Formation of 2,4-Diarylallophanate from Arylthionocarbamate in an Iodine-DMSO System

The structures of cotylenins, leaf growth substances produced by a fungus, have been assigned on the basis of degradative and spectroscopic evidence. They are novel glycosides with a common aglycone, cotylenol: cotylenins A and C have unusual sugar moieties consisting of an 6-O-methyl-α-d-glucosyl derivative with an oxygenated C₅-isoprene unit.     

Structure of a secoisolariciresinol diester from Salvia plebeia seed

The structure of a unique lignan diester from Salvia plebeia seed has been determined. Hydrolysis of the diester yields ferulic acid, (+)-l2 l-methyltetradecanoic acid and secoisolariciresinol.     

SDH全基因的拼接及原核表达

目的:构建闭联异松树脂醇二脂脱氢酶(SDH)全基因片段与原核表达质粒载体,在大肠杆菌中进行表达.方法:将SDH3'端和5'端序列通过PCR方法拼接获得全基因后,然后将其插入到相应的原核表达质粒载体PGEX-6p-1中,形成了重组载体,并使其在大肠杆菌BL21中经IFFG诱导其表达蛋白,再用SDS-PAGE电泳检测表达结果.结果:成功的将SDH基因片段拼接成全基因,并将其构建入原核表达载体PGEX-6p-1中.测序结果表明载体构建成功,无插入和移码突变.经1.5mmol/LIPTC诱导后获得与预测大小(29.253kDa)完全一致的目的蛋白即闭联异松树脂醇二脂脱氢酶蛋白,并测得在37℃、250r/min的实验条件下的最佳诱导时间为4h.结论:成功获得了重组质粒并在大肠杆菌中较好的诱导表达得到了闭联异松树脂醇二脂脱氢酶的蛋白.     

Bacterial conversion of secoisolariciresinol and anhydrosecoisolariciresinol

Aims:  It has been investigated whether secoisolariciresinol (SECO) and anhydrosecoisolariciresinol (AHS), an acid degradation product of SECO, could be fermented in a similar way, and to a similar extent, by members of the intestinal microbiota.
Methods and Results:  AHS and SECO were demethylated by Peptostreptococcus productus , Eubacterium limosum and Clostridium methoxybenzovorans . These bacteria have been identified as members of the human intestinal flora or closely related species. Demethylated AHS and demethylated SECO were purified by preparative RP-HPLC, and subsequently subjected to fermentation with Eggerthella lenta , Clostridium scindens and Clostridium hiranonis . Eggerthella lenta efficiently dehydroxylated demethylated SECO to enterodiol, whereas the other bacteria showed no dehydroxylation activity.
Conclusions:  The conversion of the diol structure of SECO into the furan ring in AHS did not influence the demethylation capability of the tested bacteria. The results also showed that the extent of dehydroxylation of demethylated AHS was much lower than that of demethylated SECO.
Significance and Impact of the Study:  Plant lignans are converted into bioactive mammalian lignans by the human intestinal bacteria. This study showed that the modification of plant lignans resulted in the formation a new type of mammalian lignan.     

A secoisolariciresinol branched fatty diester from Salvia plebeia seed

The structure of a second new lignan diester from Salvia plebeia seed has been determined. Hydrolysis of this diester yields two compounds, 12-methyltetradecanoic acid and secoisolariciresinol.     

