Seven new secondary metabolites isolated from roots of Lespedeza bicolor.

Chemical investigation of a methanol extract obtained from the roots of Lespedeza bicolor identified one new pterocarpene (1), three new pterocarpans (2-4), and three new arylbenzofurans (5-7), and two known compounds (8 and 9). Their structures were determined by interpretations obtained from combined UV, NMR, and HRTOFMS spectroscopic data. Furthermore, the absolute configurations of compounds 2 and 3 were established by the combination of electronic circular dichroism (ECD) calculations and NMR calculations with DP4+ probability analysis. All isolated compounds (1-9) were evaluated for cytotoxicity against the human lung carcinoma cell line A549 and the human hepatoma cell line Huh-7. Compound 4 showed antiproliferative activity against A549 cell line with IC50 value of 24.9 µM. Furthermore, compound 9 exhibited cytotoxicity against Huh-7 cell line with IC50 value of 68.7 µM.

Lespedeza bicolor extract supplementation reduced hyperglycemia-induced skeletal muscle damage by regulation of AMPK/SIRT/PGC1α-related energy metabolism in type 2 diabetic mice.

Lespedeza bicolor (LB) is known to have antidiabetic activities; however, the underlying molecular mechanisms of LB in hyperglycemia-induced skeletal muscle damage is unclear. Inflammation and oxidative stress caused by type 2 diabetes mellitus (T2DM) not only contributes to insulin resistance, but also promotes muscle atrophy via decreased muscle protein synthesis and increased protein degradation, leading to frailty and sarcopenia. In this study, we hypothesized that LB extract (LBE) supplementatin has an ameliorative effect on hyperglycemia-induced skeletal muscle damage by activation of 5' adenosine monophosphate-activated protein kinase (AMPK)/sirtuin (SIRT)/proliferator-activated receptor γ coactivator 1α (PGC1α)-associated energy metabolism in mice with T2DM. Diabetes was induced by a high-fat diet with a 2-time streptozotoxin injection (30 mg/kg body weight) in male C57BL/6J mice. After diabetes was induced (fasting blood glucose level ≥140 mg/dL), the mice were administered with LBE at a low dose (100 mg/kg/d) or high dose (250 mg/kg/d) by gavage for 12 weeks. LBE supplementation ameliorated glucose tolerance and hemoglobin A1c (%) in mice with T2DM. Moreover, LBE supplementation upregulated protein levels of insulin receptor subunit-1 and Akt accompanied by increased translocation of glucose transporter 4 in mice with T2DM. Furthermore, LBE increased mitochondrial biogenesis by activating SIRT1, SIRT3, SIRT4, and peroxisome PGC1α in diabetic skeletal muscle. Meanwhile, LBE supplementation reduced oxidative stress and inflammation in mice with T2DM. Taken together, the current study suggested that LBE could be a potential therapeutic to prevent skeletal muscle damage by regulation AMPK/SIRT/PGC1α-related energy metabolism in T2DM.

Biologically active polyphenolic compounds from Lespedeza bicolor.

We investigated the ability of six prenylated prerocarpans, stilbenoid, and a new dimeric flavonoid, lespebicolin B, from stem bark as well as two 3-O-rutinosides and a mixture of 3-O-ß-D-glucosides of quercetin and kaempferol from flowers of Lespedeza bicolor to inhibit HSV-1 replication in Vero cells. Pretreatment of HSV-1 with polyphenolic compounds (direct virucidal effect) showed that pterocarpans lespedezol A2 (1), (6aR,11aR)-6a,11a-dihydrolespedezol A2 (2), (6aR,11aR)-2-isoprenyldihydrolespedezol A2 (4), and (6aR,11aR,3'R)-dihydrolespedezol A3 (5) significantly inhibited viral replication, with a selective index (SI) ≥10. Compound 4 possessed the lowest 50% - inhibiting concentration (IC50) and the highest SI values (2.6 µM and 27.9, respectively) in this test. (6aR,11aR)-2-Isoprenyldihydrolespedezol A2 (4) also had a moderate effect under simultaneous treatment of Vero cells with the tested compound and virus (IC50 and SI values were 5.86 µM and 12.4, respectively). 3-O-rutinosides of quercetin and kaempferol and a mixture of 3-O-ß-D-glucosides of quercetin and kaempferol (10 and 12) also showed significant virucidal activity, with SI values of 12.5, 14.6, and 98.2, respectively, and IC50 values of 8.6, 12.2, and 3.6, respectively. We also performed a quantitative structure-activity relationship (QSAR) analysis of data on the virucidal activity of polyphenolics with 4 < pIC50 < 6. It was found that the virucidal activity of these compounds depended on both the structure of the aromatic part and the conformation of geranyl and isoprenyl side chains of their molecules. These findings are correlated with the largest value of the principal moment of inertia (pmi) descriptor describing the geometry of molecules.

