An analytical framework combining online high-performance liquid chromatography methodologies and biological properties of different extracts of Leonurus cardiaca.

Little or no information is available concerning online high-performance liquid chromatography (HPLC) antioxidants and the antibiofilm effect of Leonurus cardiaca. Five distinct extractions of methanolic, ethyl acetate, dichloromethane, hexane, and water were obtained from L. cardiaca. In the online-HPLC-antioxidant analysis of all examined samples, rosmarinic acid emerged as the primary antioxidant, registering concentrations ranging from 6 to 15 ppm at wavelengths of 517 and 734 nm. Notably, the water extract exhibited robust antioxidant activity In vitro. Regarding acetylcholinesterase and butrylcholinesterase inhibition, the n-hexane extract exhibited superior inhibition with values of 3.08 and 5.83 galanthamine equivalent, respectively. Except for the water extract, all tested extracts (at a concentration of 20 µg/mL) exhibited substantial inhibitory activity against biofilm formation, in many cases superior to 80%, and reached even 94.52% against Escherichia coli. Although less vigorous, the extracts also acted against the mature biofilm (inhibition up 76.50% against Staphylococcus aureus). They could work against the metabolism inside an immature and mature biofilm, with inhibition percentages up to 93.18% (vs. Pseudomonas aeruginosa) and 76.50% (vs. Acinetobacter baumannii), respectively. Considering its significant antioxidants, enzyme inhibition, and antimicrobial activity, L. cardiaca emerges as a promising candidate for therapeutic potential.

Protective effect of the mixture of Lactiplantibacillus plantarum KC3 and Leonurus Japonicas Houtt extract on respiratory disorders.

Air pollutants, such as particulate matter (PM) and diesel exhaust particles (DEP), are associated with respiratory diseases. Therefore, preventive and therapeutic strategies against PM-and DEP (PM10D)-induced respiratory diseases are needed. Herein, we evaluate the protective effects of a mixture of Lactiplantibacillus plantarum KC3 and Leonurus Japonicas Houtt (LJH) extract against airway inflammation associated with exposure to PM10D. To determine the anti-inflammatory effects of the LJH extract, reactive oxygen species (ROS) production and the expression of inflammatory pathways were determined in PM10-induced MH-S cells. For the respiratory protective effects, BALB/c mice were exposed to PM10D via intranasal injection, and a mixture of L. plantarum KC3 and LJH extract was administered orally for 12 days. LJH extract inhibited ROS production and the phosphorylation of downstream factors of NF-κB in PM10-stimulated MH-S cells. The mixture of L. plantarum KC3 and LJH repressed the infiltration of neutrophils, reduced the immune cells number, and suppressed the proinflammatory mediators and cyclooxygenase (COX)-2 expressions in PM10D-induced airway inflammation with reduced phosphorylation of downstream factors of NF-κB. In addition, these effects were not observed in an alveolar macrophage depleted PM10D-induced mouse model using clodronate liposomes. The extract mixture also regulated gut microbiota in feces and upregulated the mRNA expression of Foxp3, transforming growth factor (TGF)-ß1, and interleukin (IL)-10 in the colon. The L. plantarum KC3 and LJH extract mixture may inhibit alveolar macrophage- and neutrophil-mediated inflammatory responses and regulate gut microbiota and immune response in PM10D-induced airway inflammation, suggesting it is a potential remedy to prevent and cure airway inflammation and respiratory disorders.

Coumarins with Different Substituents from Leonurus japonicus Have Opposite Effects on Uterine Smooth Muscle.

