Structural determinants of plant lignans for the formation of enterolactone in vivo

The quantity of mammalian lignans enterolactone (ENL) and enterodiol (END) and of plant lignans secoisolariciresinol (SECO) and 7-hydroxymatairesinol (HMR) excreted in a 24-h rat urine sample was measured after a single p.o. dose of an equivalent quantity of secoisolariciresinol diglycoside (SDG), secoisolariciresinol (SECO), matairesinol (MR), 7-hydroxymatairesinol (HMR) and ENL. Plant lignans (SECO and HMR) were partially absorbed as such. The aglycone form of SECO was more efficiently converted into mammalian lignans END and ENL than the glycosylated form, SDG. Of plant lignans, MR produced the highest quantities of ENL: the quantity was over twofold compared with HMR or SDG. The majority of the animals, which had been given SECO, excreted higher quantities of END than ENL into urine, but ENL was the main lignan metabolite after SDG. The highest quantities of ENL in urine were measured after the administration of ENL as such. The (-)SECO isolated from Araucaria angustifolia was converted into (-)ENL only. The administration of (-)SDG, which was shown to produce (+)SECO, resulted in excretion of (+)ENL only and (-)HMR was converted into (-)ENL only. This confirmed that the absolute configurations at C8 and C8' are not changed during the microbial metabolism. Whether the biological effects are enantiomer-specific, remains to be resolved.     

Urinary excretion of lignans after administration of isolated plant lignans to rats: the effect of single dose and ten-day exposures

The difference in urinary excretion of mammalian and plant lignans in rats was determined after oral administration of equivalent doses (25 mg/kg of body weight) of 7-hydroxymatairesinol (HMR), lariciresinol (LAR), matairesinol (MR), and secoisolariciresinol (SECO). Twenty-four hours-urine samples were collected after a single dose and after administration of one dose/day for 10 days. Eight lignans were analysed in urine extracts using a high-performance liquid chromatography-tandem mass spectrometry method showing good sensitivity and repeatability. After a single dose of HMR, LAR, MR, and SECO, the main metabolites were 7-hydroxyenterolactone (HENL), cyclolariciresinol (CLAR), enterolactone (ENL), and enterodiol (END), respectively, but after 10-day exposure ENL was the main metabolite of all the tested lignans, showing a considerably higher excretion than after a single dose. Metabolic transformations of plant lignans into each other could also be observed.     

Anticancer effects of a plant lignan 7-hydroxymatairesinol on a prostate cancer model in vivo

Clinical intervention studies and experimental studies with lignan-rich diets suggest that lignans may have inhibitory effects on prostate cancer, but no clinical or experimental studies with purified lignans have been published. The purpose of this study was to investigate the effect of a plant lignan 7-hydroxymatairesinol (HMR) on LNCaP human prostate cancer xenografts in athymic mice. Athymic nude male mice were injected subcutaneously with LNCaP cells. Starting 3 days after tumor cell injections, a control diet or a control diet supplemented with 0.15% or 0.30% of HMR was administered to mice and the tumor take rate and growth was observed for 9 weeks. HMR diet inhibited the growth of LNCaP tumors. Mice treated with HMR had smaller tumor volume, lower tumor take rate, increased proportion of nongrowing tumors, and higher tumor cell apoptotic index compared with controls. Furthermore, the cell proliferation index was reduced in mice receiving the 0.30% HMR diet compared with mice receiving the control diet. Our results suggest that dietary HMR started at the early phase of the tumor development inhibits the growth of the LNCaP human prostate cancer xenografts in athymic male mice.     

Knots in trees – A new rich source of lignans

Recent research in our group has revealed that knots, i.e. the branch bases inside tree stems, commonly contain 5–10% (w/w) of lignans. Norway spruce (Picea abies) knots contain as much as 6–24% of lignans, with 7-hydroxymatairesinol (HMR) as the predominant (70–85%) lignan. Some other spruce species also contain HMR as the main lignan, but some spruce species have also other dominating lignans. Most fir (Abies) species contain secoisolariciresinol and lariciresinol as the main lignans. Lignans occur also in knots of pines (Pinus spp.), although in lower amounts than in spruces and firs. Scots pine (Pinus silvestris) knots were found to contain 0.4–3% of lignans with nortrachelogenin as the main lignan. Lignans have been identified also in knots of some hardwoods, although flavonoids are more abundant in hardwoods. Knots are detrimental in the manufacture of pulp and paper and should preferably be removed before pulping. This is possible using a recently developed industrially applicable process called ChipSep. Recent research has also established novel synthetic routes to several lignans, such as matairesinol, secoisolariciresinol, lariciresinol and cyclolariciresinol, starting from hydroxymatairesinol by applying fairly straight-forward chemical transformations. We conclude that wood knots in certain spruce and fir species constitute the richest known source of lignans in nature. The lignans occur in knots in free form and are easily extracted by aqueous ethanol, or even by water. Not only HMR, but also other potentially valuable lignans, could be produced in a scale of hundreds of tons per year by extraction of knots separated from wood chips at pulp and paper mills.     

