Main Lignan Research Articles

오미자 열매 부위별 이화학적 특성

오미자 열매를 과피, 과육 및 씨 등을 부위별로 분리한 후 일반성분을 분석한 결과 모든 부분에서 가용성 무질소물의 함량이 각각 20.72, 6.88 및 24.42%로 가장 많이 함유되어 있으며 열매에서 씨 부분이 차지하는 비율은 40% 이상이었다. 또한, 오미자의 유효성분인 schizandrin을 포함한 리그난 화합물의 총량이 씨 부분에서 15.55 mg/g으로 가장 많이 함유되어 있고 schizandrin이 5.68 mg/g, gomisin N이 2.90 mg/g, gomisin A 1.55 mg/g, schizandrin C가 1.53 mg/g 함유되어 있었으며 열풍 건조한 오미자 열매 전과와 거의 비슷한 함량을 함유하고 있었다. 오미자의 유기산은 주로 과피 쪽에 있었으며 총유기산 함량은 12.99%를 함유하고 있었고 주된 유기산은 citric acid로 4.84%를 함유하고 있었다. 오미자의 지방 함량은 씨 부위 및 열풍 건조한 오미자 열매에서 각각 19.04%와 15.11%를 함유하고 있었고 지방을 구성하고 있는 주된 지방산은 linoleic acid로 각각 72.66%와 74.10%를 차지하고 있었으며, 그다음은 oleic acid로 각각 14.78%와 17.46%를 차지하고 있었다. 오미자의 주된 무기이온은 씨에서는 P로 725.35 mg/100 g을, 열풍 건조한 열매 전과에서는 K로 2,024.10 mg/100 g을 차지하고 있었다. 결론적으로 오미자를 활용하기 위해서는 리그난 화합물이 많이 함유되어 있는 씨 부분을 충분히 활용할 수 있는 방법을 고려하여야 할 것으로 생각된다.

Gamma-irradiation produces active chlorine species (ACS) in physiological solutions: Secoisolariciresinol diglucoside (SDG) scavenges ACS - A novel mechanism of DNA radioprotection

BackgroundSecoisolariciresinol diglucoside (SDG), the main lignan in whole grain flaxseed, is a potent antioxidant and free radical scavenger with known radioprotective properties. However, the exact mechanism of SDG radioprotection is not well understood. The current study identified a novel mechanism of DNA radioprotection by SDG in physiological solutions by scavenging active chlorine species (ACS) and reducing chlorinated nucleobases. MethodsThe ACS scavenging activity of SDG was determined using two highly specific fluoroprobes: hypochlorite-specific 3′-(p-aminophenyl) fluorescein (APF) and hydroxyl radical-sensitive 3′-(p-hydroxyphenyl) fluorescein (HPF). Dopamine, an SDG structural analog, was used for proton 1H NMR studies to trap primary ACS radicals. Taurine N-chlorination was determined to demonstrate radiation-induced generation of hypochlorite, a secondary ACS. DNA protection was assessed by determining the extent of DNA fragmentation and plasmid DNA relaxation following exposure to ClO− and radiation. Purine base chlorination by ClO− and γ-radiation was determined by using 2-aminopurine (2-AP), a fluorescent analog of 6-aminopurine. Results: Chloride anions (Cl−) consumed >90% of hydroxyl radicals in physiological solutions produced by γ-radiation resulting in ACS formation, which was detected by 1H NMR. Importantly, SDG scavenged hypochlorite- and γ–radiation-induced ACS. In addition, SDG blunted ACS-induced fragmentation of calf thymus DNA and plasmid DNA relaxation. SDG treatment before or after ACS exposure decreased the ClO− or γ-radiation-induced chlorination of 2-AP. Exposure to γ-radiation resulted in increased taurine chlorination, indicative of ClO− generation. NMR studies revealed formation of primary ACS radicals (chlorine atoms (Cl) and dichloro radical anions (Cl2¯)), which were trapped by SDG and its structural analog dopamine. ConclusionWe demonstrate that γ-radiation induces the generation of ACS in physiological solutions. SDG treatment scavenged ACS and prevented ACS-induced DNA damage and chlorination of 2-aminopurine. This study identified a novel and unique mechanism of SDG radioprotection, through ACS scavenging, and supports the potential usefulness of SDG as a radioprotector and mitigator for radiation exposure as part of cancer therapy or accidental exposure.

