Microscopic and Phytochemical Comparison of the Three Leonurus Species L. cardiaca, L. japonicus, and L. sibiricus.

At least three Leonurus species, Leonurus cardiaca, Leonurus japonicus, and Leonurus sibiricus, are used in various traditional medicinal systems for different therapeutic purposes. The plant names "L. sibiricus" and "L. japonicus" are often used as synonyms, which causes confusion in literature and implies that several studies may be based on improperly identified plant material. To avoid further confusion, the current status of the identification of these three species is illustrated. Characteristics for their unequivocal identification are presented using stereo and light microscopy as well as HPTLC.The establishment of the species-specific anatomical, morphological, and phytochemical characters was based on reference specimens in comparison with wild collected or commercially obtained material. Morphologically, the species differed in shape and length ratio of the upper and lower lip of the corolla. Differentiating anatomical characters involve the presence, density, and dimension of trichomes on different organs. Detailed anatomical descriptions of the leaves and corollae of L. japonicus and L. sibiricus are given and compared to L. cardiaca. These three sets of characters facilitate fast and reliable identification. Complementary HPTLC fingerprints show type-specific patterns that allow the differentiation of L. japonicus from the other two species. For the distinction of L. cardiaca and L. sibiricus, flower morphology has to be considered.The presented data contribute to the quality control of the three Leonurus species and additionally meet a definite and timely need regarding the introduction of L. japonicus to the European Pharmacopoeia in addition to the already existing monograph of L. cardiaca.

Quercetin Stimulates Insulin Secretion and Reduces the Viability of Rat INS-1 Beta-Cells.

BACKGROUND/AIMS: Previously we described insulinotropic effects of Leonurus sibiricus L. plant extracts used for diabetes mellitus treatment in Traditional Mongolian Medicine. The flavonoid quercetin and its glycoside rutin, which exert anti-diabetic properties in vivo by interfering with insulin signaling in peripheral target tissues, are constituents of these extracts. This study was performed to better understand short- and long-term effects of quercetin and rutin on beta-cells. METHODS: Cell viability, apoptosis, phospho-protein abundance and insulin release were determined using resazurin, annexin-V binding assays, Western blot and ELISA, respectively. Membrane potentials (Vmem), whole-cell Ca2+ (ICa)- and ATP-sensitive K+ (IKATP) currents were measured by patch clamp. Intracellular Ca2+ (Cai) levels were measured by time-lapse imaging using the ratiometric Ca2+ indicator Fura-2. RESULTS: Rutin, quercetin and the phosphoinositide-3-kinase (PI3K) inhibitor LY294002 caused a dose-dependent reduction in cell viability with IC50 values of ∼75 µM, ∼25 µM and ∼3.5 µM, respectively. Quercetin (50 µM) significantly increased the percentage of Annexin-V+ cells within 48 hrs. The mean cell volume (MCV) of quercetin-treated cells was significantly lower. Within 2 hrs, quercetin significantly decreased basal- and insulin-stimulated Akt(T308) phosphorylation and increased Erk1/2 phosphorylation, without affecting P-Akt(S473) abundance. Basal- and glucose-stimulated insulin release were significantly stimulated by quercetin. Quercetin significantly depolarized Vmem by ∼25 mV which was prevented by the KATP-channel opener diazoxide, but not by the L-type ICa inhibitor nifedipine. Quercetin significantly stimulated ICa and caused a 50% inhibition of IKATP. The effects on Vmem, ICa and IKATP rapidly reached peak values and then gradually diminished to control values within ∼1 minute. With a similar time-response quercetin induced an elevation in Cai which was completely abolished in the absence of Ca2+ in the bath solution. Rutin (50 µM) did not significantly alter the percentage of Annexin-V+ cells, MCV, Akt or Erk1/2 phosphorylation, insulin secretion, or the electrophysiological behavior of INS-1 cells. CONCLUSION: We conclude that quercetin acutely stimulates insulin release, presumably by transient KATP channel inhibition and ICa stimulation. Long term application of quercetin inhibits cell proliferation and induces apoptosis, most likely by inhibition of PI3K/Akt signaling.

Quantitation of phenylpropanoids and iridoids in insulin-sensitising extracts of Leonurus sibiricus L. (Lamiaceae).

