Leonurus japonicus Houtt. modulates neuronal apoptosis in intracerebral hemorrhage: Insights from network pharmacology and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonurus japonicus Houtt. (Labiatae), commonly known as Chinese motherwort, is a herbaceous flowering plant that is native to Asia. It is widely acknowledged in traditional medicine for its diuretic, hypoglycemic, antiepileptic properties and neuroprotection. Currently, Leonurus japonicus (Leo) is included in the Pharmacopoeia of the People's Republic of China. Traditional Chinese Medicine (TCM) recognizes Leo for its myriad pharmacological attributes, but its efficacy against ICH-induced neuronal apoptosis is unclear. AIMS OF THE STUDY: This study aimed to identify the potential targets and regulatory mechanisms of Leo in alleviating neuronal apoptosis after ICH. MATERIALS AND METHODS: The study employed network pharmacology, UPLC-Q-TOF-MS technique, molecular docking, pharmacodynamic studies, western blotting, and immunofluorescence techniques to explore its potential mechanisms. RESULTS: Leo was found to assist hematoma absorption, thus improving the neurological outlook in an ICH mouse model. Importantly, molecular docking highlighted JAK as Leo's potential therapeutic target in ICH scenarios. Further experimental evidence demonstrated that Leo adjusts JAK1 and STAT1 phosphorylation, curbing Bax while augmenting Bcl-2 expression. CONCLUSION: Leo showcases potential in mitigating neuronal apoptosis post-ICH, predominantly via the JAK/STAT mechanism.

De novo chromosome-level genome assembly of Chinese motherwort (Leonurus japonicus).

Chinese motherwort (Leonurus japonicus), a member of Lamiaceae family, is a commonly used medicinal herb for treating obstetrical and gynecological diseases, producing over 280 officinal natural products. Due to limited genomic resources, little progress has been made in deciphering the biosynthetic pathway of valuable natural products in L. japonicus. Here, we de novo assembled the L. japonicus genome using high-coverage ONT long reads and Hi-C reads. The chromosome-level genome assembly contained ten chromosomes representing 99.29% of 489.34 Mb genomic sequence with a contig and scaffold N50 of 7.27 Mb and 50.86 Mb, respectively. Genome validations revealed BUSCO and LAI score of 99.2% and 21.99, respectively, suggesting high quality of genome assembly. Using transcriptomic data from various tissues, 22,531 protein-coding genes were annotated. Phylogenomic analysis of 13 angiosperm plants suggested L. japonicus had 58 expanded gene families functionally enriched in specialized metabolism such as diterpenoid biosynthesis. The genome assembly, annotation, and sequencing data provide resources for the elucidation of biosynthetic pathways behind natural products of pharmaceutical applications in L. japonicus.

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.

Multiomics analyses of two Leonurus species illuminate leonurine biosynthesis and its evolution.

The Lamiaceae family is renowned for its terpenoid-based medicinal components, but Leonurus, which has traditional medicinal uses, stands out for its alkaloid-rich composition. Leonurine, the principal active compound found in Leonurus, has demonstrated promising effects in reducing blood lipids and treating strokes. However, the biosynthetic pathway of leonurine remains largely unexplored. Here, we present the chromosome-level genome sequence assemblies of Leonurus japonicus, known for its high leonurine production, and Leonurus sibiricus, characterized by very limited leonurine production. By integrating genomics, RNA sequencing, metabolomics, and enzyme activity assay data, we constructed the leonurine biosynthesis pathway and identified the arginine decarboxylase (ADC), uridine diphosphate glucosyltransferase (UGT), and serine carboxypeptidase-like (SCPL) acyltransferase enzymes that catalyze key reactions in this pathway. Further analyses revealed that the UGT-SCPL gene cluster evolved by gene duplication in the ancestor of Leonurus and neofunctionalization of SCPL in L. japonicus, which contributed to the accumulation of leonurine specifically in L. japonicus. Collectively, our comprehensive study illuminates leonurine biosynthesis and its evolution in Leonurus.

Rapid classification and identification of chemical constituents in Leonurus japonicus Houtt based on UPLC-Q-Orbitrap-MS combined with data post-processing techniques.