Determination of Afzelin and Astragalin from Lespedeza cuneata on Aldose Reductase Inhibition.

The bioactive chemicals in L. cuneata were investigated by repeated column chromatography and their effect on aldose reductase (AR), obtained from rat lenses, was examined. Results showed that the ethyl acetate and n-butanol fractions of L. cuneata exhibited potential inhibitory effect against AR with IC50 values of 0.57 and 0.49 µg/mL, respectively. Phytochemical analysis of these two fractions resulted in the isolation of five flavonoids namely, acacetin (1), afzelin (2), astragalin (3), kaempferol (4) and scutellarein 7-O-glucoside (5). The AR inhibitory effect of compounds 1-5 was explored; compounds 2, 3 and 5 showed potential AR-inhibitory effects with IC50 values of 2.20, 1.91 and 12.87 µM, respectively. Quantitative analysis of afzelin (2) and astragalin (3) in L. cuneata by high performance liquid chromatography with ultraviolet detection revealed its content to be 0.722-11.828 and 2.054-7.006 mg/g, respectively. Overall, this study showed that L. cuneata is rich in flavonoids with promising AR-inhibitory activities, which can be utilized for the development of natural therapies for treating and managing diabetic complications.

Quebracho tannins aid in the control of Eimeria spp. and gastrointestinal nematodes in lambs and goat kids.

The objective was to examine the effects of supplementary quebracho on control of coccidiosis and gastrointestinal nematodes in lambs and kids. In Exp. 1, naturally infected lambs weaned (87.8 ± 0.4 days of age; day 0) in January (winter) were blocked by sex and randomly assigned (n = 10/treatment) to receive supplement with or without 100 g/lamb of quebracho for 28 days. In Exp. 2, single or twin rearing ewes were randomly assigned into two groups, and naturally infected lambs were fed control (n = 28) or quebracho (100 g/lamb of quebracho tannins in feed; n = 27) between -28 and 21 days (weaning = day 0; 70.8 ± 0.1 days of age). In Exp. 3, weaned doe kids (57.6 ± 2.0 days of age) were randomly assigned to receive alfalfa (Medicago sativa) supplement with (n = 9) or without (n = 8) 50 g/kid quebracho or sericea lespedeza (Lespedeza cuneata) with quebracho (n = 8) for 21 days. Fecal oocyst count (FOC), nematode egg counts (FEC), fecal score, dag score (soiling around rear quarters), and blood packed cell volume (PCV) were determined every 7 days. Data were analyzed as repeated measures using mixed models. In Exp. 1, FOC decreased in quebracho-fed lambs (diet × time, P < 0.001) but FEC was similar between treatments during the feeding period (P = 0.19). Packed cell volume (P = 0.19) and fecal score (P = 0.42) were similar between groups. Quebracho-fed lambs had a greater dag score initially (diet × time, P = 0.02), but were similar by day 42 (P = 0.72). In Exp. 2, FOC remained low (P = 0.02), PCV tended to decrease (P = 0.06), but FEC increased on days 14 and 21 (diet × time; P < 0.001) in quebracho compared with control-fed lambs. Quebracho-fed lambs had lower fecal score (diet × time; P = 0.005) but higher dag score (diet × time; P < 0.001). In Exp. 3, FOC of kids fed quebracho (alfalfa or sericea lespedeza supplement) was lower than control (P < 0.001). Fecal score of kids fed sericea lespedeza compared with alfalfa were lower regardless of quebracho (P = 0.01). There were no differences among treatments for dag, FEC, PCV, or body weight (P> 0.10). Quebracho was effective in reducing FOC but not clinical signs of coccidiosis in both lambs and kids, and may not be highly digestible in lambs as it caused loose stools.