In silico Analysis and Experimental Validation of Lignan Extracts from Kadsura longipedunculata for Potential 5-HT1AR Agonists.

Objectives Kadsura longipedunculata (KL) has been widely used for the treatment of insomnia in traditional Chinese medicine. The aim of this study was to explore the mechanism of the sedative and hypnotic effects of KL.Materials and MethodsThe content of KL was evaluated by HPLC-TOF-MS, and a potential target was found and used to construct its 3D structure to screen for potential ligands among the compounds in KL by using bioinformatics analysis, including similarity ensemble approach (SEA) docking, homology modeling, molecular docking and ligand-based pharmacophore. The PCPA-induced insomnia rat model was then applied to confirm the potential targets related to the sedative effects of KL by performing the forced swimming test (FST), the tail suspension test (TST) and the measurement of target-related proteins using western blotting and immunofluorescence.ResultsBioinformatics analysis showed that most of lignan compounds in KL were optimal ligands for the 5-HT1A receptor (5-HT1AR), and they were found to be potential targets related to sedative effects; the main lignan content of KL extracts was characterized by HPLC-TOF-MS, with 7 proposed lignans detected. Administration of KL could significantly reduce FST and TST immobility time in the PCPA-induced 5HT-depleted insomnia rat model. The expressions of proteins related to the 5-HT1AR pathway were regulated by extracts of KL in a concentration-dependent manner, indicating that extracts of KL had 5-HT1AR agonist-like effects.Conclusion In silico analysis and experimental validation together demonstrated that lignan extracts from KL can target 5-HT1AR in insomniac rats, which could shed light on its use as a potential 5-HT1AR agonist drug.

Validation of an Electrochemical Detection–High-Performance Liquid Chromatography Method for Simultaneous Determination of Lignans in Flaxseed (Linum usitatissimum L.)

Flaxseed is a major source of lignans, which are important bioactive compounds. The aims of this work were to validate a liquid chromatographic method for the rapid and simultaneous determination of the main lignans in flaxseed using high-performance liquid chromatography coupled with electrochemical detection and to analyze the composition of commercial samples of flaxseed. The performance criteria of the proposed method demonstrate that the method can be used for the analysis of secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO), matairesinol (MATA), pinoresinol (PINO), lariciresinol (LARI), hydroxymatairesinol (HYDROXY), and isolariciresinol (ISOLARI) in flaxseeds at suitable levels. Calibration curves were determined for six different concentrations of standard solutions injected in triplicate. The sensitivity of the calibration curve was evaluated considering the confidence intervals of the intercept and slope. The limit of detection and limit of quantification were 7.4 and 10.9 μg/l, respectively, for LARI and 17.7 and 37.5 μg/l, respectively, for MATA. The relative standard deviation of repeatability values were lower than 2.59 %, which are acceptable because the Horwitz ratio values were 0.1 for all of the lignans. The recoveries of lignans were in the range of 74–100 % of SECO, which are consistent with the literature. The precision of the proposed method was determined by analyzing four flaxseed samples of different years and varieties. SDG was the main lignan present in all the samples, followed by ISOLARI and HYDROXY.

Seasonal Variations in the Deoxypodophyllotoxin Content and Yield of Anthriscus sylvestris L. (Hoffm.) Grown in the Field and under Controlled Conditions

Deoxypodophyllotoxin (DPT) is the main lignan in Anthriscus sylvestris . For this study two sets of experiments with 16 plants and seeds, collected from a wide geographical range, were carried out. The DPT content in roots was significantly lower (p < 0.05) when the plants were cultivated in a non-native environment. For field-grown plants the highest DPT content was found in March (second year): 0.15% w/w (dry weight) in roots; 0.03% w/w in aerial parts. For plants grown in the climate room, the highest concentration (0.14% w/w) was observed in April (second year) in the roots and in July (first year) in the aerial parts (0.05% w/w). For the isolation of DPT, roots are the most suitable part. The best harvest times are March (second year) for outdoor plants and April (second year) for indoor plants when height content and adequate biomass give the optimal DPT yield.

