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.

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.

Analysis of Antibiofilm Activities of Bioactive Compounds from Honeyweed (Leonurus sibiricus) Against P. aeruginosa: an In Vitro and In Silico Approach.

Leonurus sibiricus (Red verticilla, honeyweed) is a type of herbaceous plant predominantly found in Asian subcontinents as weed in crop fields and is widely used for treating diabetes, bronchitis, and menstrual irregularities. However, there is a dearth of study in the application of the plant phytocompounds for treating biofilm-associated chronic infections. The bioactive compounds mainly comprise of tri-terpenes, di-terpenes, phenolic acid, and flavonoids which may have potential role as antimicrobial and antibiofilm agents. Acute and chronic infection causing microbes usually form biofilm and develop virulence factors and antibiotic resistance through quorum sensing (QS). In this study, the bioactive compounds leosibirin, sibiricinone A, leosibirone A, leonotin, quercetin, lavandulifolioside, and myricetin were identified using GC-MS analysis. These were used for analyzing the antibiofilm and anti-quorum sensing activities (rhamnolipid, AHL assay, swarming motility assay) against the biofilm formed by Pseudomonas aeruginosa, the most significant nosocomial disease-causing bacteria. The compounds were able to bring about maximum inhibition in biofilm formation and QS. Although the antibiofilm activity of the phytoextract was found to be higher than that of individual phytocompounds at a concentration of 250 µg/mL, quercetin and myricetin showed highest antibiofilm activity against Pseudomonas aeruginosa, respectively, at MIC values of 135 µg/mL and 150 µg/mL against P aeruginosa. FT-IR study also revealed that the active ingredients were able to bring about the destruction of exopolysaccharides (EPS). These observations were further validated by molecular docking interactions that showed the active ingredients inhibit the functioning of QS sensing proteins by binding with them. It was observed that myricetin showed better interactions with the QS proteins of P. aeruginosa. Myricetin and quercetin show considerable inhibition of biofilm in comparison to the phytocompounds. Thus, the present study suggests that the active compounds from L. sibiricus can be used as an alternate strategy in inhibiting the biofilm formed by pathogenic organisms.

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.

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.

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].

Over-Expression of AtPAP1 Transcriptional Factor Enhances Phenolic Acid Production in Transgenic Roots of Leonurus sibiricus L. and Their Biological Activities.

This study examines the production of five phenolic acids (chlorogenic acid, neochlorogenic acid, ferulic acid, caffeic acid and p-coumaric acid) following over-expression of AtPAP1 transcription factor by four transgenic root clones of Leonurus sibiricus after Agrobacterium rhizogenes transformation. The AtPAP1 expression level was estimated by quantitative real-time PCR. High levels of phenolic acids were found in the transgenic roots of L. sibiricus and were determined by high-performance liquid chromatography-mass spectrometry analysis. Additionally, transgenic roots showed antimicrobial potential and cytotoxic activity on glioma cells in IV grade. Our results suggest that L. sibiricus transformed roots with AtPAP1 gene over-expression may represent a potential source of phenolic acids.

Metabolic Profile of and Antimicrobial Activity in the Aerial Part of Leonurus turkestanicus V.I. Krecz. et Kuprian. from Kazakhstan.

An HPLC quadrupole time-of-flight (Q-TOF) MS method was developed for the identification of secondary metabolites in Leonurus turkestanicus V.I. Krecz. et Kuprian. Ethanolic and chloroform extracts from the plant's aerial parts were tested. A total of 16 compounds (iridoid glycosides, phenylpropanoids, flavonoids, and nitrogen-containing compounds, as well as diterpene acetate derivatives) were identified and tentatively characterized based on (or using) their retention times and UV and Q-TOF-MS data. Previously reported aucubin (1), 6-deoxy-8-acetylharpagid (2), and stachydrine (13, 15) and homostachydrine isomers (14, 16) were identified, along with a lavandulifolioside isomer (3), verbascoside (4), rutin (5), 3-O-kaempferol rutinoside (6), and an unknown diterpene acetate (8). Compounds 3-6 were detected for the first time in this plant. Additionally. antimicrobial activity was evaluated. No significant differences were found between ethanolic and chloroform extracts of L. turkestanicus; however, the alcoholic extract showed stronger antifungal activity [minimal inhibitory concentration (MIC) of 2.5-5 mg/mL], whereas the chloroform extract showed stronger activity against the tested spore-forming Bacillus species (MIC 1.25-2.5 mg/mL).