Preparation, characterization and anti-cancer activity of graphene oxide-­silver nanocomposite.

This work reported the preparation, characterization, cytotoxicity of green synthesized Lespedeza cuneate mediated silver nanoparticles (Lc-AgNPs) and graphene oxide­silver nanocomposite (GO-AgNComp) using Lc-AgNPs. The UV absorption spectrum at 419 nm indicated the successful formation of GO-AgNComp. The TEM analysis displayed the thin sheet of graphene decorated Lc-AgNPs in GO-AgNComp. Zeta potential was -13.2 mV for Lc-AgNPs and -30.5 mV for GO-AgNComp. The photothermal conversion efficiency was calculated as 31.09% for GO-AgNComp. The negatively charged zeta potential of GO-AgNComp enhanced its cellular penetration through enhanced permeability and retention (EPR) effect. The near-infrared laser (NIR) induced the anticancer activity of Lc-AgNPs and GO-AgNComp in human lung cancer cells (A549) and brain tumour (LN229). The results indicated that about 50% of A549 cells and LN229 cells were ablated by treatment of 24.73 ± 2.98 µg/mL and 27.34 ± 1.62 µg/mL of Lc-AgNPs, as well by 15.46 ± 2.31 µg/mL and 20.95 ± 1.35 µg/mL of GO-AgNComp respectively. Moreover, GO-AgNComp was not cytotoxic to normal mouse fibroblast cells (NIH3T3), but it caused the cancer cell death in A549 and LN229 through ROS generation, nuclear damage, and mitochondrial membrane potential (∆ψm) loss. This work reported the anticancer potential of GO-AgNComp, which deserves further study on the molecular elucidation of GO-AgNComp mediated human lung and tumour therapy.

Aviculin Isolated from Lespedeza cuneata Induce Apoptosis in Breast Cancer Cells through Mitochondria-Mediated Caspase Activation Pathway.

The global incidence of breast cancer has increased. However, there are many impediments to the development of safe and effective anticancer drugs. The aim of the present study was to evaluate the effect of aviculin isolated from Lespedeza cuneata (Dum. Cours.) G. Don. (Fabaceae) on MCF-7 human breast cancer cells and determine the underlying mechanism. Using the bioassay-guided isolation by water soluble tetrazolium salt (WST-1)-based Ez-Cytox assay, nine compounds (four lignan glycosides (1-4), three flavonoid glycosides (5-7), and two phenolic compounds (8 and 9)) were isolated from the ethyl acetate (EA) fraction of the L. cuneata methanolic extract. Of these, aviculin (2), a lignan glycoside, was the only compound that reduced metabolic activity on MCF-7 cells below 50% (IC50: 75.47 ± 2.23 µM). The underlying mechanism was analyzed using the annexin V Alexa Fluor 488 binding assay and Western blotting. Aviculin (2) was found to induce apoptotic cell death through the intrinsic apoptosis pathway, as indicated by the increased expression of initiator caspase-9, executioner caspase-7, and poly (ADP-ribose) polymerase (PARP). Aviculin (2)-induced apoptotic cell death was accompanied by an increase in the Bax/Bcl-2 ratio. These findings demonstrated that aviculin (2) could induce breast cancer cell apoptosis through the intrinsic apoptosis pathway, and it can therefore be considered an excellent candidate for herbal treatment of breast cancer.

Polyphenolic Compounds from Lespedeza Bicolor Root Bark Inhibit Progression of Human Prostate Cancer Cells via Induction of Apoptosis and Cell Cycle Arrest.

From a root bark of Lespedeza bicolor Turch we isolated two new (7 and 8) and six previously known compounds (1-6) belonging to the group of prenylated polyphenols. Their structures were elucidated using mass spectrometry, nuclear magnetic resonance and circular dichroism spectroscopy. These natural compounds selectively inhibited human drug-resistant prostate cancer in vitro. Prenylated pterocarpans 1-3 prevented the cell cycle progression of human cancer cells in S-phase. This was accompanied by a reduced expression of mRNA corresponding to several human cyclin-dependent kinases (CDKs). In contrast, compounds 4-8 induced a G1-phase cell cycle arrest without any pronounced effect on CDKs mRNA expression. Interestingly, a non-substituted hydroxy group at C-8 of ring D of the pterocarpan skeleton of compounds 1-3 seems to be important for the CDKs inhibitory activity.