Investigation of lignan accumulation in developing Linum usitatissimum seeds by immunolocalization and HPLC

Lignans are widely distributed plant metabolites associated with a large range of biological activities. An immunological probe was previously developed to immunolocalize lignans in mature flaxseed ( Linum usitatissimum) and allowed to show that they are mainly localized in the secondary wall of the sclerite cells layer of the outer integument of the seed. In order to precise the temporal regulation of these secondary metabolites, L. usitatissimum plants were cultivated and their developing seeds were investigated according to their SDG (secoisolariciresinol diglucoside) content (main lignan in seeds) by immunolocalization and HPLC techniques. The HPLC results showed that SDG content increased as a function of the developmental stage to reach a maximum of about 9 mg/g at the end of seed maturation. In addition, the seed dissection during seed maturation allowed to observe a similar pattern of evolution of SDG content in the outer integument whereas this content decreased in the inner integument and the endosperm. These results were confirmed by immunolocalization experiments.

Production of podophyllotoxin in Linum linearifolium in vitro cultures

For the first time, callus and suspension cultures of Linum linearifolium were initiated. Podophyllotoxin (PTOX), a strong antitumor precursor, was isolated from the calli and suspension, as a main lignan besides smaller amount of 6-methoxypodophyllotoxin (6MPTOX). L. linearifolium is now the third Linum species of section Syllinum, with PTOX as the main lignan. The amounts of lignans, especially PTOX, found in L. linearifolium cell cultures are quite high within the studied Linum species until now. The antiproliferative effects of extracts were tested in a panel of human tumor cell lines, using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]-dye reduction assay. The lignan mixtures caused concentration-dependent inhibition of malignant cell proliferation and showed moderate cytotoxic activity. The results clearly demonstrate that the lignan mixture of L. linearifolium exerts inhibitory effects against malignant cells.

Selection of optimal conditions for separating lignan-containing extract from oil flax seed by thin-layer chromatography

The optimum conditions for separating a lignan-containing extract from seeds of the oil flax (Linum usitatissimum) by thin-layer chromatography were identified. Elution using the method proposed here allowed identification of a brilliant blue fluorescent spot associated with the main lignan, which was secoisolariciresinol diglucoside (SDG), giving clear identification of this component in mixtures even without a standard. This method can be used for qualitative express analysis of phytopreparations based on lignans, with the aim of detecting adulterated phytopreparations and biologically active additives.

Antioxidants and dairy production: the example of flax

This manuscript reports on the main problems decreasing productivity of dairy cows (e.g. fatty liver syndrome and poor fertility) and how antioxidants could enhance it. High producing dairy cows are prone to oxidative stress, and the situation can be exacerbated under certain environmental, physiological, and dietary conditions. Antioxidants have important effects on the expression of genes involved in the antioxidant status, which may enhance animal health and reproduction. Moreover, antioxidants may contribute to decrease the incidence of spontaneous oxidized flavour in milk enriched in polyunsaturated fatty acids. Plant lignans are strong antioxidants and flax is the richest source of plant lignans. Flax lignans are converted in the mammalian lignans enterolactone and enterodiol. The main mammalian lignan in milk is enterolactone and flax lignans are converted in enterolactone mainly under the action of ruminal microbiota. Therefore, ruminal microbiota may be the most important flora to target for plant lignan metabolism in order to increase concentration of mammalian lignan antioxidants in milk of dairy cows. However, more research is required to improve our knowledge on metabolism of other antioxidants in dairy cows and how they can contribute in decreasing milk oxidation.

Bioactive phenols in leaves of Forsythia species

Nowadays a number of lignans (arctigenin, matairesinol, pinoresinol and phillygenin) have come to the fore in research due to their various biological activities. In this paper the accumulation of these constituents in leaf extracts of Forsythia plants (F. intermedia, F. ovata 'Robusta’ and `Tetragold', F. suspensa, F. viridissima) was quantified using a new isolation method, supercritical CO2 fluid extraction. The total phenolic and flavonoid contents, the antioxidant capacity and the aglycone lignan profile were determined in leaf extracts of Forsythia species. Within the phenols, the flavonoids were only present in small quantities, but the amount of aglycone lignans was extremely high. F. ovata `Robusta' had the highest total lignan content (103.8 mg/g) of all the Forsythia species. The main lignan in this species is arctigenin, which normally makes up about 60% of the total lignan content, but in the case of F. ovata `Robusta' this value was 96.1%. Since this arctigenin content is outstanding compared to that of other Forsythia species, it could be promising to develop a fermentation technology for the production of this natural compound.

