flow and Si uptake. Within the roots, more than 80% of the total Si was located in the cell wall and less than 10% in the cytoplasmic fractions in tomato. In bitter gourd, between 60% and 70% of the total root Si was attributed to the cell‐wall fraction whereas the proportion of the cytoplasmic fraction reached more than 30%. Our results clearly confirm that tomato belongs to the Si excluders and bitter gourd to the Si‐accumulator plant species for which high Si concentrations in the cytoplasmic root fraction appear to be characteristic.     

Determination of benzo(a)pyrene in foods

An analytical method was developed for determining benzo(a)pyrene in foods, suitable for routine use. The method consists of 4 cleanup steps: (1) alkali cleavage of sample, (2) preliminary silica gel column chromatography, (3) selective extraction with concentrated sulfuric acid, and (4) further silica gel column chromatography. Recoveries of benzo(a)pyrene added to 50 g (or 10 g) food at levels of 0.4 ppb (or 2 ppb) ranged from 70% for short-necked clam and mackeral to 85% for chicken meat. The sulfuric acid extraction step affords a simple method for isolating benzo(a)pyrene from various kinds of interfering substances which could not be separated by existing methods.     

Identification of two polyphenolic compounds with antioxidant activities in longan pericarp tissues

Longan fruits contain a significant amount of polyphenols. In the present study, polyphenols were extracted from longan pericarp tissues, and then two representative polyphenols were separated and purified by polyamide column chromatography, Sephadex LH-20 column chromatography, and silica gel column chromatography. On the basis of 1H NMR, 13C NMR, and electrospray ionization mass spectrometric (ESI-MS) data, the two compounds were identified as 4-O-methylgallic acid and (-)-epicatechin, respectively. In terms of reaction with longan polyphenol oxidase (PPO), (-)-epicatechin was further identified as the PPO substrate that caused longan fruit to brown. The results of antioxidant activity showed that 4-O-methylgallic acid had higher reducing power and 2,2-diphenyl-1-picrylhydrazyl- (DPPH-), hydroxyl radical-, and superoxide radical-scavenging activities than (-)-epicatechin.     

Thin layer chromatographic determination of aflatoxin in corn dust

Methods adopted by the AOAC and the American Association of Cereal Chemists for determining aflatoxin in corn were modified, and techniques were developed for application to samples of less than 1 to 10 g instead of the specified 50 g samples. Analysis included chloroform extraction of dust samples or dust collected from glass fiber filters, purification of extracts on a silica gel column of appropriate size, and measurement of aflatoxin by either 1- or 2-dimensional thin layer chromatography (TLC). The solvent for 1-dimensional TLC was chloroform-acetone-water (91 + 9 + 1). Solvents for 2-dimensional TLC were, first direction, ether-methanol-water (95 + 4 + 1, lined tank) and second direction, chloroform-acetone-water (91 + 9 + 1, unlined tank), or first direction, chloroform-acetone-water (91 + 9 + 1, unlined tank) and second direction, toluene-ethyl acetate-formic acid (60 + 30 + 10, unlined tank). When samples weighed less than or equal to 0.1 g, the entire concentrated extract was applied to the TLC plate. About 0.5-1.0 ng aflatoxin B1 could be detected on the plate, making the limit of detection about 9 ng/g for 0.1 g samples.     

Isolation and identification of cucurbitane-type triterpenoids with partial agonist/antagonist potential for estrogen receptors from Momordica charantia

This study aims at investigating the estrogenic activity and active cucurbitane-type triterpenoid compounds of bitter gourd (Momordica charantia, MC) using a transactivation assay for estrogen receptors (ER) α and β. The lyophilized fruits of MC were exhaustively extracted with ethyl acetate (EA) and 95% ethanol (EtOH), sequentially. The nonsaponifiable fraction (NS) of the EA extract as well as the acid hydrolyzed EtOH extract (AH) was fractionated and isolated by repeated column chromatography and further purified by preparative HPLC or RP-HPLC. One known compound, 5β,19-epoxycucurbita-6,24-diene-3β,23ξ-diol (6), was isolated from the NS, and five new compounds (1-5) were isolated from AH and identified as cucurbita-6,22(E),24-trien-3β-ol-19,5β-olide (1), 5β,19-epoxycucurbita-6,22(E),24-triene-3β,19-diol (2), 3β-hydroxycucurbita-5(10),6,22(E),24-tetraen-19-al (3), 19-dimethoxycucurbita-5(10),6,22(E),24-tetraen-3β-ol (4), and 19-nor-cucurbita-5(10),6,8,22(E),24-pentaen-3β-ol (5). In the noncytotoxic concentration range, compounds 1, 2, 5 and 6 showed weak agonistic activity via ER α and β. Compounds 1, 2, 3 and 6 significantly antagonized the transactvation of 17β-estradiol (E(2)) via both ER α and β. In conclusion, this study demonstrates, for the first time as far as we know, the partial agonist/antagonist activity via ER of four new and one known cucurbitane-type triterpenoids from MC. Further studies are worthy to explore the selective estrogen receptor modulator (SERM) activity of MC.     