Antiproliferative Pterocarpans and Coumestans from Lespedeza bicolor.

Chromatographic purification of a methanol extract of the roots of Lespedeza bicolor led to the isolation of four new pterocarpans (1-4), two new coumestans (6 and 7), two new arylbenzofurans (8 and 9), and the known pterocarpan 1-methoxyerythrabyssin II (5). Their structures were identified using NMR spectroscopy, UV spectroscopy, and mass spectrometry. Cytotoxicity assays showed that compounds 1-9 exerted antiproliferative effects on blood cancer cells. Of these compounds, 1 and 6 induced mitochondrial depolarization and induced apoptosis in Jurkat cells. These compounds promoted cell death by inducing cell-cycle arrest at the G1 stage, reducing levels of BCL2, and increasing cleavage of PARP-1. These findings indicate that 1 and 6 are possible lead compounds for the treatment of human leukemia cells via intracellular signaling.

Lespedeza bicolor Extract Improves Amyloid Beta25 - 35-Induced Memory Impairments by Upregulating BDNF and Activating Akt, ERK, and CREB Signaling in Mice.

Lespedeza bicolor, a traditional herbal medicine widely used in Australia, North America, and Eastern Asia, has various therapeutic effects on inflammation, nephritis, hyperpigmentation, and diuresis. In this study, to evaluate the effects of L. bicolor on cognitive function, we examined whether L. bicolor improved amyloid beta-induced memory impairment and assessed the possible mechanisms in mice. Catechin, rutin, daidzein, luteolin, naringenin, and genistein were identified in the powdered extract of L. bicolor by HPCL-DAD analyses. In behavioral experiments, L. bicolor (25 and 50 mg/kg, p. o.) significantly improved amyloid beta25 - 35 (6 nmol, intracerebroventricular)-induced cognitive dysfunction in the Y-maze, novel recognition, and passive avoidance tests. Our molecular studies showed L. bicolor (25 and 50 mg/kg, p. o.) significantly recovered the reduced glutathione content as well as increased thiobarbituric acid reactive substance and acetylcholinesterase activities in the hippocampus. Furthermore, we found that L. bicolor significantly increased the expression of brain-derived neurotrophic factor, and phospho-Akt, extracellular signal-regulated kinase, and cAMP response element binding caused by amyloid beta25 - 35 in the hippocampus. In conclusion, L. bicolor exerts a potent memory-enhancing effect on cognitive dysfunction induced by amyloid beta25 - 35 in mice.

Cytotoxic polyphenolic compounds from Lespedeza bicolor stem bark.

Four new pterocarpans (6aR,11aR)-6a,11a-dihydrolespedezol A2 (2), (6aR,11aR)-2-isoprenyl-6a,11a-dihydrolespedezol A2 (3), (6aR,11aR,3'R)-6a,11a-dihydrolespedezol A3 (4), (6aR,11aR,3'S)-6a,11a-dihydrolespedezol A3 (5) and one new stilbenoid with 1,2-diketone fragment named bicoloketone (6) along with one previously known pterocarpen lespedezol A2 (1) have been isolated from Lespedeza bicolor stem bark using multistage column chromatography on polyamide and silica gel. The structures of the isolated polyphenolic compounds were determined by spectroscopic methods. The absolute configurations of 4 and 5 were determined by comparison of their electronic circular dichroism (ECD) spectra obtained experimentally and the spectra calculated using time-dependent density functional theory (TDDFT). The isolated compounds exhibited a moderate DPPH scavenging effect and ferric reducing power compared to the reference antioxidant quercetin. The cytotoxicity of compounds against three human cancer cell lines, HTB-19, Kyse-30, and HEPG-2, and two normal cell lines, RPE-1 and HEK-293, was tested using the MTT assay. Compound 3 showed the strongest cytotoxic activity against all cell lines (IC50 6.0-19.1 µM) compared with the positive control cisplatin. The other tested compounds possessed moderate cytotoxic activity against cancer cells.

Effects of Lespedeza Cuneata aqueous extract on testosterone-induced prostatic hyperplasia.