REGIONAL DISTRIBUTION IN THE FATTY ACIDS OF TRIACYLGLYCEROLS AND PHOSPHOLIPIDS OF SESAME SEEDS (SESAMUM INDICUM)

ABSTRACT The fatty acid distribution of triacylglycerols (TAGs) and major phospholipids (PLs) in sesame (Sesamum indicum) seeds was investigated in relation to the endogenous antioxidants. γ‐Tocopherol was present in the highest concentration, and δ‐ and α‐tocopherols were found in very small amounts. Sesamin and sesamolin were the main lignan components. The lipids extracted from three cultivars were separated by thin‐layer chromatography. The major lipid components were TAGs and PLs, while steryl esters, free fatty acids and sn‐1,3‐ and sn‐1,2‐diacylglycerols were also present in minor proportions. Significant differences (P &lt; 0.05) in fatty acid distribution existed when different sesame seed cultivars were examined. However, the principal characteristics of the fatty acid distribution in the TAGs, as well as the major PLs, were evident among the three cultivars; unsaturated fatty acids were predominantly concentrated in the sn‐2‐position, and saturated fatty acids primarily occupied the sn‐1‐ or sn‐3‐position. These results suggest that there are no essential differences (P &gt; 0.05) in the oil components among three cultivars.PRACTICAL APPLICATIONSThis paper describes the fatty acid compositions of glycerides, particularly the positional distribution of the fatty acids of triacylglycerols along with those of the main PL in sesame seeds in relation to the endogenous antioxidants. These results were similar to the positional fatty acid distributions in the lipid classes of peanut, corn and soybeans among the plants seeds in the literature, and suggest that there are no essential differences (P &gt; 0.05) in the oil components among the three cultivars. The data obtained would be contributed to both producers and consumers to investigate the functional properties of sesame seed oil.

Microwave‐assisted extraction of the main phenolic compounds in flaxseed

A microwave-assisted extraction (MAE) method has been applied for the first time to the extraction of the main lignan, secoisolariciresinol diglucoside (SDG), and the two most concentrated hydroxycinnamic acid glucosides in flaxseed. The effects of microwave power, extraction time and alkaline treatment were investigated. It was shown that a 3 min MAE resulted in an SDG content of 16.1+/-0.4 mg/g, a p-coumaric acid glucoside content of 3.7+/-0.2 mg/g and a ferulic acid glucoside content of 4.1+/-0.2 mg/g. These values were compared with those obtained using conventional extraction methods and the results demonstrated that MAE was more effective in terms of both yield and time consumption.

Antioxidant Distributions and Triacylglycerol Molecular Species of Sesame Seeds (Sesamum indicum)

AbstractExtracted lipids from sesame (Sesamum indicum) seeds of three varieties were determined by high‐performance liquid chromatography (HPLC) for endogenous antioxidants. The molecular species and fatty acid (FA) distribution of triacylglycerol (TAG) isolated from total lipids in sesame seeds were analyzed by a combination of argentation thin‐layer chromatography (TLC) and gas chromatography (GC), and were investigated in relation to their antioxidant distribution. γ‐Tocopherol was present in highest concentration, and δ‐, and α‐tocopherols were very small amounts. Sesamin and sesamolin were the main lignan components. A modified argentation‐TLC procedure, developed to optimize the separation of the complex mixture of total TAG, provided 12 different groups of TAG, based on both the degree of unsaturation and the total acyl‐chain length of FA groups. With a few exceptions, the major TAG components were SM2 (6.5–6.7%), SMD (19.8–20.7%), M2D (15.0–26.3%), MD2 (23.6–35.0%), and D3 (7.7–10.7%) (where S denotes a saturated FA, M denotes a monoene, D denotes a diene, and T denotes a triene). It seems that the three varieties were highly related to each other based on the FA composition of the TAG as well as the distribution pattern in the different TAG molecular species. These results suggest that there are no essential differences in the oil components among the three varieties.