INTRODUCTION: Leonurus sibiricus L. is regularly used in traditional Mongolian medicine including for the treatment of symptoms of diabetes mellitus. OBJECTIVES: To provide a validated quantitation method for the quality control of Leonurus sibiricus and to prove in vitro insulin-sensitisation, thereby supporting the traditional use of Leonurus sibiricus. METHODOLOGY: Pulverised Leonurus sibiricus material was either extracted with methanol or methanol:water (25:75, v/v). HPLC-CAD (charged aerosol detector) separations were performed on a Luna Phenyl-Hexyl column with water and acetonitrile (both modified with 0.1% formic acid) as mobile phase. Gradient elution was employed using theophylline as internal standard. Tentative peak identification was facilitated by HPLC-MS. Validation was carried out according to ICH (International Conference on Harmonisation) guidelines. Potential insulin-sensitisation of accordant extracts was assessed in glucose uptake experiments in C2C12 myocytes and protein tyrosine phosphatase 1B (PTP1B) enzyme assays. RESULTS: Thirty-six compounds were tentatively identified based on their retention times, UV spectra, MS fragments and data from literature. They comprise phenolcarboxylic acids, flavonoids, iridoid glycosides, and phenylpropanoids, among which acetylharpagide, ajugoside, lavandulifolioside, and verbascoside were selected for quantitation. The methanol extract contained 0.42% combined iridoids, and 1.58% combined phenylpropanoids. Validation showed good accuracy, intermediate precision and robustness. The methanol extract of Leonurus sibiricus led to a 1.5 fold increase in insulin-stimulated cellular glucose uptake and inhibition of PTP1B by 40% at a concentration of 10 µg/mL. CONCLUSION: HPLC-CAD analysis allowed sensitive quantitation of the selected marker compounds in Leonurus sibiricus, thereby providing a reliable tool for its quality control.

Resveratrol-zinc combination for prostate cancer management.

Zinc, an essential trace element, plays a critical role in cell signaling, and defect(s) in zinc homeostasis may contribute to adverse physiological and pathological conditions, including cancer. Zinc is present in healthy prostate at a very high concentration, where it is required for important prostatic functions. However, zinc levels are significantly diminished in cancerous tissue, and intracellular zinc level is inversely correlated with prostate cancer progression. During neoplastic transformation, zinc-accumulating, citrate-producing normal prostate cells are metabolically transformed to citrate oxidizing cells that lose the ability to accumulate zinc. Interestingly, zinc has been shown to function as chemopreventive agent against prostate cancer, albeit at high doses, which may lead to many adverse effects. Therefore, novel means to enhance bioaccumulation of sufficient zinc in prostate cells via increasing zinc transport could be useful against prostate cancer. On the basis of available evidence, we present a possibility that the grape antioxidant resveratrol, when given with zinc, may lead to retuning the zinc homeostasis in prostate, thereby abolishing or reversing malignancy. If experimentally verified in in vivo model(s) of prostate cancer, such as transgenic mouse models, this may lead to novel means toward management of prostate cancer and other conditions with compromised zinc homeostasis.

Walnut leaf extract inhibits PTP1B and enhances glucose-uptake in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Walnut, Juglans regia L. (Juglandaceae), is one of the medicinal plants used to treat diabetic symptoms in Austrian folk medicine. The air-dried green leaves are either used as aqueous decoctions or liquor preparations and are consumed on a daily basis. We investigated the hypoglycemic effect of a methanolic Juglans regia leaf extract on glucose uptake, protein tyrosine phosphatase 1B (PTP1B) inhibition and peroxisome proliferator-activated receptor gamma (PPARγ) activation. MATERIAL AND METHODS: Hypoglycemic activity was assessed by glucose-uptake in C2C12 myocytes, inhibition of PTP1B and activation of PPARγ. Phytochemical characterization of the extract was carried out by LC-MS and GC-MS. RESULTS: Methanolic Juglans regia leaf extract enhanced the glucose uptake rate in C2C12 myocytes at concentrations of 25µg/mL compared to untreated cells. This activity may partly be explained by the inhibition of PTP1B but not PPARγ agonism. LC-MS analyses revealed chlorogenic acid (1), 3-p-coumaroylquinic acid (2), a trihydroxynaphthalene-hexoside (3), as well as eight flavonoids (4-11) as main phenolic constituents in the active extract. CONCLUSIONS: The finding that Juglans regia leaf extract enhances glucose uptake and inhibits PTP1B provides an in vitro-based rationale for the traditional use of walnut leaf preparations against elevated blood-glucose levels.