Leonurus japonicus Houtt is an exceptional medicinal herb used to treat obstetrical and gynecological diseases in traditional Chinese medicine, and it has significant effects on the treatment of dysmenorrhea and postpartum hemorrhage. This study investigated the effects of coumarins with diverse substituent groups from L. japonicus on isolated uterine smooth muscle and the preliminary mechanism of the most effective compound. Eight coumarins isolated from L. japonicus were assessed for their effects on the isolated uterine smooth muscle of nonpregnant rats in vitro. Coumarins 1 and 2 significantly promoted the contraction of rat uterine smooth muscle strips, whereas coumarins 3-5 showed remarkable relaxing effects against oxytocin (OT)-induced rat uterine smooth muscle contraction. Further mechanism investigations revealed that bergapten (coumarin 1) significantly increased the level of Ca2+ in uterine tissues by promoting extracellular Ca2+ influx and intracellular Ca2+ release, which were related to the activation of L-type Ca2+ channels and α-receptors. By contrast, osthole (coumarin 5), an α receptor antagonist, inhibited OT-induced uterine smooth muscle contraction by decreasing the level of Ca2+ in uterine tissues via inhibition of extracellular Ca2+ influx and intracellular Ca2+ release. This study demonstrates that the coumarins from L. japonicus are effective substances for regulating uterine smooth muscle contraction, but different coumarins with diverse substituent groups have different, even opposite effects. It can be inferred that coumarins are closely related to the efficacy of L. japonicus in the treatment of dysmenorrhea and postpartum hemorrhage.

Genetic diversity and population divergence of Leonurus japonicus and its distribution dynamic changes from the last interglacial to the present in China.

BACKGROUND: Leonurus japonicus, a significant medicinal plant known for its therapeutic effects on gynecological and cardiovascular diseases, has genetic diversity that forms the basis for germplasm preservation and utilization in medicine. Despite its economic value, limited research has focused on its genetic diversity and divergence. RESULTS: The avg. nucleotide diversity of 59 accessions from China were 0.00029 and hotspot regions in petN-psbM and rpl32-trnL(UAG) spacers, which can be used for genotype discrimination. These accessions divided into four clades with significant divergence. The four subclades, which split at approximately 7.36 Ma, were likely influenced by the Hengduan Mountains uplift and global temperature drop. The initial divergence gave rise to Clade D, with a crown age estimated at 4.27 Ma, followed by Clade C, with a crown age estimated at 3.39 Ma. The four clades were not showed a clear spatial distribution. Suitable climatic conditions for the species were identified, including warmest quarter precipitation 433.20 mm ~ 1,524.07 mm, driest month precipitation > 12.06 mm, and coldest month min temp > -4.34 °C. The high suitability distribution showed contraction in LIG to LGM, followed by expansion from LGM to present. The Hengduan Mountains acted as a glacial refuge for the species during climate changes. CONCLUSIONS: Our findings reflected a clear phylogenetic relationships and divergence within species L. japonicus and the identified hotspot regions could facilitate the genotype discrimination. The divergence time estimation and suitable area simulation revealed evolution dynamics of this species and may propose conservation suggestions and exploitation approaches in the future.

Diterpene synthases from Leonurus japonicus elucidate epoxy-bridge formation of spiro-labdane diterpenoids.

Spiro-9,13-epoxy-labdane diterpenoids are commonly found in Leonurus species, particularly in Leonurus japonicus Houtt., which is a medicinal herb of long-standing use in Asia and in which such spiro-heterocycles are present in at least 38 diterpenoids. Here, through generation of a transcriptome and functional characterization of six diterpene synthases (diTPSs) from L. japonicus, including three class II diTPSs (LjTPS1, LjTPS3, and LjTPS4) and three class I diTPSs (LjTPS5, LjTPS6, and LjTPS7), formation of the spiro-9,13-epoxy-labdane backbone was elucidated, along with identification of the relevant diTPSs for production of other labdane-related diterpenes. Similar to what has been found with diTPSs from other plant species, while LjTPS3 specifically produces the carbon-9 (C9) hydroxylated bicycle peregrinol diphosphate (PPP), the subsequently acting LjTPS6 yields a mixture of four products, largely labda-13(16),14-dien-9-ol, but with substantial amounts of viteagnusin D and the C13-S/R epimers of 9,13-epoxy-labda-14-ene. Notably, structure-function analysis identified a critical residue in LjTPS6 (I420) in which single site mutations enable specific production of the 13S epimer. Indeed, extensive mutagenesis demonstrated that LjTPS6:I420G reacts with PPP to both specifically and efficiently produce 9,13S-epoxy-labda-14-ene, providing a specialized synthase for further investigation of derived diterpenoid biosynthesis. The results reported here provide a strong foundation for future studies of the intriguing spiro-9,13-epoxy-labdane diterpenoid metabolism found in L. japonicus.