Growth suppression of human cancer cells by polyphenolics from sweetpotato (Ipomoea batatas L.) leaves

Sweetpotato leaves (Ipomoea batatas L.) contain a high content of polyphenolics that consist of caffeic acid, chlorogenic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and 3,4,5-tri-O-caffeoylquinic acid. We investigated the suppression of the proliferation of selected human cancer cells by phenolic compounds isolated from sweetpotato leaf. The human cancer cells used in this research included a stomach cancer (Kato III), a colon cancer (DLD-1), and a promyelocytic leukemia cell (HL-60). Caffeic acid and di- and tricaffeoylquinic acids dose-dependently depressed cancer cell proliferation, and the difference in sensitivity between caffeoylquinic acid derivatives and each kind of cancer cell was observed. Specifically, 3,4,5-tri-O-caffeoylquinic acid effectively depressed the growth of three kinds of cancer cells, and caffeic acid had an exceptionally higher effect against HL-60 cells than other di- and tricaffeoylquinic acids. In attempting to clarify the mechanism of growth suppression with the addition of the apoptotic inhibitor N-ethylmaleimide, we observed that the nuclear granulation in 3,4,5-tri-O-caffeoylquinic acid-treated HL-60 cells suggested apoptosis induction. This effect was confirmed by DNA fragmentation, an increase of caspase-3 activity, and expression of c-Jun. Growth suppression of HL-60 cells by 3,4,5-tri-O-caffeoylquinic acid was determined to be the result of apoptotic death of the cells. These results indicate that 3,4,5-tri-O-caffeoylquinic acid may have potential for cancer prevention.     

Isolation and identification of organosulfur compounds oxidizing canine erythrocytes from garlic (Allium sativum)

Five compounds oxidizing canine erythrocytes were isolated from an aqueous ethanol garlic extract by silica gel column chromatography and preparative thin-layer chromatography. On the basis of nuclear magnetic resonance, infrared spectroscopy, and mass spectrometry, they were identified as three known compounds: bis-2-propenyl trisulfide (1), bis-2-propenyl tetrasulfide (2), and bis-2-propenyl pentasulfide (3) as well as two novel compounds, bis-2-propenyl thiosulfonate (4) and trans-sulfuric acid allyl ester 3-allylsulfanyl-allyl ester (5). A mixture of compounds 1-3 and compounds 4 and 5 induced methemoglobin formation in canine erythrocyte suspension in vitro resulting in the oxidation of canine erythrocytes. These groups of characteristic organosulfur compounds contained in garlic probably contribute to oxidations in blood. The constituents of garlic have the potential to oxidize erythrocytes and hemoglobin, suggesting that foods containing quantities of garlic should be avoided for feeding dogs.     

Synthesis and analysis of symmetrical and nonsymmetrical disaturated/monounsaturated triacylglycerols

Symmetrical disaturated triacylglycerols of the structure SUS, where S is stearic acid (18:0) and U is an unsaturated fatty acid, either oleic (O; 9cis-18:1), linoleic (L; 9cis,12cis-18:2), or linolenic (Ln; 9cis,12cis,15cis-18:3), are important components providing functionality to interesterified fat blends and structurally modified oils. Nonsymmetrical triacylglycerols of the structure SSU can significantly change melting point and solid fat content profiles. To characterize the physical properties of pure and symmetrical and nonsymmetrical triacylglycerol mixtures, the same reaction sequence has been used to prepare multigram quantities of triacylglycerols SUS and SSU. Tristearin was converted to a mixture of mono-, di-, and triacylglycerols, and the 1,3- and 1,2-diacylglycerol fraction was isolated by silica column chromatography. The 1,3-diacylglycerols were removed by crystallization from acetone and esterified with the appropriate fatty acid to form the symmetrical triacylglycerols with >99% SUS structure. The more difficult to obtain 1,2-diacylglycerols were prepared by esterification of the enriched 1,2-diacylglycerol fraction (80-86% 1,2-diacylglycerols) remaining after removal of much of the 1,3-isomer by crystallization, but silver resin or silver nitrate impregnated silica gel chromatography was required to isolate the nonsymmetrical triacylglycerols. SSL and SSLn were prepared in purities of >98% by this procedure, but not SSO. Silver ion HPLC was found to be as accurate as, and more rapid than, lipolysis/gas chromatography for the determination of the isomeric purities of the synthesized triacylglycerols.     