Leonurus japonicus Houtt (LJH) is a bulk medicinal material commonly used in clinical practice, but its complex constituents have not been completely understood, posing challenges to pharmacology, pharmacokinetic research, and scientific and rational drug use. As a result, it is critical to develop an efficient and accurate method for classifying and identifying the chemical composition of LJH. In this study, ultra-performance liquid chromatography-quadrupole electrostatic field-orbital trap high resolution mass spectrometry (UPLC-Q-Orbitrap-MS) was successfully established, along with two data post-processing techniques, characteristic fragmentations (CFs) and neutral losses (NLs), to quickly classify and identify the chemical constituents in LJH. As a result, 44 constituents of LJH were identified, including four alkaloids, 20 flavonoids, two phenylpropanoids, 17 organic acids, and one amino acid. The method in this paper enables classification and identification of chemical compositions rapidly, providing a scientific foundation for further research on the effective and toxic substances of LJH.

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.

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.

New di-spirocyclic labdane diterpenoids from the aerial parts of Leonurus japonicus.

Phytochemical investigation on the ethanol extract of a well-known medicinal herb Leonurus japonicus, led to the separation of 18 labdane type diterpenoids (1-18). Through comprehensive spectroscopic analyses and quantum chemical calculations, these compounds were structurally characterized as six new interesting 5,5,5-di-spirocyclic ones (1-6), two new (7 and 8) and six known (13-18) interesting 6,5,5-di-spirocyclic ones, a new rare 14,15-dinor derivative (9), and three new ones incorporating a γ-lactone unit (10-12). An in vitro neuroprotective assay in RSC96 cells revealed that compounds 7 and 12 exhibited neuroprotective activity in a concentration-dependent way, comparable to the reference drug N-acetylcysteine.

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.

Anti-inflammatory labdane diterpenoids from the aerial parts of Leonurus japonicus.

Twenty-two labdane-type diterpenoids, including ten pairs of 15-epimers and a pair of 13,15-epimers, were obtained from the aerial parts of a well-known medicinal plant Leonurus japonicus Houtt. While these epimers were separated by chiral HPLC, their structures were established mainly via spectroscopic methods especially NMR, X-ray crystallography and ECD techniques. Among them, seventeen compounds, encompassing three pairs of solvolysis artefacts likely due to the use of ethanol as extracting solvent, were reported for the first time in the current work. Our preliminary anti-inflammatory screening demonstrated that seven diterpenoids displayed noteworthy inhibitory effect on the NO production in LPS-induced RAW264.7 cells. In addition, the release of pro-inflammatory factors TNF-α, IL-1ß and IL-6, as well as the expression of iNOS and COX-2 proteins, was also suppressed by the unreported 15,16-epoxy-6ß-hydroxy-15α-methoxy-7,16-dioxolabd-8,13-diene. Further investigation into the preliminary anti-inflammatory mechanism of this compound indicated that it could block the activation of NF-κB signaling pathway.

Stachydrine, a potential drug for the treatment of cardiovascular system and central nervous system diseases.

Cardiovascular disease (CVD) is the leading cause of death globally and poses at significant challenge in terms of effective medical treatment. Leonurus japonicus Houtt, a traditional Chinese herb, is widely used in China for the treatment of obstetrical and gynecological disorders, including menstrual disorders, dysmenorrhea, amenorrhea, blood stasis, postpartum bleeding, and blood-related diseases such as CVD. Stachydrine, the main alkaloid component of Leonurus, has been shown to exhibit a wide range of biological activities including anti-inflammatory, antioxidant, anti-coagulant, anti-apoptotic, vasodilator, angiogenic promoter. Additionally, it has been demonstrated to have unique advantages in the prevention and treatment of CVD through regulation of various disease-related signaling pathways and molecular targets. In this comprehensive review, we examine the latest pharmacological effects and molecular mechanisms of Stachydrine in treating cardiovascular and cerebrovascular diseases. Our aim is to solid scientific basis for the development of new CVD drug formulations.

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.

New di-spirocyclic labdane diterpenoids from the aerial parts of Leonurus japonicus / 中国天然药物

Phytochemical investigation on the ethanol extract of a well-known medicinal herb Leonurus japonicus, led to the separation of 18 labdane type diterpenoids (1-18). Through comprehensive spectroscopic analyses and quantum chemical calculations, these compounds were structurally characterized as six new interesting 5,5,5-di-spirocyclic ones (1-6), two new (7 and 8) and six known (13-18) interesting 6,5,5-di-spirocyclic ones, a new rare 14,15-dinor derivative (9), and three new ones incorporating a γ-lactone unit (10-12). An in vitro neuroprotective assay in RSC96 cells revealed that compounds 7 and 12 exhibited neuroprotective activity in a concentration-dependent way, comparable to the reference drug N-acetylcysteine.