Preliminary Evaluation of Wound Healing Potential of Leonurus japonicus Houtt. Extracts.

Leonurus japonicus Houtt. is a medicinal plant popular in Brazil as "rubim", used in local folk medicine for several applications as an anti-inflammatory, antioxidant, analgesic, and antimicrobial phytomedicine. The traditional use for wound healing is related; however, few studies have evaluated the wound healing activity. Thus, this study aimed to analyse the popular indication of the hydroalcoholic and aqueous extracts of L. japonicus aerial parts in a rat wound healing model. The initial chemical characterization was performed using flavonoid quantification and complemented with mass spectroscopy/chemometrics analysis. The wound's lesion contraction and tissue regeneration (histological study stained with hematoxylin-eosin and picrosirius) were determined. Hydroalcoholic and aqueous extracts presented high flavonoid content, and mass spectrometry analysis of the extracts demonstrated the presence of compounds with a mass between 100-650, reinforcing the presence of polyphenolic constituents. The extracts of L. japonicus improve various wound healing phases, like inflammatory modulation, wound contraction, and collagen synthesis, resulting in faster healing in rats. These effects could be related to the extracts' polyphenolic compounds.

Activating Blood Circulation, Anti-Inflammatory and Diuretic Effects of Leonurus japonicus Extract on a Rat Model of Trauma Blood Stasis and Its Phytochemical Profiling.

Leonurus japonicus Houtt. has been traditionally used to treat many ailments. This study evaluated the activating blood circulation, anti-inflammatory, and diuretic effects of L. japonicus extract (LJ) and identified its phytochemicals. In this work, the phytochemicals in LJ were identified using liquid chromatography mass spectrometry. Rats were randomly assigned to three groups (n=8): Control group was treated with saline, while the Model group (saline) and LJ group (426 mg/kg) had induced traumatic injury. All rats were treated with once by daily oral gavage for one week. The biochemical indices and protein expression were measured. Herein, 79 constituents were identified in LJ, which were effective in elevating body weight, food consumption, water intake, and urinary excretion volume, as well as in ameliorating traumatic muscle tissues in model rats. In addition, LJ prominently decreased the contents of plasma viscosity, platelet aggregation rate, thrombin time, prothrombin time, activated partial thromboplastin time, fibrinogen, thromboxane B2 (TXB2), TXB2/6-keto-prostaglandin F1α (6-keto-PGF1α), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor 1 (PAI-1), PAI-1/tissue-type PA (t-PA), and PAI-1/u-PA, while significantly increasing antithrombin III, 6-keto-PGF1α, and t-PA contents. Furthermore, LJ notably inhibited tumor necrosis factor alpha, interleukin 6 (IL-6), IL-8, angiotensin II, antidiuretic hormone, aldosterone, aquaporin 1 (AQP1), AQP2, and AQP3 levels, and markedly elevating IL-10 and natriuretic peptide levels. Finally, LJ markedly reduced the protein expression of AQP1, AQP2, and AQP3 compared to the model group. Collectively, LJ possessed prominent activating blood circulation, anti-inflammatory, and diuretic effects, thus supporting the clinical application of L. japonicus.

In Vitro Evaluation and In Silico Calculations of the Antioxidant and Anti-Inflammatory Properties of Secondary Metabolites from Leonurus sibiricus L. Root Extracts.