Mammalian phytoestrogens: enterodiol and enterolactone

The mammalian phytoestrogens enterodiol (END) and enterolactone (ENL) are produced in the colon by the action of bacteria on the plant precursors matairesinol (MAT), secoisolariciresinol (SECO), their glycosides, and other precursors in the diet. Both END and ENL have been shown to possess weakly estrogenic and antiestrogenic activities, and it has been suggested that the high production of these antiestrogenic mammalian lignans in the gut may serve to protect against breast cancer in women and prostate cancer in men. Various in vitro experiments suggested END and ENL significantly inhibited the growth of human colon tumor cells, and the E2-induced proliferation of MCF-7 breast cancer cells was inhibited by ENL. The protective effects of mammalian lignans may be due to their ability to compete with E2 for the type II estrogen receptor, to induce sex hormone binding globulin (SHBG), to inhibit placental aromatase, and to act as antioxidants. This review mainly deals with the chemistry, quantitative analysis, biological properties and health effects of END and ENL.     

Determination of lignans in human plasma by liquid chromatography with coulometric electrode array detection

The presence of mammalian lignans, mainly enterolactone, in human plasma has been related to lower incidence of certain cancers and cardiovascular disease. The plant lignans secoisolariciresinol and matairesinol have been reported to be precursors of mammalian lignans, but recently other plant lignans relatively abundant in the diet have also been identified as precursors. To evaluate the importance and contribution of these new dietary precursors to the mammalian lignan formation in vivo, metabolic studies in human subjects must be carried out. For this purpose a method based on high-performance liquid chromatography using coulometric electrode array detection for the simultaneous determination of nine plant lignans and two mammalian lignans in human plasma was developed, validated, and shown to fulfill the reliability criteria.     

Antidiabetic effect of enterolactone in cultured muscle cells and in type 2 diabetic model db/db mice

Enterolactone (ENL) is formed by the conversion of dietary precursors like strawberry lignans via the gut microbiota. Urinary concentrations of lignan metabolites are reported to be significantly associated with a lower risk of Type 2 diabetes (T2D). In the present study, antidiabetic effect of ENL and its modes of action were studied in vitro and in vivo employing a rat skeletal muscle-derived cell line, L6 myocytes in culture, and T2D model db/db mice. ENL dose-dependently increased glucose uptake in L6 myotubes under insulin absent condition. This increase by ENL was canceled by compound C, an inhibitor of 5′-adenosine monophosphate-activated protein kinase (APMK). Activation (=phosphorylation) of AMPK and translocation of glucose transporter 4 (GLUT4) to plasma membrane in L6 myotubes were demonstrated by Western blotting analyses. Promotion by ENL of GLUT4 translocation to plasma membrane was also visually demonstrated by immunocytochemistry in L6 myoblasts that were transfected with glut4 cDNA-coding vector. T2D model db/db mice were fed the basal 20 % casein diet (20C) or 20C supplemented with ENL (0.001 or 0.01 %) for 6 weeks. Fasting blood glucose (FBG) levels were measured every week and intraperitoneal glucose tolerance test (IPGTT) was conducted. ENL at a higher dose (0.01 % in 20C) suppressed the increases in FBG levels. ENL was also demonstrated to improve the index of insulin resistance (HOMA-IR) and glucose intolerance by IPGTT in db/db mice. From these results, ENL is suggested to be an antidiabetic chemical entity converted from dietary lignans by gut microbiota.     

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.     

Biological activities of lignans

Lignans are widely distributed in angiosperms and gymnosperms. The range of their structures and biological activities is broad. Various lignans are known to have anti-tumour, antimitotic and antiviral activity and to specifically inhibit certain enzymes. Toxicity to fungi, insects and vertebrates is observed for some lignans and a variety of physiological activities have been documented. This review summarizes what is presently known about the biological activities of lignans.     

Metabolism of isoflavones, lignans and prenylflavonoids by intestinal bacteria: producer phenotyping and relation with intestinal community

Many studies have investigated the importance of the intestinal bacterial activation of individual phytoestrogens. However, human nutrition contains different phytoestrogens and the final exposure depends on the microbial potential to activate all different groups within each individual. In this work, interindividual variations in the bacterial activation of the different phytoestrogens were assessed. Incubation of feces from 100 individuals using SoyLife EXTRA, LinumLife EXTRA and isoxanthohumol suggested that individuals could be separated into high, moderate and low O-desmethylangolensin (O-DMA), equol, enterodiol (END), enterolactone (ENL) or 8-prenylnaringenin producers, but that the metabolism of isoflavones, lignans and prenylflavonoids follows separate, independent pathways. However, O-DMA and equol production correlated negatively, whereas a positive correlation was found between END and ENL production. In addition, END production correlated negatively with Clostridium coccoides-Eubacterium rectale counts. Furthermore, O-DMA production was correlated with the abundance of methanogens, whereas equol production correlated with sulfate-reducing bacteria, indicating that the metabolic fate of daidzein may be related to intestinal H(2) metabolism.     

