Identification and bioactivity evaluation of ring-opening and lactone forms of enterolactone from sheep fed flaxseed cake.

The previously undescribed lactone ring-opening enterolactone and its sulphate were purified along with the lactone counterparts from the urine of dairy sheep fed flaxseed cake. The structures were determined by NMR and MS analyses. The ring-opening and lactone forms underwent mutual transformation with changes in pH and milk could protect the lactone form. Enterolactone exhibited more effective anti-proliferation activity on MDA-MB-231 breast cancer cells than its ring-opening counterpart, while the ring-opening enterolactone demonstrated more effective anti-osteoporosis activity than the lactone form. The results indicated the potential for targeting biological functions through pH and medium manipulation.

Anti-osteoporosis effects of mammalian lignans and their precursors from flaxseed and safflower seed using zebrafish model.

Secoisolariciresinol diglucoside (SDG) and tracheloside (TCL) are the main lignan components of flaxseed cake and safflower seed cake, which are by-products of oil extraction. Both SDG and TCL are metabolized into mammalian lignan enterolactone (EL) with the involvement of intestinal bacteria. In this research, we evaluated the anti-osteoporosis effects of SDG and the in vivo metabolites EL and enterodiol (ED) prepared in our previous work, as well as the newly isolated chemical constituents from safflower seed, including TCL, the lactone ring opening product of TCL (OTCL) and two alkaloids on the alloxan-induced zebrafish model. All the compounds showed significant anti-osteoporosis effects at 80 µM, with p < 0.05 for EL and p < 0.001 for other compounds compared with the model. SDG and TCL showed the most significant and concentration-dependent effects, with p < 0.001 compared with model at 20 µM. The alkaloids, N-coumaroylserotonin glucoside and N-feruloylserotonin glucoside, also showed anti-osteoporosis at 20 µM with p < 0.01, whereas EL, ED, and OTCL showed no significant effects. Quantitative real-time polymerase chain reaction revealed that SDG and TCL upregulated the expression of osteogenic genes Runx2, SP7, OPG, Col1a1a, Alp, ON, OPN, and OCN in alloxan-treated zebrafish. The in vivo metabolite of lignans, EL, showed significant anti-inflammatory effect (p < 0.01) at 20 µM, which might also help to combat osteoporosis and other complications caused by excessive immune response in the body. The results provided scientific data for using the oil extraction by-products as sources of anti-osteoporosis compounds. PRACTICAL APPLICATION: This study found that lignans in flaxseed cake and safflower seed cake exhibited anti-osteoporosis effects by upregulating the expression of osteogenic genes, making the oil extraction by-products sources of anti-osteoporosis compounds.

Effects and mechanisms of 6-hydroxykaempferol 3,6-di-O-glucoside-7-O-glucuronide from Safflower on endothelial injury in vitro and on thrombosis in vivo.

Background: The florets of Carthamus tinctorius L. (Safflower) is an important traditional medicine for promoting blood circulation and removing blood stasis. However, its bioactive compounds and mechanism of action need further clarification. Objective: This study aims to investigate the effect and possible mechanism of 6-hydroxykaempferol 3,6-di-O-glucoside-7-O-glucuronide (HGG) from Safflower on endothelial injury in vitro, and to verify its anti-thrombotic activity in vivo. Methods: The endothelial injury on human umbilical vein endothelial cells (HUVECs) was induced by oxygen-glucose deprivation followed by reoxygenation (OGD/R). The effect of HGG on the proliferation of HUVECs under OGD/R was evaluated by MTT, LDH release, Hoechst-33342 staining, and Annexin V-FITC apoptosis assay. RNA-seq, RT-qPCR, Enzyme-linked immunosorbent assay and Western blot experiments were performed to uncover the molecular mechanism. The anti-thrombotic effect of HGG in vivo was evaluated using phenylhydrazine (PHZ)-induced zebrafish thrombosis model. Results: HGG significantly protected OGD/R induced endothelial injury, and decreased HUVECs apoptosis by regulating expressions of hypoxia inducible factor-1 alpha (HIF-1α) and nuclear factor kappa B (NF-κB) at both transcriptome and protein levels. Moreover, HGG reversed the mRNA expression of pro-inflammatory cytokines including IL-1ß, IL-6, and TNF-α, and reduced the release of IL-6 after OGD/R. In addition, HGG exhibited protective effects against PHZ-induced zebrafish thrombosis and improved blood circulation. Conclusion: HGG regulates the expression of HIF-1α and NF-κB, protects OGD/R induced endothelial dysfunction in vitro and has anti-thrombotic activity in PHZ-induced thrombosis in vivo.

Technical note: Quantification of lignans in the urine, milk, and plasma of flaxseed cake-fed dairy sheep.

Mammalian lignans are phytoestrogens with important bioactivities, and their concentrations in livestock milk may influence the health of consumers. This research aimed to establish a method to quantify multiple mammalian lignans in the biofluids of dairy sheep using ultra-HPLC-triple quadropole mass spectrometry with multiple-reaction monitoring. Secoisolariciresinol, 2-[(4-hydroxy-3-methoxyphenyl)methyl]-3-[(3-hydroxyphenyl)methyl]-1,4-butanediol, enterodiol (ED), enterolactone (EL), ED-sulfate (ED-S), and EL-sulfate (EL-S) were purified from the urine of flaxseed cake-fed dairy sheep. The structures of these lignans were identified by a combination of mass and nuclear magnetic resonance spectra. These purified lignans were used as standards to optimize their quantification conditions in urine, milk, and plasma of dairy sheep. On this basis, the lignan metabolites in biofluids were quantified. To improve analysis sensitivity, plasma and milk were pretreated with acetonitrile containing 1% formic acid and passed through a HybridSPE-PL 55261-U column (Supelco, Bellefonte, PA). The limit of quantification of the lignans ranged from 1.43 to 18.3 ng/mL in plasma, and from 1.01 to 18.7 ng/mL in milk. The linearity of the calibration curves ranged from their limit of quantification to at least 217 ng/mL in plasma, and 217 ng/mL in milk. Regression coefficient of the calibration curves were above 0.99 for secoisolariciresinol, 2-[(4-hydroxy-3-methoxyphenyl)methyl]-3-[(3-hydroxyphenyl)methyl]-1,4-butanediol, ED, EL, ED-S, and EL-S, indicating satisfactory relationships between the peak areas and concentrations in the quantification range. The relative concentrations of ED-glucuronide and EL-glucuronide (EL-G) in different biofluids were compared based on their chromatogram peak areas. The sheep plasma contained all forms of mammalian lignans (i.e., ED, EL, ED-S, EL-S, ED-glucuronide, and EL-G.); the urine contained ED, EL, ED-S, and EL-S; and the milk contained ED, EL, ED-S, EL-S, and EL-G. Milk-to-plasma concentration ratios of the mammalian lignans indicated that the free forms were more permeable than the sulfated conjugates. Mammalian lignans found in sheep plasma and milk may provide health benefits to the sheep and sheep-product consumers. The analytical method established in this work could be used to quantify mammalian lignans in livestock products.