Leonurus sibiricus herb extract suppresses oxidative stress and ameliorates hypercholesterolemia in C57BL/6 mice and TNF-alpha induced expression of adhesion molecules and lectin-like oxidized LDL receptor-1 in human umbilical vein endothelial cells.

In Leonurus sibiricus herb extract (LHE)-supplemented animals, plasma cholesterol decreased and high-density lipoprotein-cholesterol increased, resulting in a lowered atherogenic index. The plasma trolox equivalent antioxidant capacity, levels of hepatic thiobarbituric acid-reactive substances, and protein carbonyl values decreased significantly in LHE-supplemented mice (p<0.05), whereas the hepatic antioxidant indicators were all significantly elevated (p<0.05). In human umbilical vein endothelial cells stimulated with tumor necrosis factor alpha, LHE significantly suppressed intracellular reactive oxygen species, LOX-1, and adhesion molecules. LHE supplementation may modulate the lipoprotein composition and attenuate oxidative stress by elevated antioxidant processes, thus suppressing the activation of inflammatory mediators. This is a possible mechanism of the anti-atherogenic effect.

Anti-inflammatory activity of Motherwort (Leonurus sibiricus L.).

Motherwort (MW), a Korean folk medicine, has been applied to treat inflammatory disease. However, its effect on inflammatory cytokine release from mast cells is not well known. We investigated the anti- inflammatory effect of MW on the secretion of inflammatory cytokine such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 and IL-8 in human mast cell line (HMC-1). MW was treated in vitro before activation of HMC-1 cells with phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187. MW had no cytotoxic effects on HMC-1 cell viability. MW (1 mg/ml) inhibited PMA plus A23187-stimulated gene expression and production of TNF-alpha, IL-6, and IL-8. Stimulation with PMA plus A23187 induced NF-kappaB activation in HMC-1 cells, which was inhibited by MW (1 mg/ml). MW inhibited secretion of TNF-alpha, IL-6, and IL-8 possibly by inhibiting NF-kappaB activation. These results indicate that MW may be helpful in regulating inflammatory diseases.

Characterization and expression of an nsLTPs-like antimicrobial protein gene from motherwort (Leonurus japonicus).

In screening for potent antimicrobial proteins (AMPs) from plant seeds, we had purified a heat-stable AMP, LJAMP2, from the seeds of a medicine herb, motherwort (Leonurus japonicus Houtt). In an in vitro assay, the protein can inhibit the growth of both fungi and bacteria. Then a cDNA encoding LJAMP2 was cloned by the rapid amplification of cDNA ends based on the N-terminal amino acid sequence determined. The deduced amino acid sequences of this cDNA show similarity to plant non-specific lipid transfer proteins. Northern blotting assay revealed that this nsLTP-like gene, designated LJAMP2, was expressed in seeds. Overexpression of LJAMP2 in tobacco enhanced resistance to the fungal pathogen Alternaria alternata and the bacterial pathogen Ralstonia solanacearum, significantly, while no visible alteration in plant growth and development. Our data confirm the antifungal and antibacterial function of LJAMP2 from motherwort seeds and suggest the potential of LJAMP2 in improving disease resistance in plants.

Expression of a novel small antimicrobial protein from the seeds of motherwort (Leonurus japonicus) confers disease resistance in tobacco.

Medicinal plants are valuable resources of natural antimicrobial materials. A novel small protein with antimicrobial activities, designated LJAMP1, was purified from the seeds of a medicinal herb, motherwort (Leonurus japonicus Houtt). LJAMP1 is a heat-stable protein with a molecular mass of 7.8 kDa and a determined isoelectric point of 8.2. In vitro assays showed that LJAMP1 inhibits the growth of an array of fungi and bacteria. The hyphal growth inhibition by LJAMP1 was more evident against hyphomycete fungi, such as Alternaria alternata, Cercospora personata, and Aspergillus niger. The N-terminal amino acid sequence of LJAMP1 was determined, and its coding gene was consequently cloned by the rapid amplification of cDNA ends. The gene LJAMP1 has no intron and encodes a polypeptide of 95 amino acids, in which the first 27 residues was deduced as a signal peptide. The mature LJAMP1 shows relatively low identity to plant napin-like storage proteins. Northern blot assays revealed that LJAMP1 is expressed preferentially in seeds. Bioassays in transgenic tobacco demonstrated that that overexpression of LJAMP1 significantly enhanced the resistance of tobacco against not only the fungal pathogen A. alternata but also the bacterial pathogen Ralstonia solanacearum, while no visible alteration in plant growth and development was observed.