Using NMR to determine the relative stereochemistry of 7,7-diaryl-8,8′-dimethylbutan-1-ol lignans

Due to their linear, freely rotatable, structure many natural 7,7-diaryl-8,8′-dimethylbutan-7′-ol lignans are reported without any stereochemical assignment. Analysis of synthetic 8,8′-dimethylbutanol lignans and analogues reveals significant differences between the NMR data of syn- and anti-isomers. This information was then used to determine the relative stereochemistry of the C-8 and C-8′ methyl groups in previously undefined natural products.     

New Lignans and Their Biological Activities

Lignans, which are widely distributed in higher plants, represent a vast and rather diverse group of phenylpropane derivatives. They have attracted considerable attention due to their pharmacological activities. Some of the lignans have been developed approved therapeutics, and others are considered as lead structures for new drugs. This article is based on our previous review of lignans discovered in the period 2000–2004, and it provides a comprehensive compilation of the 354 new naturally occurring lignans obtained from 61 plant families between 2005 and 2011. We classified five main types according to their structural features, and provided the details of their sources, some typical structures, and diverse biological activities. A tabular compilation of the novel lignans by species is presented at the end. A total of 144 references were considered for this review.     

The American mayapple revisited—podophyllum peltatum—still a potential cash crop?

Podophyllum peltatum, was reexamined for its potential use in the commercial production of podophyllotoxin, a lignan used in the semisynthesis of important anticancer drugs. A survey of the natural population of the American mayapple, Podophyllum peltatum, was conducted in order to identify high-yielding genotypes. Plants were collected from the eastern and central United States. The lignan content of leaf blades and rhizome material of the collected specimens was characterized by aqueous extraction followed by HPLC analysis. Podophyllotoxin and α-peltatin appeared most prominently among the lignans obtained. Leaf blades were generally richer in podophyllotoxin than rhizomes. Several high-yielding accessions were identified, the blades of which contained 4.0–5.6% podophyllotoxin. A negative correlation was observed between podophyllotoxin and peltatin content in the blades. The combination of high biosynthetic capacity and preferential accumulation of podophyllotoxin in leaves of mayapple makesthis plant an excellent candidate for agricultural production of podophyllotoxin.     

Liquid chromatography method for plant and mammalian lignans in human urine

Recently new mammalian lignan precursors were identified but no analysis methods are available for assay of those compounds in human urine. Previously published methods were developed for GC-MS about only two plant lignans were included. Consequently, a method for HPLC equipped with a coulometric electrode array detector was developed to measure plant and mammalian lignans in human urine. The plant lignans, secoisolariciresinol (Seco), matairesinol (Mat), lariciresinol (Lar), pinoresinol (Pin), syringaresinol (Syr) and isolariciresinol (IsoL) were included into the new method together with two mammalian lignans, enterolactone (Enl) and enterodiol (End). Validation of the method demonstrated that it could be applied to normal urine containing low amounts of plant lignans and moderate amounts of mammalian lignans, but the method was also applicable for samples from study subjects in supplementation studies, i.e. sample with very high concentrations of mammalian lignans. The method was found to be a useful tool for studies on plant lignan intake and the activity of micro flora in the metabolism of plant lignans.     

Hydroxycinnamic acids are ester-linked directly to glucosyl moieties within the lignan macromolecule from flaxseed hulls

In flaxseed hulls, lignans are present in an oligomeric structure. Secoisolariciresinol diglucoside (SDG), ester-linked to hydroxy-methyl-glutaric acid (HMGA), forms the backbone of this lignan macromolecule. The hydroxycinnamic acids p-coumaric acid glucoside (CouAG) and ferulic acid glucoside (FeAG) are also part of the lignan macromolecule. However, their position and type of linkage are still unknown. The aim of this study was to investigate how CouAG and FeAG are linked within the lignan macromolecule from flaxseed hulls. Fragments of the lignan macromolecule were obtained by partial saponification. After isolation of the fragments by preparative RP-HPLC, several key structures were identified by MS and NMR. Within the lignan macromolecule, CouAG is attached to the C-6 position of a glucosyl moiety of SDG. FeA is linked to the C-2 position of a glucosyl moiety of SDG. FeAG is ester-linked within the lignan macromolecule with its carboxyl group, but it remains unclear whether FeAG links to the C-2 or C-6 position of SDG. Attachment of HMGA to the glucosyl moiety of CouAG or FeAG was not observed. The results clearly show that within the lignan macromolecule, the hydroxycinnamic acids are linked directly via an ester bond to the glucosyl moiety of SDG.     