Leonurus sibiricus L. has great ethnobotanical and ethnomedicinal significance. This study aimed to assess the antioxidant and anti-inflammatory properties of Leonurus sibiricus L. transgenic roots extracts transformed by Rhizobium rhizogenes, with and without the AtPAP1 transcriptional factor. The study determined the total phenolic and flavonoid contents, as well as in vitro antioxidant assays, including hydrogen peroxide and nitric oxide scavenging activity. In addition, in silico computational studies and molecular docking were conducted to evaluate the antioxidant and anti-inflammatory potential of the identified compounds. The ligands were docked to NADPH oxidase, cyclooxygenase 2,5-lipoxygenase, inducible nitric synthase and xanthine oxidase: enzymes involved in the inflammatory process. The total phenolic and flavonoid contents ranged from 85.3 ± 0.35 to 57.4 ± 0.15 mg/g GAE/g and 25.6 ± 0.42 to 18.2 ± 0.44 mg/g QUE/g in hairy root extracts with and without AtPAP1, respectively. H2O2 scavenging activity (IC50) was found to be 29.3 µg/mL (with AtPAP1) and 37.5 µg/mL (without AtPAP1 transcriptional factor), and NO scavenging activity (IC50) was 48.0 µg/mL (with AtPAP1) and 68.8 µg/mL (without AtPAP1 transcriptional factor). Leonurus sibiricus L. transformed root extracts, both with and without AtPAP1, are a source of phytochemicals belonging to different classes of molecules, such as flavonoids (catechin and rutin), phenolic compounds (caffeic acid, coumaric acid, chlorogenic acid, ferulic acid) and phenylpropanoid (verbascoside). Among the radicals formed after H removal from the different -OH positions, the lowest bond dissociation enthalpy was observed for rutin (4'-OH). Rutin was found to bind with cyclooxygenase 2, inducible nitric synthases and xanthine oxidase, whereas chlorogenic acid demonstrated optimal binding with 5-lipoxygenase. Therefore, it appears that the Leonurus sibiricus L. transformed root extract, both with and without the AtPAP1 transcriptional factor, may serve as a potential source of active components with antioxidant and anti-inflammatory potential; however, the extract containing AtPAP1 demonstrates superior activities. These properties could be beneficial for human health.

Anticancer Activity of Leonurus sibiricus L.: Possible Involvement of Intrinsic Apoptotic Pathway.

Liver cancer is the most common cause of mortality in men and women. The aim of this study was to evaluate the methanolic extract of aerial parts of Leonurus sibiricus L (LS) for its anticancer activity on HCC cell lines. Human HCC cell lines, Huh-7 and HSC-T6 were used for cytotoxicity assay, determination of ROS and gene expression analysis (p53, Bcl-2, Bax, caspase-3, caspase-8, and caspase-9). Western blotting was used to assess ERK, AKT, and Caspase-3 activation. HPLC-MS analysis was also performed to determine the phytochemicals present in LS-M extract. LS-M extract has increased the expression of proapoptotic genes, including p53, Bax, and caspase-9 and down-regulated the activation of ERK and Akt. The caspase-3 activity as well as the ROS generation were significantly increased in a dose-dependent manner compared to control. The carotenoids, lutein and beta carotene present in LS-M extract exhibited anticancer activity. In overall, the methanolic extract of LS induces apoptosis in Huh-7 as well as in HSC-T6 cells possibly involving a ROS-mediated mitochondrial signaling pathway. Graphical abstract - The methanolic extract of LS induces apoptosis in Huh-7 as well as in HSC-T6 cells possibly involving a ROS-mediated mitochondrial signaling pathway[Formula: see text].

Foliar-applied nanoscale zero-valent iron (nZVI) and iron oxide (Fe3O4) induce differential responses in growth, physiology, antioxidative defense and biochemical indices in Leonurus cardiaca L.