[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.

Total alkaloid fraction of Leonurus japonicus Houtt. Promotes angiogenesis and wound healing through SRC/MEK/ERK signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonurus japonicus Houtt., also known as motherwort, is a traditional Chinese medicine that was first identified in Shennong Bencao Jing, the first and essential pharmacy monograph in China. L. japonicus has been regarded as a good gynecological medicine since ancient times. It has been widely used in clinical settings for treatment of gynecological diseases and postnatal rehabilitation with good efficacy and low adverse effects. AIM OF THE STUDY: The main purpose of this study was to determine the angiogenic and wound healing effects of total alkaloid fraction from L. japonicus Houtt. (TALH) in vivo and in vitro. In addition, the main bioactive components of total alkaloids were to be identified and analyzed in this study. MATERIALS AND METHODS: First, the UHPLC/Q-TOF-MS method was used to identify and quantify the major components in the TALH extract. The wound healing activity was evaluated in vivo using a rat full-thickness cutaneous wound model. Histological study of wound healing in rat model was performed via immunohistochemistry and immunofluorescence. Cell proliferation was determined by MTT assay. Wound healing and transwell assays were used for detection of cell migration. The effect on tube formation was determined by tube formation assay in HUVECs. Western blot and RT-PCR were used to detect the expressions of relative proteins and genes respectively. Knock-down of SRC by siRNA was done to verify the crucial role of SRC in promotion of angiogenesis induced by TALH. RESULTS: Seven characteristic peaks were recognized in the UHPLC/Q-TOF-MS spectrum, while four of the main components were quantified. The wound model in rats showed that treatment of TALH promoted wound healing by stimulating cellular proliferation and collagen deposition. In vitro experiments showed that co-treatment of TALH and VEGF increased cell proliferation, migration and tube formation in HUVECs. Mechanistic studies suggested that the co-treatment increased gene expressions of SRC, MEK1/2 and ERK1/2, as well as the phosphorylation levels of these proteins. Furthermore, the effect of co-treatment was attenuated after SRC knockdown, suggesting that SRC plays an important role in angiogenesis and wound healing induced by TALH and VEGF co-treatment. CONCLUSION: Our results showed that TALH was one of the main active components of L. japonicus that promoted angiogenesis and wound healing by regulating the SRC/MEK/ERK pathway. Our study provided scientific basis for better clinical application of L. japonicas.

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.

Leonurus japonicus Houtt. (Motherwort): Systematic research through chemical profiling, stability under controlled conditions and pharmacokinetic analysis on screening Q-markers for quality control.

Leonurus japonicus Houtt. (Motherwort) is the fresh or dried aerial part of Leonurus japonicus Houtt. (Labiaceae), which is widely used in clinical practice and daily life, used to treat gynecological diseases. However, the differences between different parts, single index component in Pharmacopoeias and the less stability of active ingredients affect its clinical efficacy. This study aimed to find the multi-active compounds between different parts of Motherwort to ensure its clinical efficacy, which related to stability and had pharmacokinetic behavior. Firstly, HPLC-Q-TOF-MS/MS was used to analyze the components in vitro and in vivo, as well as multivariate statistical analysis and network pharmacology analysis was conducted to find the significant different components related to activity. Secondly, the content determination methods were established to study the stability of effective components during storage in order to establish the content limit for quality control of Motherwort. Thirdly, UFLC-MS/MS was used to analyze the pharmacokinetic behavior of active components in Motherwort. The results showed that a total of 131 chemical constituents were identified in vitro and 21 prototype absorption compounds and 72 metabolites were found in vivo. Meantime, multivariate statistical analysis and network pharmacology analysis was combined to find that leonurine, stachydrine and trigonelline were activity-related substance, which could be used as active components related to pharmacodynamics in different parts. Then the stability variation trend and content limit of three alkaloids were found, which could be used for the quality control of Motherwort. Furthermore, the results showed that three alkaloids had pharmacokinetic behavior in vivo. 3 alkaloids were screened, which could be used as active components most closely related to pharmacodynamics among different parts. The stable stage, assay tolerance and pharmacokinetic characteristics were studied by the active substances, which could provide a basis for quality control and clinical medication of Motherwort.