Pinoresinol–lariciresinol reductase gene expression and secoisolariciresinol diglucoside accumulation in developing flax (Linum usitatissimum) seeds

The transcription activity of the pinoresinol-lariciresinol reductase (PLR) gene of Linum usitatissimum (so-called LuPLR), a key gene in lignan synthesis, was studied by RT-PCR and promoter-reporter transgenesis. The promoter was found to drive transcription of a GUSint reporter gene in the seed coats during the flax seed development. This fitted well with the tissue localization monitored by semi-quantitative RT-PCR of LuPLR expression. Accumulation of the main flax lignan secoisolariciresinol diglucoside was coherent with LuPLR expression during seed development. This three-way approach demonstrated that the LuPLR gene is expressed in the seed coat of flax seeds, and that the synthesis of PLR enzyme occurs where flax main lignan is found stored in mature seeds, confirming its involvement in SDG synthesis.

In vivo anti-osteoporotic activity of isotaxiresinol, a lignan from wood of Taxus yunnanensis

Isotaxiresinol, the main lignan isolated from the water extract of wood of Taxus yunnanensis, was investigated for its effect on bone loss, on serum biochemical markers for bone remodeling and on uterine tissue, using ovariectomized (OVX) rats as the model of postmenopausal osteoporosis. After oral administration of isotaxiresinol (50 and 100mg/kg/d) for 6 weeks, bone mineral content (BMC) and bone mineral density (BMD) in total and cortical bones were increased as compared to those of OVX control rats, and decreases of three bone strength indexes induced by OVX surgery were prevented. Serum biochemical markers for bone remodeling revealed that isotaxiresinol slightly increased bone formation and significantly inhibited bone resorption without side effect on uterine tissue. These results suggest that isotaxiresinol may be useful for treatment of postmenopausal osteoporosis, especially for prevention of bone fracture induced by estrogen deficiency.

Cytotoxic activity of extracts from Linum cell cultures

Callus and suspension cultures of Linum narbonense and Linum leonii were developed to study the production of lignans and their cytotoxic activity. Justicidin B was determined to be the main lignan. The maximal yield of justicidin B up to 2.22 mg/g of the cell dry weight was detected in the callus cultures of L. leonii, followed by the callus cultures of L. narbonense (1.57 mg/g dwt). The cytotoxicity of the obtained extracts was measured using the MTT-dye assay. L. narbonense and L. leonii both showed cytotoxic activity.

Lignans in selected wines

Wines have been studied a lot and several different groups of phenolic compounds have been analysed in wine samples, but lignans in wines have been only preliminary studied. In this study, we analysed 10 different wines of which eight were red wines and two white wines. Samples were selected among the most consumed wines in Finland in year 1999. Eight different lignans were analysed with HPLC using coulometric electrode array detector. The amount of lignans in red wines ranged from 0.812 to 1.406 mg/l. The main lignan in all studied wines was isolariciresinol and percentage amount of mammalian lignan precursors varied from 34 to 43% in the red wines. The amount of lignans was much lower in the studied white wines compared with the red wines.

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.

Accumulation of lignans in suspension cultures of Linum mucronatum ssp. armenum (Bordz.) Davis.

For the first time callus and suspension cultures of Linum mucronatum ssp. annenum were initiated, grown in darkness at 25 degrees C and analyzed for lignans. 6-Methoxypodophyllotoxin was the main lignan besides smaller amounts of podophyllotoxin isolated and identified by chromatographic methods as well as by 1H NMR.

An oligomer from flaxseed composed of secoisolariciresinoldiglucoside and 3-hydroxy-3-methyl glutaric acid residues

A straight-chain oligomeric structure composed of five secoisolariciresinoldiglucoside (SDG) residues interconnected by four 3-hydroxy-3-methyl glutaric acid (HMGA) residues (molecular weight ca. 4000 Da) was assigned to the main lignan of flaxseed on the basis of nuclear magnetic resonance spectroscopy (NMR).