The impacts of nZVI and iron oxides on growth, physiology and elicitation of bioactive antioxidant metabolites in medicinal aromatic plants must be critically assessed to ensure their safe utilization within the food chain and achieve nutritional gains. The present study investigated and compared the morpho-physiological and biochemical changes of Leonurus cardiaca L. plants as affected by various concentrations (0, 250, 500 and 1000 mg L-1) of nZVI and Fe3O4. The foliar uptake of nZVI was verified through Scanning Electron Microscopy (SEM) images and Energy Dispersive X-ray (EDX) analytical spectra. Plants exposed to nZVI at low concentration showed comparatively monotonic deposition of NPs on the surface of leaves, however, the agglomerate size of nZVI was raised as their doses increased, leading to remarkable changes in anatomical and biochemical traits. 250 mg L-1 nZVI and 500 mg L-1 Fe3O4 significantly (P < 0.05) increased plant dry matter accumulation by 37.8 and 27% over the control, respectively. The treatments of nZVI and Fe3O4 at 250 mg L-1 significantly (P < 0.01) improved chlorophyll a content by 22.4% and 15.3% as compared to the control, and then a rapid decrease (by 14.8% and 4.1%) followed at 1000 mg L-1, respectively. Both nZVI and Fe3O4 at 250 mg L-1 had no significant impact on malondialdehyde (MDA) formation, however, at an exposure of 500-1000 mg L-1, the MDA levels and cellular electrolyte leakage were increased. Although nZVI particles could be utilized by plants and enhanced the synthesis of chlorophylls and secondary metabolites, they appeared to be more toxic than Fe3O4 at 1000 mg L-1. Exposure to nZVI levels showed positive, negative and or neutral impacts on leaf water content compared to control, while no significant difference was observed with Fe3O4 treatments. Soluble sugar, total phenolics and hyperoside content were significantly increased upon optimum concentrations of employed treatments-with 250 mg L-1 nZVI being most superior. Among the extracts, those obtained from plants treated with 250-500 mg L-1 nZVI revealed the strong antioxidant activity in terms of scavenging free radical (DPPH) and chelating ferrous ions. These results suggest that nZVI (at lower concentration) has alternative and additional benefits both as nano-fertilizer and nano-elicitor for biosynthesis of antioxidant metabolites in plants, but at high concentrations is more toxic than Fe3O4.

Isolation of three glucaric acids from Leonurus japonicus Houtt. by using high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography.

The isomerism of glucaric acids and the complexity of the composition of Leonurus japonicus Houtt. increased the difficulty of the separation of glucaric acids from the herb. In the present study, three glucaric acids were isolated from Leonurus japonicus Houtt. by using high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography. Cation exchange resin chromatography was applied to remove the alkaloids and enrich the glucaric acid fractions. Preliminary separation of the glucaric acid extract by high-speed countercurrent chromatography was carried out at 45℃ by using an optimized solvent system of ethyl acetate/n-butanol/formic acid/water (1:1:0.01:2, v/v/v/v) with satisfied stationary phase retention and separation factor. The semi-preparative high-performance liquid chromatography was used for further separation and purification of the target fractions, and three monomeric compounds were obtained with purities of 90.0, 91.0, and 95.3%. UV spectroscopy, NMR spectroscopy, and mass spectrometry were employed to identify their structures, which were assigned as 2-syringyl glucaric acid, 2,4-disyringyl glucaric acid, and 3,4-disyringyl glucaric acid, respectively, and 2,4-disyringyl glucaric acid was reported for the first time.

Leonurine Preconditioning Attenuates Ischemic Acute Kidney Injury in Rats by Promoting Nrf2 Nuclear Translocation and Suppressing TLR4/NF-κB Pathway.

Despite the precise mechanisms for renal ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) are poorly understood, nuclear factor erythroid 2 related factor 2 (Nrf2) and Toll-like receptor 4 (TLR4) pathways were considered as the important targets. Leonurine (LEO) is a special alkaloid extracted from Chinese motherwort (Leonurus japonicus Houtt), which has an anti-inflammatory effect and reduces oxidative stress. We conducted the study to explore the efficacy of LEO against I/R-induced AKI in rats and further investigated the underlying mechanisms. Ischemic renal injury was induced by temporary vascular clamping for 45 min. We have measured the levels of inflammation-related biomarkers and antioxidative stress markers. Next, Western blot analysis and Real-time PCR were performed to analyze whether the Nrf2 and TLR4/nuclear factor-kappaB (NF-κB) pathways were involved in this process. We found that LEO pretreatment remarkably decreased serum creatinine and blood urea nitrogen (BUN) in I/R rats and attenuated acute tubular damage. In addition, LEO markedly increased the expression of antioxidant proteins and decreased the levels of inflammatory factors. Further study revealed that LEO promoted Nrf2 into the nucleus, promoted the expression of heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO-1), and suppressed the TLR4/NF-κB signal pathway in kidney tissues of ischemic AKI rats. The study reveals that LEO has a protective effect to prevent ischemic AKI through activation of Nrf2 nuclear translocation resisting oxidative stress injury and inhibition of the TLR4/NF-κB pathway mediated inflammatory gene expression.