Phylogeny of human intestinal bacteria that activate the dietary lignan secoisolariciresinol diglucoside

The human intestinal microbiota is essential for the conversion of the dietary lignan secoisolariciresinol diglucoside (SDG) via secoisolariciresinol (SECO) to the enterolignans enterodiol (ED) and enterolactone (EL). However, knowledge of the species that catalyse the underlying reactions is scant. Therefore, we focused our attention on the identification of intestinal bacteria involved in the conversion of SDG. Strains of Bacteroides distasonis, Bacteroides fragilis, Bacteroides ovatus and Clostridium cocleatum, as well as the newly isolated strain Clostridium sp. SDG-Mt85-3Db, deglycosylated SDG. Demethylation of SECO was catalysed by strains of Butyribacterium methylotrophicum, Eubacterium callanderi, Eubacterium limosum and Peptostreptococcus productus. Dehydroxylation of SECO was catalysed by strains of Clostridium scindens and Eggerthella lenta. Finally, the newly isolated strain ED-Mt61/PYG-s6 catalysed the dehydrogenation of ED to EL. The results indicate that the activation of SDG involves phylogenetically diverse bacteria, most of which are members of the dominant human intestinal microbiota.     

Protective effect of secoisolariciresinol diglucoside against streptozotocin-induced diabetes and its mechanism

Objectives: Reactive oxygen species (ROS) have been implicated in the development of streptozotocin (STZ)-induced diabetes mellitus. Secoisolariciresinol diglucoside (SDG) isolated from flaxseed is an antioxidant. An investigation was made of the effects of SDG on the development of STZ-induced diabetes in rat, to determine if SDG can prevent/reduce the development of diabetes and if this prevention/reduction is associated with reduction in oxidative stress.Design and Methods: The rats were divided into 4 groups: Group I, Control; Group II, SDG (22 mg/kg body wt, orally) for 24 days; Group III, STZ (80 mg/kg intraperitoneally); Group IV, SDG in the dose similar to Group II three days prior to STZ and 21 days thereafter. Oxidative stress was assessed by measuring serum and pancreatic lipid peroxidation product malondialdehyde (MDA), pancreatic antioxidant reserve (pancreatic-CL) and oxygen free radical producing activity of white blood cells (WBC-CL). A diagnosis of diabetes was made on the basis of glucosuria and was confirmed at the time of sacrifice (21 days after STZ treatment) by the presence of hyperglycemia. At the end of the protocol blood samples were collected for estimation of glucose, MDA and WBC-CL, and pancreas were removed for estimation of MDA and antioxidant reserve.Results: Incidence of diabetes was 100% in Group III and 25% in Group IV. SDG prevented the development of diabetes by 75%. Development of diabetes was associated with an increase in serum and pancreatic MDA, and in WBC-CL, and a decrease in pancreatic antioxidant reserve. Prevention of diabetes by SDG was associated with a decrease in serum and pancreatic MDA and WBC-CL and an increase in pancreatic antioxidant reserve.Conclusions: These results suggest that STZ-induced diabetes is mediated through oxidative stress and that SDG is effective in reducing the STZ-induced diabetes mellitus.     

The effects of dietary flaxseed on the Fischer 344 rat: II. Liver gamma-glutamyltranspeptidase activity

The effect of 10% flax chow consumption from the 30th to the 130th day after birth was examined in male Fischer 344 rats. The effects of both the high lignan/high oil Norlin strain and a high lignan/low oil Solin strain of flaxseed were compared. Physically and behaviourally there were no differences in rats belonging to the three dietary groups at any time. At 50 and 100 days of dietary exposure, blood glucose levels were the same in Norlin and Solin flax chow-fed and as well as regular chow-fed rats; there were no signs of toxicity in the Norlin and Solin flax-fed rats since their plasma levels of alanine aminotransferase were the same and equal to those of regular chow-fed rats. The activity of gamma-glutamyltranspeptidase (gammaGT) displayed an increase in the liver homogenates of flax chow-fed rats. This increase was the same in Norlin and Solin flax-fed rats at 50 and 100 days. Thus the liver effect was not oil, but lignan, likely secoisolariciresinol diglucoside (SDG), induced and was effected early on, and sustained, after flax exposure. The degree of heat activation of liver homogenate gammaGT was the same in regular chow-fed and flax chow-fed rats. Compared to liver homogenate gammaGT activity, the soluble form of gammaGT was expressed at very low levels while the plasma membrane-bound form of gammaGT was expressed at very high levels in rat liver in both regular chow-fed and flax chow-fed rats. There was no effect of flax feeding on the soluble form of liver gammaGT which was expressed at a very low level. Flax feeding effected an increase in the activity of gammaGT in isolated plasma membrane fractions which mirrored that in liver homogenates: the same degree of increase was seen in Norlin flax chow-fed and Solin flax chow-fed rats. Flax consumption effects an increase in the activity of liver gammaGT at the level of the plasma membrane which is lignan dependent, physiologically relevant and may be linked to hepatoprotection against injury through an increase in reduced glutathione.     