Isolation and characterization of a novel thermostable non-specific lipid transfer protein-like antimicrobial protein from motherwort (Leonurus japonicus Houtt) seeds.

In screening for potent antimicrobial proteins from plant seeds, a novel heat-stable antimicrobial protein, designated LJAMP2, was purified from seeds of the motherwort (Leonurus japonicus Houtt), a medicine herb, with a procedure involving cation exchange chromatography on a CM FF column, and reverse phase HPLCs on C8 column and C18 column. LJAMP2 exhibited a molecular mass of 6.2 kDa determined. Automated Edman degradation determined the partial N-terminal sequence of LJAMP2 to be NH2-AIGCNTVASKMAPCLPYVTGKGPLGGCCGGVKGLIDAARTTPDRQAVCNCLKTLAKSYSG, which displays homology with plant non-specific lipid transfer proteins (nsLTPs). In vitro bioassays showed that LJAMP2 inhibits the growth of a variety of microbes, including filamentous fungi, bacteria and yeast. The growth of three phytopathogenic fungi, Alternaria brassicae, Botrytis maydis, and Rhizoctonia cerealis, are inhibited at 7.5 microM of LJAMP2, whereas Bacillus subtilis is about 15 microM. The IC(50) of LJAMP2 for Aspergillus niger, B. maydis, Fusarium oxysporum, Penicillium digitatum and Saccharomyces cerevisiae are 5.5, 6.1, 9.3, 40.0, and 76.0 microM, respectively.

[Leonurus cardiaca in vitro]. / Leonurus cardiaca in vitro.

Segments of sterile germinating plants were used to derive the tissue culture. Tests of the optimal hormonal composition of the medium from the standpoint of the growth of cultures revealed that the highest growth in biomass was achieved in the medium with an addition of 2.4-D (0.1, 1.0 mg.l-1), NAA (10.0 mg.l-1), and a combination of IAA (1.0 mg.l-1) + K (1.0 mg.l-1). The difference between these values was statistically insignificant. Other tested concentrations of growth regulators showed statistically significantly lower increases. The effects of different types of growth regulators and their concentrations on the production of selected metabolites in the tissue cultures were also examined and compared with the results obtained from the extract of the drug. Of alkaloids, stachydrine was demonstrated both in the extract of the drug and the extracts from all tested calluses regardless of the type and concentration of the growth regulator. Of flavonoid substances, two stains were demonstrated in the extract from the drug as well as in the extracts from all tested calluses. Neither of them was identical with the employed standards of rutin and quercetin. The results of TLC of iridoid substances were different in the extract from the drug (12 stains) and in the extracts from calluses (4 stains--3 identical with the stains in the extract from the drug). As to saccharides, three of them--glucose, fructose, and sucrose--were identified in the extract of the drug as well as in the extracts from all calluses.

[Regulation on growth and accumulation of total alkaloid in Leonurus artemisia (Lour.)S.Y. Hu by various microfertilizers].

OBJECTIVE: To trace the effects of various microfertilizers on the growth and accumulation of total alkaloid in Leonurus artemisia. METHOD: The total alkaloid was determined by spectrophotometry. RESULT: Microfertilizers of different concentrations could facilitate the growth of L. artemisia in various degrees and Mn-fertilizer of standard concentration and Cu-fertilizer of excessive concentration could help raise the output of the plant markedly by up to 43.6% and 55.2% respectively. Microfertilizers of different concentrations could improve the content of total alkaloid in L. artemisia: Mn-fertilizer of standard concentration and B-fertilizer of excessive concentration could enhance the contents of total alkaloid up to 2.15% and 2.09% respectively. Mn-fertilizer and Cu-fertilizer of standard concentration have been proved very useful in improving the resistibility and ability of the plant to survive summer safely, and Fe-fertilizer of standard concentration could bring the plant an obvious increase of output after surviving summer. CONCLUSION: Application of Mn-fertilizer of standard concentration is very important for cultivating L. artemisia of excellent quality and high output.