[Effects of pH value on stachydrine biosynthesis of hydroponic Leonurus japonicus and its physiological mechanism].

The present study explored the physiological mechanism of the effects of different pH treatments on the growth, physiological characteristics, and stachydrine biosynthesis of Leonurus japonicus to provide references for the cultivation and quality control of L. japonicus. Under hydroponic conditions, different pH treatments(pH 5,6,7,8) were set up. The growth, physiology, and the content of stachydrine and total alkaloids of L. japonicus, as well as the content of key intermediate products in stachydrine biosynthesis pathway(i.e., pyruvic acid, α-ketoglutaric acid, glutamic acid, and ornithine) were monitored to explore the physiological mechanism of the effects of pH on the growth and active components of L. japonicus. The results showed that L. japonicus. could grow normally in the pH 5-8 solution. The pH treatment of neutral acidity was more conducive to the accumulation of photosynthetic pigments and the increase in soluble protein in leaves of L. japonicus. to promote its growth and yield. However, since stachydrine is a nitrogen-containing pyrrolidine alkaloid, its synthesis involves the two key rate-limiting steps of nitrogen addition: reductive ammoniation reaction and Schiff base formation reaction. High pH treatments promote the synthesis and accumulation of substrates and products of the above two reactions, indicating that the alkaline environment can promote the nitrogen addition reaction, thereby promoting the biosynthesis and accumulation of stachydrine.

Comparative transcriptome analysis of sesquiterpene biosynthesis and functional characterization of sesquiterpene synthases in Leonurus sibiricus L.

MAIN CONCLUSION: Two sesquiterpene synthases were identified through comparative transcriptome analysis of Leonurus sibiricus. LsSqTPS2 could produce high titer of δ-cadinene in vivo which suggests the terpene specificity of L. sibiricus. Leonurus sibiricus L., a medicinal herb, is widely used in China due to its pharmacological activities. Cadinene type sesquiterpenes, one of major bioactive components mainly present in aerial parts of L. sibiricus, showed antibacterial, anti-inflammatory, antioxidant and antiproliferative properties. However, there is no report about the sesquiterpene biosynthesis in L. sibiricus. This study identified L. sibiricus sesquiterpene synthases (LsSqTPSs) through comparative transcriptome analysis of L. sibiricus leaf and root samples using the BGISEQ-500 sequencing technique. A total of 83,244 unigenes were obtained with an average length of 1025 bp. Among them, 50,356 unigenes (60.49%) acquired annotations according to the BLAST searching results. A total of 68 differentially expressed genes (DEGs) were potentially involved in the sesquiterpene biosynthesis. Furthermore, four candidate DEGs encoding LsSqTPSs were characterized. The enzymatic characterization in engineered yeast showed that LsSqTPS1 produced α-farnesene as the single product and LsSqTPS2 mainly produced 76.23 mg/L of δ-cadinene, which constituted the major component of L. sibiricus leaf essential oil. This work contributes to the investigation of sesquiterpene biosynthesis in L. sibiricus.

New lignans from the fruits of Leonurus japonicus and their hepatoprotective activities.

Twelve tetrahydrofuran lignans (1-12), including six new compounds (1-6), were isolated from the 70% EtOH extract of the fruits of Leonurus japonicus. Spectroscopic analyses and ECD and OR calculations were used to determine their structures. Compounds 5 and 6 were unusual alkaloidal lignans with a butyrolactam unit. Based on the beneficial effects of the fruits of L. japonicus (Chongweizi in Chinese) on the liver in traditional Chinese medicine (TCM), the hepatocyte protective activities of the isolates were studied by MTT, Hoechst 33,342 staining, and western blotting. The MTT results revealed that compounds 1, 2, 7, and 8 significantly increased the survival rates of HL-7702 cells injured by acetaminophen, with EC50 values of 10.41 ± 0.90 µM, 19.86 ± 3.13 µM, 9.68 ± 1.93 µM, and 21.35 ± 3.58 µM, respectively. In the Hoechst 33,342 fluorescence staining, compounds 1 and 7 suppressed the apoptosis of the injured HL-7702 cells. Furthermore, the western blot analysis showed that compounds 1 and 7 increased the Bcl-2/Bax protein expression ratio and procaspase-3 protein expression, indicating that compounds 1 and 7 may exert hepatoprotective activity by regulating the mitochondrial apoptotic pathway.