亚麻木酚素的微波辅助提取工艺研究

采用微波辅助提取法从脱脂亚麻籽壳中提取亚麻木酚素,以磷钼酸显色的方法定量测定亚麻木酚素,通过单因素试验、中心组合试验及响应面分析,确定微波辅助提取的最优工艺条件为:乙醇浓度40.9%(v/v)、液固比21.9:1(mL/g)、超声处理5 min进行预浸、辐照时间90.5 s、微波功率为130 W。与常规溶剂提取法和索氏提取方法相比,微波辅助提取法显著提高了亚麻木酚素的得率,大大缩短了提取时间,并节省了能耗。     

Biarylheptanoids and other constituents from wood of Alnus japonica

The wood of Alnus japonica has been shown to contain a number of biarylheptanoids as well as other phenolics, including secoisolariciresinol diferulate. The co-occurrence of cyclized biarylheptanoids with their corresponding acyclic biarylheptanoids has been demonstrated and this fact may have biosynthetic significance. The possible chemotaxonomic importance of biarylheptanoids in members of the Betulaceae is discussed. The isolation and identification of several steroids and triterpenoids are also described.     

Oxidative stress as a mechanism of diabetes in diabetic BB prone rats: Effect of secoisolariciresinol diglucoside (SDG)

Secoisolariciresinol diglucoside (SDG) isolated from flaxseed has antioxidant activity and has been shown to prevent hypercholesterolemic atherosclerosis. An investigation was made of the effects of SDG on the development of diabetes in diabetic prone BioBreeding rats (BBdp rats), a model of human type I diabetes [insulin dependent diabetes mellitus (IDDM)] to determine if this type of diabetes is due to oxidative stress and if SDG can prevent the incidence of diabetes. The rats were divided into three groups: Group I, BioBreeding normal rats (BBn rats) (n = 10); group II, BBdp untreated (n = 11); and group III, BBdp treated with SDG 22 mg/kg body wt, orally) (n = 14). Oxidative stress was determined by measuring lipid peroxidation product malondialdehyde (MDA) an index of level of reactive oxygen species in blood and pancreas; and pancreatic chemiluminescence (Pancreatic-CL), a measure of antioxidant reserve. Incidence of diabetes was 72.7% in untreated and 21.4% in SDG-treated group as determined by glycosuria and hyperglycemia. SDG prevented the development of diabetes by approximately 71%. Development of diabetes was associated with an increase in serum and pancreatic MDA and a decrease in antioxidant reserve. Prevention in development of diabetes by SDG was associated with a decrease in serum and pancreatic-MDA and an increase in antioxidant reserve. These results suggest that IDDM is mediated through oxidative stress and that SDG prevents the development of diabetes.     

Lignans of Araucaria angustifolia and 13C NMR analysis of some phenyltetralin lignans

Secoisolariciresinol monomethyl ether and lariciresinol-4-methyl ether were isolated from the knots of dead trees of Araucaria angustifolia. On the basis of spectral evidence, the position of the OH group was located in these compounds. The ¹³C NMR spectra of the phenyltetralin lignans galbulin, galcatin, isogalcatin and cyclogalgravin have also been recorded and the signals assigned, based on the methyl shifts of cyclogalgravin.     