Development of antibodies against secoisolariciresinol--application to the immunolocalization of lignans in Linum usitatissimum seeds

Lignans are widely distributed plant metabolites associated with a large range of biological activities. In order to gain insight into their biosynthesis and their spatio-temporal accumulation an immunological probe was developed. Secondary metabolites generally have too small molecular weight to be antigenic and have to be associated with a carrier protein. Secoisolariciresinol was chosen as the hapten and was linked to bovine serum albumin via a spacer arm, the p-aminohippuric acid. The artificial antigen was injected to New Zealand rabbits. The successful production of polyclonal antibodies against secoisolariciresinol was assessed with indirect enzyme immunosorbent assay (ELISA) by comparison with pre-immune serum and by competitive assays using dilutions of secoisolariciresinol standards. The antibodies had an IC(50) value of 94 μg/ml and showed moderate cross-reactivities with structurally related compounds. They were thus used to immunolocalize lignans in flaxseed (Linum usitatissimum), one of the richest sources of lignans. The immunohistochemical labeling allowed us to localize for the first time lignans in planta. They are mainly localized in the secondary wall of the sclerite cells of the outer integument of the seed. A very light labeling is also observed in cytoplasmic inclusions of the endosperm. The results were correlated with HPLC analytical results which enabled to evaluate the relative lignan quantities: in flaxseed about 90% of the metabolites are localized in the outer integument.     

New lignans in Conocarpus erectus

The wood of Conocarpus erectus contains conocarpol and 2′-methoxyconocarpol, simple 1,4-diarylbutane-type lignans, and conocarpan, a lignan of the dehydrodi-isoeugenol type.     

Lignans in plant cell and organ cultures: An overview

Lignans are found in a wide variety of plant species. The lignan podophyllotoxin is of special interest, since its derivatives like e.g. etopophos® are used in anticancer therapy. As chemical synthesis of podophyllotoxin is not yet economic, it still has to be isolated from wild growing Podophyllum species, some of which are considered to be endangered species. Therefore plant in vitro cultures may serve as alternative sources for podophyllotoxin and for other types of lignans as well. This review describes the establishment of plant cell and tissue cultures for lignan production and the experiments to improve product yields by changing the cultivation parameters, addition of elicitors and feeding of precursors. It also summarizes the use of plant cell and organ cultures to study the biosynthesis of lignans on enzymological level. Abbreviations: DOP – deoxypodophyllotoxin; LARI – lariciresinol; MATAI – matairesinol; 6MPTOX – 6-methoxypodophyllotoxin; PINO – pinoresinol; PTOX – podophyllotoxin; SECO – secoisolariciresinol     

Biosynthesis of antimalarial lignans from Holostylis reniformis

Holostylis reniformis biosynthesizes 8-8′ linked lignans without 9,9′-oxygenation. To elucidate the biosynthetic pathways to these lignans, the reputed precursors [U-¹⁴C]phenylalanine, [9-³H₁]coniferyl alcohol, and [9-³H₁]isoeugenol were administered to roots of the plant, which led to the incorporation of ³H and ¹⁴C into ten 2,7′ linked-lignans (aryltetralone lignans) and two 7,7′-epoxylignans (furan lignans). These administration experiments demonstrated that the lignans were propenylphenol-derived and that H. reniformis can exhibit regioselective control over radical-radical coupling (via isoeugenol radicals). Regiospecific control over propenylphenol-derived lignan biosynthesis was observed, together with diastereoselective control of C2-C7′ bond formation for the aryltetralone lignans (7′R). These experiments provide evidence that isoeugenol is a biosynthetic intermediate to the aryltetralone and furan lignans.     

Six new dihydrobenzofuran lignans from the branches and leaves of Illicium wardii and their cytotoxic activities

Six new dihydrobenzofuran lignans, named illiciumlignans A⿿F (compounds 1⿿6), along with 15 known compounds (7⿿21) were isolated from the branches and leaves of Illicium wardii. The structures of 1⿿6 were determined using a combination of 1D and 2D NMR, HR-ESI⿿-MS, and CD spectroscopic data. Illiciumlignan D (4) is the first reported dihydrobenzofuran lignan arabinofuranoside that is derivatized with the arabinofuranose moiety on C-9⿲. Compounds 1⿿21 were evaluated for cytotoxic activity against four human cancer cell lines. Compounds 8, 12 and 20 exhibited significant activity against human cancer cell lines (A549, SKOV3, HepG2 and HCT116), with IC₅₀ values ranging from 2.7 to 14.9 μM.