TLC-MS identification of alkaloids in Leonuri Herba and Leonuri Fructus aided by a newly developed universal derivatisation reagent optimised by the response surface method.

INTRODUCTION: Dragendorff's reagent has low sensitivity and non-specificity for some alkaloids. A new alkaloid derivatisation reagent has been developed and optimised by using a Box-Behnken design method. This new reagent is applicable for structurally diverse natural alkaloids, and is proposed as a universal alkaloid staining reagent. OBJECTIVE: To establish an efficient and sensitive thin-layer chromatography (TLC) identification method for Leonuri Herba and Leonuri Fructus to characterise their differences and similarities. METHOD: Three key components (bismuth subnitrate, potassium iodide and iodine) in the derivatisation reaction were re-constructed and optimised using a response surface method. Different inorganic acids, essential additives of the staining reaction, were compared by a single-factor experiment design. RESULTS: This newly optimised reagent for alkaloids reported in this study, named the enhanced Dragendorff-Wagner's reagent, is composed of 0.82% bismuth subnitrate, 11.1% potassium iodide and 0.76% iodine in 38% phosphoric acid solution. Validation results indicate that the TLC spot of stachydrine stained with the enhanced Dragendorff-Wagner's reagent had a limit of detection of 2.0 µg, good intra- and inter-plate, and intra- and intra-day precisions with relative standard devition values less than 5.0%, and stability over 90 min. CONCLUSION: This enhanced Dragendorff-Wagner's reagent was applied for TLC identification of 16 reference alkaloids representing 11 structural skeletons, and two closely related herbs (Leonuri Herba and Leonuri Fructus). This newly enhanced Dragendorff-Wagner's reagent is a universal, effective, and sensitive staining reagent for alkaloids.

Leonurus sibiricus root extracts decrease airway remodeling markers expression in fibroblasts.

Bronchial asthma is believed to be provoked by the interaction between airway inflammation and remodeling. Airway remodeling is a complex and poorly understood process, and controlling it appears key for halting the progression of asthma and other obstructive lung diseases. Plants synthesize a number of valuable compounds as constitutive products and as secondary metabolites, many of which have curative properties. The aim of this study was to evaluate the anti-remodeling properties of extracts from transformed and transgenic Leonurus sibiricus roots with transformed L. sibiricus roots extract with transcriptional factor AtPAP1 overexpression (AtPAP1). Two fibroblast cell lines, Wistar Institute-38 (WI-38) and human fetal lung fibroblast (HFL1), were incubated with extracts from transformed L. sibiricus roots (TR) and roots with transcriptional factor AtPAP1 over-expression (AtPAP1 TR). Additionally, remodeling conditions were induced in the cultures with rhinovirus 16 (HRV16). The expressions of metalloproteinase 9 (MMP)-9, tissue inhibitor of metalloproteinases 1 (TIMP-1), arginase I and transforming growth factor (TGF)-ß were determined by quantitative polymerase chain reaction (qPCR) and immunoblotting methods. AtPAP1 TR decreased arginase I and MMP-9 expression with no effect on TIMP-1 or TGF-ß mRNA expression. This extract also inhibited HRV16-induced expression of arginase I, MMP-9 and TGF-ß in both cell lines (P < 0·05) Our study shows for the first time to our knowledge, that transformed AtPAP1 TR extract from L. sibiricus root may affect the remodeling process. Its effect can be attributed an increased amount of phenolic acids such as: chlorogenic acid, caffeic acid or ferulic acid and demonstrates the value of biotechnology in medicinal research.

The Effect of Leonurine on Multiterritory Perforator Flap Survival in Rats.