13C NMR spectral analysis of lignans from Araucaria angustifolia

Pinoresinol dimethyl ether, secoisolariciresinol, lariciresinol, isolariciresinol and isolariciresinol-4′-methyl ether were isolated from the knots of dead trees of Araucaria angustifolia. The ¹³C NMR spectra of these compounds, their methyl and acetyl derivatives, and the corresponding one of matairesinol, have been recorded and the signals assigned. On the basis of these assignments, the structure of the new monomethyl ether of isolariciresinol has been established.     

Cadmium induced MTs synthesis via oxidative stress in yeast Saccharomyces cerevisiae

Lignan complex has been isolated from flaxseed. It has been shown to reduce serum lipids and the extent of hypercholesterolemic atherosclerosis. However, it is not known whether the chronic use of lignan complex has any adverse effects on the hemopoietic system. The effects of lignan complex (40 mg/kg body wt orally daily for 2 months) on the red blood cells (RBC) count, mean corpuscular volume (MCV), red cell distribution width (RDW), hematocrit (Hct), hemoglobin (Hb), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and counts of white blood cell (WBC), granulocytes, lymphocytes, monocytes and platelet, and platelet volume were investigated in normo- and hypercholesterolemic rabbits. The results show that lignan complex had no adverse effects of counts of RBC, WBC, granulocytes, lymphocytes, monocytes and platelet in both the normo- and hyper-cholesterolemic rabbits. The values for MCV, RDW, Hct, Hb, MCH, MCHC, and platelet volume were similar in lignan complex-treated or untreated normo- and hypercholesterolemic rabbits. It is concluded that chronic use of lignan complex had no adverse effects on the hemopoietic system. (Mol Cell Biochem 270: 139–145, 2005)     

Flax seed lignan in disease prevention and health promotion

Lignans have been part of both diet and herbal medicines for centuries. It is only in the last half century that phytochemists have described the structures of the lignans. Pharmacologists have only become interested in the biological activity of lignans in the last few decades. Much of the early interest focused on podophyllotoxin type lignans and their derivatives. Recent literature has recorded very many new lignans or lignan derivatives with a diverse range of biological activities. In 1955, the isolation from flax seed and the structure of the lignan derivative secoisolariciresinol diglucoside (SDG) was reported. Possible biological activity of SDG, and the mammalian lignan metabolites, enterolactone and enterodiol, was initially reported about 20 years later. During the next 30 years, there has been extensive research on the biological effects of both flax seed and rye lignans since both are metabolized into the mammalian lignans. Research on the activity of lignans on breast, colon, prostate and thyroid cancer has generally shown beneficial effects although there are some studies with either no conclusive or negative effect. Lignans have been shown to have positive effects in lowering relative risk factors for heart disease. Use of flax seed or SDG has been shown to have positive effects in both lupus and polycystic kidney disease models. Studies of both type I and II diabetes models have reported positive results when using SDG. Flax seed has also been reported to be hepatoprotective. Reproductive effects have been observed with flax seed or SDG and have been found to be dose and time related. There are many possible mechanistic explanations for the observed bioactivities including involvement in hormonal metabolism or availability, angiogenesis, anti-oxidation and gene suppression. Abbreviations: ALA – alpha linolenic acid; ApcMin – adenomatous polyposis coli multiple intestinal neoplasia; BBdp – BioBreeding diabetic prone; CCl₄– carbon tetrachloride; CDC – Crop Development Centre; CHD – cardiovascular heart disease; DMBA – dimethylbenzanthracene; DNA – deoxyribonucleic acid; ER – estrogen receptor; λGT –γ-glutamyltranspeptidase; HDL – high-density lipoprotein; HMGA – hydroxymethyl glutaric acid; IDDM – insulin dependent diabetes mellitus; LDL – low-density lipoprotein; MRL/lpr, Murine Lupus/lymphoproliferative; MDA – malondiadehyde; NIDDM – non-insulin dependent diabetes mellitus; ORF – oxygen free radical; PAF – platelet activating factor; PKD – Polycystic kidney disease; PEPCK – phosphoenolpyruvate carboxykinase; PMNL – polymorphonuclear monocytes; STZ – streptozoticin; TC – total cholesterol; TG – triglycerides; SDG – secoisolariciresinol diglucoside; ZDF – Zucker diabetic fatty. This revised version was published online in June 2006 with corrections to the Cover Date.     