CONTEXT: Lespedeza cuneata G. Don (Fabaceae), has been used as a traditional treatment of various diseases. There is a report L. cuneata effects on hormone replacement therapy for endocrine-related disease. However, studies related to benign prostatic hyperplasia (BPH) have not been investigated. OBJECTIVE: The effects of L. cuneata aqueous extract (LCW) on testosterone-induced prostatic hyperplasia (TPH) were examined. MATERIALS AND METHODS: Male Wistar rats (10 weeks, 330-350 g) were randomly divided to 6 groups (n = 6): Control group; TPH group (3 mg/kg, s.c, daily); TPH + LCW (25, 50, 100 mg/kg); TPH + Finasteride 10 mg/kg for 6 weeks. At the end of treatment, histological change of prostate, serum dihydrotestosterone (DHT) level, mRNA expression of 5α-reductase, inflammatory factors, proliferating cell nuclear antigen (PCNA) and fibroblast growth factor-2 (FGF-2) in prostate were examined. Then, LCW was treated with BPH-1, a human BPH cell line, at 25, 50, 100 µg/mL for 24 h and examine mRNA level of androgen receptor (AR) and prostate-specific antigen (PSA). In addition, the content of vicenin-2 was analyzed. RESULTS: LCW treatment of TPH inhibited serum DHT levels by 54.5, 51.2 and 54.1% and mRNA expression of 5α-reductase were inhibited 54.3, 61.3 and 73.6%, respectively. In addition, mRNA expression of inflammatory factors, PCNA and FGF-2 were decreased in the prostate of rats. Also, LCW attenuated mRNA level of AR and PSA in BPH-1 cell. The content of vicenin-2 in the LCW was analyzed to 0.89 mg/g. DISCUSSION AND CONCLUSIONS: Based on the results, LCW is a potential pharmacological candidate for the treatment of prostatic hyperplasia.

Effects of lespedeza condensed tannins alone or with monensin, soybean oil, and coconut oil on feed intake, growth, digestion, ruminal methane emission, and heat energy by yearling Alpine doelings.

Fifty-four Alpine doelings (initial BW and age of 31.7 ± 0.38 kg and 306 ± 1.9 d, respectively) were allocated to nine treatments individually fed for ad libitum intake of 25% concentrate and 75% forage diets (DM basis). Alfalfa was the forage in the control diet. Other diets contained Sericea lespedeza as the forage, with 1.25% DM of quebracho extract included in the concentrate fraction for a dietary condensed tannin level of 8.4%. Lespedeza treatments were no additive (L) and inclusion of monensin (I) at 22 mg/kg DM (L-I), soybean oil at 3% (L-S), coconut oil at 3% (L-N), I and 3% soybean oil (L-I-S), I and 3% coconut oil (L-I-N), 1.5% soybean oil and 1.5% coconut oil (L-S-N), and I, 1.5% soybean oil, and 1.5% coconut oil (L-I-S-N). The experiment was 12 wk with two 6-wk periods. Gas exchange was determined in weeks 6 and 12, and other measures occurred in weeks 5 and 11. The control diet offered averaged 2.67% nitrogen, 43.8% neutral detergent fiber, and 8.8% acid detergent lignin, and the L diet offered averaged 2.03% nitrogen, 42.8% neutral detergent fiber, and 13.2% acid detergent lignin. There were no treatment × period interactions for digestibilities (P ≥ 0.770) or methane emission (P ≥ 0.324). There were differences (P < 0.001) between the control treatment and diets with lespedeza in intake of DM (1.46, 1.23, 1.30, 1.18, 1.32, 1.10, 1.02, 1.20, and 1.01 kg/d; SEM = 0.059), digestibility of OM (57.4%, 50.9%, 51.8%, 52.7%, 50.3%, 52.1%, 52.1%, 51.9%, and 49.8%; SEM = 1.42), and digestibility of nitrogen (59.1%, 31.2%, 32.5%, 37.1%, 31.6%, 38.3%, 30.4%, 38.4%, and 34.1% for control, L, L-I, L-S, L-N, L-I-S, L-I-N, L-S-N, and L-I-S-N, respectively; SEM = 2.21). Ruminal methane emission was less (P < 0.001) for diets with lespedeza than for the control in MJ/d (1.36, 0.76, 0.84, 0.71, 0.71, 0.66, 0.65, 0.68, and 0.68; SEM = 0.048) and relative to intake of gross energy (5.92%, 3.27%, 3.49%, 3.19%, 2.84%, 2.91%, 3.20%, 3.20%, and 3.27%; SEM = 0.165) and digestible energy (11.19%, 6.98%, 7.40%, 6.38%, 5.90%, 5.69%, 6.37%, 6.38%, and 6.70% for control, L, L-I, L-S, L-N, L-I-S, L-I-N, L-S-N, and L-I-S-N, respectively; SEM = 0.400). In conclusion, the magnitude of effect of condensed tannins from lespedeza and quebracho extract on ruminal methane emission by Alpine doelings did not diminish over time and was not markedly influenced by dietary inclusion of monensin, soybean oil, or coconut oil.