超长钝顶螺旋藻的选育及形态和生长特性初步研究

用组织匀浆破碎和离心沉降法制备和分离出钝顶螺旋藻(Spirulina platensis)Sp-Z 的单细胞或少于5个细胞的片段,并用60Coγ射线辐照处理,半致死剂量为1.2 kGy.在2.4kGy的处理组中,筛选出4株藻丝长度、螺旋数、螺旋长分别为Sp-Z的15、10和1.8倍的超长钝顶螺旋藻突变体Sp-Z(E.L).Sp-Z的生长曲线几乎呈直线型,而Sp-Z(E.L)的生长曲线则呈较明显的"S"形.在室外培养时,Sp-Z(E.L)的产量比Sp-Z高11.7%.     

Isolation and characterization of a new compound from Prunus mume fruit that inhibits cancer cells

An active compound that inhibits cancer cells was isolated from the fruit of Prunus mume, and its structure and in vitro activities were characterized. The n-hexane fraction obtained from methanol extracts exhibited the strongest inhibitory effect on the growth of cancer cells. From the n-hexane fraction, a new compound named B-1 was purified through preparative thin-layer chromatography, ODS column chromatography, and reverse phase high-performance liquid chromatography and its structure was analyzed by fast atom bombardment mass spectrometry and 1H and 13C NMR. The molecular formula of B-1 was C19H22O6 {2-hydroxy-1-[(7-hydroxy-2-oxo-2H-chromen-6-yl)methyl]-2-methylpropyl-(2Z)-3-methyl-but-e-enoate:prunate}, and the IC50 value was in the range of 39-58 microg/mL in descending order of the cancer cell lines Hep-2, SW-156, HEC-1-B, and SK-OV-3. B-1 exhibited 81-96% inhibition at a concentration level of 100 microg/mL against all cells, based on an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. However, B-1 showed little effect against normal cells with only 23% or less growth inhibition at 100 microg/mL. Thus, B-1 has a highly specific inhibitory effect against cancer cells but little effect against normal cells. When the cancer cell lines Hep-2 and SK-OV-3 were incubated with B-1 for 72 h, most of the tested cells suffered strong growth inhibition. The compound has the potential to be developed as a nutraceutical.     

Wheat bran oil and its fractions inhibit human colon cancer cell growth and intestinal tumorigenesis in Apc(min/+) mice

This study was designed to investigate the cancer preventive activities of wheat bran (WB) oil. We studied the colon cancer preventive effects of WB oil and its subfractions in the Apc(min/+) mouse model, a recognized mouse model for human colorectal cancer, and used human colon cancer cell lines (HCT-116 and HT-29) to identify possible active fractions in WB oil. Our results showed that the oil fraction of WB was more active than the water fraction against the growth of human colon cancer cell lines and that 2% WB oil significantly inhibited the overall tumorigenesis by 35.7% (p < 0.0001) in the Apc(min/+) mouse model. The WB oil was further fractioned into nonpolar lipids and phytochemicals and the phytochemical fraction was fractionated into phytosterols and phytosterol ferulates, 5-alk(en)ylresorcinols, and unidentified constituents by normal phase silica gel column chromatography. Results on cell culture showed that the phytochemical fraction had a higher inhibitory effect on HCT-116 human colon cancer cells than that of WB oil, whereas the nonpolar lipid fraction had less growth inhibitory effectiveness. However, neither fractions showed a stronger inhibition than WB oil in the Apc(min/+) mouse model. The current results demonstrate, for the first time, the intestinal cancer preventive activity of WB oil. The active ingredients, however, remain to be identified.     

苦瓜果实酵母双杂交文库构建及McRPF互作蛋白筛选

为研究苦瓜果实成熟的分子调控机制,以苦瓜自交系E12201-e1为材料,取不同成熟期苦瓜果肉组织等量混合,采用All-Direct方法构建酵母双杂交cDNA文库。经质量鉴定,该文库库容为1.25×10⁷ CFU,平均插入片段大于1 200 bp,阳性率为100%,文库质量较高,符合建库标准。同时,以苦瓜果实成熟关键调控因子McRPF为诱饵,构建诱饵表达载体pGBKT7-McRPF。经鉴定该诱饵蛋白无自激活活性。利用共转化法,从文库中筛选到29个初始阳性菌落,经过DNA测序和BLAST比对分析,最终筛选得到12个可能与McRPF互作的蛋白,为进一步探究McRPF调控苦瓜果实成熟的分子机制奠定了理论基础。