(+)-Pinoresinol/(-)-lariciresinol reductase from Linum perenne Himmelszelt involved in the biosynthesis of justicidin B

A cDNA encoding a pinoresinol-lariciresinol reductase PLR (PLR-Lp1) was isolated from a cell culture of Linum perenne Himmelszelt accumulating the arylnaphthalene lignan justicidin B. The recombinant PLR-Lp1 prefers (+)-pinoresinol in the first reaction step, but (-)-lariciresinol in the second step. Therefore, it is the first PLR described with opposite enantiospecificity within the two reaction steps catalysed by PLRs. Hairy root lines transformed with an ihpRNAi construct to suppress plr gene expression show less mRNA accumulation for the plr-Lp1 gene and PLR enzyme activity. Justicidin B accumulation was reduced down to 24% in comparison to control lines showing the involvement of PLR-Lp1 in the biosynthesis of justicidin B.     

Antioxidant activity of the flaxseed lignan secoisolariciresinol diglycoside and its mammalian lignan metabolites enterodiol and enterolactone

The antioxidant activities of the flaxseed lignan secoisolariciresinol diglycoside (SDG) and its mammalian lignan metabolites, enterodiol (ED) and enterolactone (EL), were evaluated in both lipid and aqueous in vitro model systems. All three lignans significantly (p 0.05) inhibited the linoleic acid peroxidation at both 10 and 100 M over a 24-48 h of incubation at 40°C. In a deoxyribose assay, which evaluates the non site-specific and site-specific Fenton reactant-induced ·OH scavenging activity, SDG demonstrated the weakest activity compared to ED and EL at both 10 and 100 M; the greatest ·OH scavenging for ED and EL was observed at 100 M in both assays. The incubation of pBR322 plasmid DNA with Fenton reagents together with SDG, ED or EL showed that the inhibition of DNA scissions was concentration dependent. The greatest non site-specific activity of lignans was at 100 M, thus, confirming the results of the deoxyribose test. In contrast, the protective effect of SDG and EL in the site-specific assay was lost and that of ED was minimal. Therefore, the results indicate a structure-activity difference among the three lignans with respect to specific antioxidant efficacy. All three lignans did not exhibit reducing activity compared to ascorbic acid, therefore, did not possess indirect prooxidant activity related to potential changes in redox state of transition metals. The efficacy of SDG and particularly the mammalian lignans ED and EL to act as antioxidants in lipid and aqueous in vitro model systems, at relatively low concentrations (i.e. 100 M), potentially achievable in vivo, is an evidence of a potential anticarcinogenic mechanism of flaxseed lignan SDG and its mammalian metabolites ED and EL.     

Impaired phosphatidylcholine biosynthesis and ascorbic acid depletion in lung during lipopolysaccharide-induced endotoxaemia in guinea pigs