Lespedeza cuneata protects the endothelial dysfunction via eNOS phosphorylation of PI3K/Akt signaling pathway in HUVECs.

BACKGROUND: Lespedeza cuneata G.Don (LCE), which belongs to the genus Lespedeza (Leguminosae), is a traditional oriental medicine known to prevent diabetes and cardiovascular diseases. However, no scientific studies about the effectiveness of LCE, their responsible bioactive constituents, and its mechanisms against endothelial dysfunction have been performed. PURPOSE: This study was performed to investigate the role of LCE and its chemical components in ameliorating endothelial dysfunction. METHODS: The production of nitric oxide (NO) was evaluated after LCE treatment in HUVECs. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent. Western blot analysis was performed to determine the protein expression of endothelial nitric oxide synthase (eNOS) and protein kinase B (PKB, also known as Akt) in human umbilical vein endothelial cells (HUVECs). RESULTS: Pretreatment with L-NAME and LY294002 significantly decreased the LCE-induced NO production, as well as eNOS and Akt phosphorylation. ß-Sitosterol and ß-Sitosterol 6'-linolenoyl-3-O-ß-D-glucopyranoside are the bioactive constituents increase NO production as well as eNOS phosphorylation. CONCLUSION: Our findings suggest that LCE increase NO production via eNOS phosphorylation of PI3K/Akt signaling pathway.

Lignan Glycosides and Flavonoid Glycosides from the Aerial Portion of Lespedeza cuneata and Their Biological Evaluations.

Lespedeza cuneata (Fabaceae), known as Chinese bushclover, has been used in traditional medicines for the treatment of diseases including diabetes, hematuria, and insomnia. As part of a continuing search for bioactive constituents from Korean medicinal plant sources, phytochemical analysis of the aerial portion of L. cuneata led to the isolation of two new lignan glycosides (1,2) along with three known lignan glycosides (3⁻7) and nine known flavonoid glycosides (8⁻14). Numerous analysis techniques, including 1D and 2D NMR spectroscopy, CD spectroscopy, HR-MS, and chemical reactions, were utilized for structural elucidation of the new compounds (1,2). The isolated compounds were evaluated for their applicability in medicinal use using cell-based assays. Compounds 1 and 4⁻6 exhibited weak cytotoxicity against four human breast cancer cell lines (Bt549, MCF7, MDA-MB-231, and HCC70) (IC50 < 30.0 µM). However, none of the isolated compounds showed significant antiviral activity against PR8, HRV1B, or CVB3. In addition, compound 10 produced fewer lipid droplets in Oil Red O staining of mouse mesenchymal stem cells compared to the untreated negative control without altering the amount of alkaline phosphatase staining.

Ultrasonicated Lespedeza cuneata extract prevents TNF-α-induced early atherosclerosis in vitro and in vivo.