Leonurus japonicus Houttuyn induces reactive oxygen species-mediated apoptosis via regulation of miR-19a-3p/PTEN/PI3K/AKT in U937 and THP-1 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonurus japonicus Houttuyn is a medicinal ingredient in more than 300 prescriptions in traditional Korean medicine. It is especially important for women's health and blood-related diseases. Recent research revealed that Leonurus japonicus Houttuyn extracts have antioxidative, anticancer, analgesic, anti-inflammatory, and neuroprotective properties. AIM OF THE STUDY: However, its underlying anti-cancerous mechanisms remain unclear. This study elucidated the anticancer mechanism of Leonurus japonicus Houttuyn in U937 and THP-1 cancer cells. MATERIALS AND METHODS: High-performance liquid chromatography (HPLC) was used for detecting main compound of Leonurus japonicus Houttuyn, rutin. EZ-Cytox cell viability assay, Western blot analysis, live and dead cell assay, 2', 7' dichlorofluorescin diacetate (DCFDA) assay, quantitative real-time PCR (qRT-PCR) analysis, and microRNA (miR) mimic transfection assay were applied to further investigate anti-cancer efficacies and underlying mechanism in U937 and THP-1 cells. RESULTS: The main compound of Leonurus japonicus Houttuyn, rutin was detected using HPLC. The cytotoxic effect of Leonurus japonicus Houttuyn was exerted in U937 and THP-1 cancer cells but not in MDBK and IEC-6 normal cells. Leonurus japonicus Houttuyn decreased mitochondria membrane potential (ΔΨm). Consistently, Leonurus japonicus Houttuyn reduced the expression of survivin and cleaved caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP). Cell death was increased in Leonurus japonicus Houttuyn treated groups. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and CCAAT-enhancer-binding protein homologous protein (CHOP) was increased and phosphatidylinositol-3-kinase (PI3K) and Protein kinase B (AKT) were decreased by Leonurus japonicus Houttuyn. Reactive oxygen speices generation was elevated by Leonurus japonicus Houttuyn and its cytotoxicity was reversed by N-acetyl-l-cysteine (NAC) pretreatment. Moreover, onco-microRNA (miR), miR-19a-3p was suppressed by Leonurus japonicus Houttuyn and transfection of miR-19a-3p mimic reversed the regulated PTEN, p-AKT, CHOP expression, attenuating Leonurus japonicus Houttuyn induced apoptosis. CONCLUSIONS: These findings indicated that Leonurus japonicus Houttuyn has anti-cancer effects by regulation of PTEN/PI3K/AKT signal pathway and ROS-related ER stress-induced apoptosis via regulation of miR-19a-3p. Leonurus japonicus Houttuyn may be an effective candidate for triggering PTEN-dependent apoptosis of cancer cells related to acute myeloid leukemia.

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.

Gastroprotective action of the ethanol extract of Leonurus sibiricus L. (Lamiaceae) in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonurus sibiricus L. (Lamiaceae) is a medicinal plant known in Brazil as "rubim" or "erva de macaé". It is used for various purposes, including stomach disorders. AIM OF THE STUDY: To evaluate the effect of the ethanol extract of the aerial parts of L. sibiricus (EELs) in models of gastric damage in mice. MATERIAL AND METHODS: The effect of EELs (50, 100 and 300 mg/kg, p.o., 1 h before induction) was tested on acidified ethanol (ACEt)-induced gastric ulcers. Additionally, we tested the effect of EELs (by intraduodenal administration) in the pylorus ligation (PL) model. RESULTS: Pretreatment with EELs, at 300 mg/kg, but not 50 and 100 mg/kg, reduced the relative area of gastric ulcers induced by ACEt (p < 0.01) and lipoperoxidation (p < 0.001), and increased the sulfhydryl content (p < 0.01) in the stomach in comparison with the vehicle group. Pretreatment with N-ethylmaleimide (a blocker of non-protein sulfhydryl groups, 10 mg/kg, i.p.) or glibenclamide (a KATP channel blocker, 10 mg/kg, i.p.) inhibited the gastroprotective response caused by EELs (300 mg/kg; p < 0.001), but there were no alterations due to pretreatments with inhibitors of the synthesis of prostaglandins (indomethacin, 10 mg/kg), nitric oxide (L-NAME, 70 mg/kg) or hydrogen sulfide (DL-propargylglycine, 10 mg/kg). Treatment with EELs (300 mg/kg) reduced mucus production (p < 0.001) and the volume of gastric secretion (p < 0.001) after PL without affecting gastric acidity or pH. CONCLUSIONS: These results provide evidence that EELs exerts gastroprotective action in mice, with the participation of oxidative stress and mediation of NP-SH, KATP channels and mucus production.