Recently there has been a moderate resurgence in the use of flax-seed in a variety of ways including bread. The scientific basis of its use is very limited. There is some claim for beneficial effects in cancer and lupus nephritis. These claims could be due to its ability to scavenge oxygen radicals. However, its antioxidant activity is not known. Recently a method has been developed to isolate secoisolariciresinol diglucoside (SDG) from defatted flax-seed in large quantity (patent pending). We investigated the ability of SDG to scavenge úOH using high pressure liquid chromatography (HPLC) method. úOH was generated by photolysis of H2O2 (1.25-10.0 \sgmaelig;moles/ml) with ultraviolet light and was trapped with salicylic acid which is hydroxylated to produce úOH-adduct products 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA. H2O2 produced a concentration-dependent úOH as estimated by 2,3-DHBA and 2,5-DHBA. A standard curve was constructed for known concentrations of 2,3-DHBA and 2,5-DHBA against corresponding area under the peaks which then was used for measurement of 2,3-DHBA and 2,5-DHBA generated by UV irradiation of H2O2 in the presence of salicylic acid. SDG in the concentration range of 25, 50, 100, 250, 500, 750, 1000 and 2000 \sgmaelig;g/ml (36.4, 72.8, 145.6, 364.0, 728.0, 1092.0, 1456.0 and 2912.0 \sgmaelig;M respectively) produced a concentration-dependent decrease in the formation of 2,3-DHBA and 2,5-DHBA, the inhibition being 4 and 4.65% respectively with 25 \sgmaelig;g/ml (36.4 \sgmaelig;M) and 82 and 74% respectively with 2000 \sgmaelig;g/ml (2912.0 \sgmaelig;M). The decrease in úOH-adduct products was due to scavenging of úOH not and by scavenging of formed 2,3-DHBA and 2,5-DHBA. SDG prevented the lipid peroxidation of liver homogenate in a concentration-dependent manner in the concentration range from 319.3-2554.4 \sgmaelig;M. These results suggest that SDG scavenges úOH and therefore has an antioxidant activity.     

An historical perspective on lignan biosynthesis: Monolignol,allylphenol and hydroxycinnamic acid coupling and downstream metabolism

This review describes discoveries from this laboratory on monolignol, allylphenol and hydroxycinnamic acid coupling, and downstream metabolic conversions, affording various lignan skeleta. Stereoselective 8-8′ coupling (dirigent protein-mediated) of coniferyl alcohol to afford (+)-pinoresinol is comprehensively discussed, as is our current mechanistic/kinetic understanding of the protein’s radical-radical binding, orientation and coupling properties, and insights gained for other coupling modes, e.g. affording (−)-pinoresinol. In a species dependent manner, (+)- or (−)-pinoresinols can also undergo enantiospecific reductions, catalyzed by various bifunctional pinoresinol-lariciresinol reductases (PLR), to afford lariciresinol and then secoisolariciresinol. With X-ray structures giving a molecular basis for differing PLR enantiospecificities, comparisons are made herein to the X-ray structure of the related enzyme, phenylcoumaran benzylic ether reductase, capable of 8-5′ linked lignan regiospecific reductions. Properties of the enantiospecific secoisolariciresinol dehydrogenase (also discovered in our laboratory and generating 8-8′ linked matairesinol) are summarized, as are both in situ hybridization and immunolocalization of lignan pathway mRNA/proteins in vascular tissues. This entire 8-8′ pathway thus overall affords secoisolariciresinol and matairesinol, viewed as cancer preventative agent precursors, as well as intermediates to cancer treating substances, such as podophyllotoxin derivatives. Another emphasis is placed on allylphenol/hydroxycinnamic acid coupling and associated downstream metabolism, e.g. affording the antiviral creosote bush lignan, nordihydroguaiaretic acid (NDGA), and the fern lignans, blechnic/brainic acids. Regiospecific 8-8′ allylphenol coupling is described, as is characterization of the first enantiospecific membrane-bound polyphenol oxidase, (+)-larreatricin hydroxylase, involved in NDGA formation. Specific [¹³C]-labeling also indicated that Blechnum lignans arise from stereoselective 8-2′ hydroxycinnamic acid coupling. Abbreviations: CD – circular dichroism; e.e. – enantiomeric excess; DP – dirigent protein; ESI-MS – electrospray ionization mass spectrometry; MALDI -TOF – matrix assisted laser desorption ionization-time of flight; MALLS – multiangle laser light scattering; PLR – pinoresinol lariciresinol reductase; SDH – secoisolariciresinol dehydrogenase. This revised version was published online in June 2006 with corrections to the Cover Date.