BACKGROUND: Leonurine (Leo), a natural active compound of Leonurus cardiaca, has been shown to possess various biological activities. However, it is not known whether Leo promotes perforator flap survival. METHODS: In this study, a perforator flap was outlined in the rat dorsum. The rats that survived surgery were divided randomly to control and Leo groups (n = 36 per group). Flap viability, flap perfusion, and level of protein linked with oxidative stress, cell apoptosis, and angiogenesis were evaluated. RESULTS: Relative to control group, the Leo group showed significantly higher the flap survival percentage (70.5% versus 90.2%, P < 0.05) and blood perfusion (197.1 versus 286.3, P < 0.05). Leo also increased 1.8-fold mean vessel density and upregulated 2.1-fold vascular endothelial growth factor protein expression compared with the control group, both of which indicate increased angiogenesis. Moreover, it significantly inhibited apoptosis by lowering caspase-3 activity. Superoxide dismutase expression was remarkably elevated in Leo group compared with the control group (56.0 versus 43.2 U/mg/protein, P < 0.01), but malondialdehyde quantities were significantly lower in the Leo group compared with control group (41.9 versus 57.5 nmol/mg/protein, P < 0.05). CONCLUSIONS: Leo may serve as an effective drug for improving perforator flap survival in rats via antioxidant and antiapoptotic mechanisms and promotion of angiogenesis.

Labdane-Type Diterpenes from the Aerial Parts of Rydingia persica: Their Absolute Configurations and Protective Effects on LPS-Induced Inflammation in Keratinocytes.

Phytochemical investigations of an extract of the aerial parts of Rydingia persica led to the isolation of 14 labdane-type diterpenoids, of which compounds 1-5, 8, and 12-14 turned out to be new natural products, while the remaining compounds were isolated for the first time from the genus Rydingia. Their structures were elucidated using 1D- and 2D-NMR and mass spectrometry, and their absolute configurations were determined by quantum chemical calculation methods. Furthermore, DP4+ NMR chemical shift probability calculations were performed for compounds 12-14, in order to elucidate the orientation of the ambiguous chiral center at C-15, prior to absolute configuration determination. The methanol extract of the aerial parts of R. persica along with subfractions obtained and selected isolated compounds were evaluated for their effects on inflammation-related factors such as nitrotyrosine formation, IL-6 release, and TNF-α release, along with tight-junction proteins claudin-1 and occludin expression in LPS-stimulated HaCaT cells. Occludin and claudin-1 are tight-junction proteins, which play a pivotal role in wound repair mechanisms. Overall, the subfractions and compounds isolated showed moderate to high activity, indicating that labdane-type diterpenoids contribute to the anti-inflammatory and wound-healing activity of R. persica.

Structurally Diverse Labdane Diterpenoids from Leonurus japonicus and Their Anti-inflammatory Properties in LPS-Induced RAW264.7 Cells.

A phytochemical study on the aerial parts of Leonurus japonicus led to the isolation and identification of 38 labdane diterpenoids, including 18 new (1, 2, 11, 12, 16-21, 24, 30-34, 37, 38) and 20 known (3-10, 13-15, 22, 23, 25-29, 35, 36) analogues. Their structures were elucidated based on physical data analysis, including 1D and 2D NMR, HRMS, UV, IR, and X-ray diffraction. The structure of the known compound 4 was confirmed by single-crystal X-ray diffraction data. These compounds can be divided into furanolabdane (1-10), tetrahydrofuranolabdane (11-15), lactonelabdane (16-23), labdane (24-29), and seco-labdane (30-38) type diterpenoids. All compounds were screened by lipopolysaccharide (LPS)-induced nitric acid (NO) production in RAW264.7 cells to evaluate anti-inflammatory effects. Compounds 1, 5, 10-13, 16-19, 31-33, and 38 inhibited NO production with IC50 values lower than 50 µM, with compound 30 being the most active, with an IC50 value of 3.9 ± 1.7 µM. Further studies show that compound 30 inhibits pro-inflammatory cytokine production and IKK α/ß phosphorylation and restores the IκB expression levels in the NF-κB signaling pathway.