This study evaluated the use of ultrasonication to extract Lespedeza cuneata as a potential nutraceutical for preventing vascular inflammation. Ultrasonicated L. cuneata extract (ULCE) was prepared using 20% ethanol and 2 h of ultrasonication at room temperature, and its effects were investigated using relevant in vitro and in vivo models. ULCE suppressed tumor necrosis factor-alpha (TNF-α)-induced adhesion capacity, vascular cell adhesion protein 1 (VCAM-1) expression, and nuclear factor kappa-B (NF-κB) activity in human umbilical vein endothelial cells (HUVECs). ULCE also suppressed TNF-α-induced NF-κB signaling pathways and p65 translocation from the cytosol to the nucleus, as well as the mRNA expression of IL-1ß, IL-6, and TNF-α in HUVECs. Oral administration of ULCE suppressed TNF-α-induced monocyte infiltration into the intima and VCAM-1 expression, as well as the IL-1ß, IL-6, TNF-α, and monocyte chemoattractant protein-1 (MCP-1) mRNA expression in the main artery in mice. Among the compounds identified in the hydrolyzed ULCE, quercetin exhibited the strongest inhibitory effect against TNF-α-induced cell adhesion capacity. These results demonstrate that ULCE contains potent preventive factors against early atherosclerosis, which act by suppressing the NF-κB and VCAM-1 signaling axis.

Protective effect of Lespedeza cuneata ethanol extract on Bisphenol A-induced testicular dysfunction in vivo and in vitro.

PURPOSE: Bisphenol A (BPA) has been regarded as a possible risk factor for reproductive health. We examined potential reproductive health benefits of Lespedeza cuneata ethanol extract (LCE). Previously, Lespedeza cuneata showed many therapeutic effects. However, the protective effect of LCE on BPA-induced testicular dysfunction and its mechanisms have not been precisely studied. METHODS: Mice were randomly divided into six groups (n = 7). Sperm counts and motility were measured by light microscope. Testosterone, total cholesterol, triglycerides, HDL, LDL-cholesterol, glucose, free fatty acids, hs-CRP, Angiotensinogen, Angiotensin II, GOT, GPT, TBARS, GSH, CAT, and SOD1 were measured in mouse serum. The potential protective effects of the LCE on mouse sertoli cells were evaluated. RESULTS: Oral administration of LCE in BPA-exposed male mice restored testis weight, sperm count, motility, and testosterone levels by inhibiting markers in serum. In addition, treatment with LCE in BPA-treated TM4 sertoli cells recovered cell viability by attenuating Bax expression and activating caspase 3 and PARP. CONCLUSIONS: These results indicate that LCE prevented BPA-induced testicular dysfunction and cell viability in BPA-treated TM4 sertoli cells. Our study also suggests that LCE has the potential to protect male reproductive health.

PER, a Circadian Clock Component, Mediates the Suppression of MMP-1 Expression in HaCaT Keratinocytes by cAMP.

Skin circadian clock system responds to daily changes, thereby regulating skin functions. Exposure of the skin to UV irradiation induces the expression of matrix metalloproteinase-1 (MMP-1) and causes DNA damage. It has been reported both DNA repair and DNA replication are regulated by the circadian clock in mouse skin. However, the molecular link between circadian clock and MMP-1 has little been investigated. We found PERIOD protein, a morning clock component, represses the expression of MMP-1 in human keratinocytes by using a PER-knockdown strategy. Treatment with siPer3 alleviated the suppression of MMP-1 expression induced by forskolin. Results revealed PER3 suppresses the expression of MMP-1 via cAMP signaling pathway. Additionally, we screened for an activator of PER that could repress the expression of MMP-1 using HaCaT cell line containing PER promoter-luciferase reporter gene. Results showed Lespedeza capitate extract (LCE) increased PER promoter activity. LCE inhibited the expression of MMP-1 and its effect of LCE was abolished in knockdown of PER2 or PER3, demonstrating LCE can repress the expression of MMP-1 through PER. Since circadian clock component PER can regulate MMP-1 expression, it might be a new molecular mechanism to develop therapeutics to alleviate skin aging and skin cancer.

(-)-9'-O-(α-l-Rhamnopyranosyl)lyoniresinol from Lespedeza cuneata suppresses ovarian cancer cell proliferation through induction of apoptosis.

Lespedeza cuneata (Dum. Cours.) G. Don. (Fabaceae), known as Chinese bushclover or sericea lespedeza, has been used in traditional medicine to treat diabetes, hematuria, and insomnia, and it has been reported that bioactive compounds from L. cuneata possess various pharmacological properties. However, there has been no study to determine the active compounds from L. cuneata with potential activity against ovarian cancer. This study aimed to isolate cytotoxic compounds from L. cuneata and identify the molecular mechanisms underlying the apoptosis pathway in ovarian cancer cells. Based on cytotoxic activity identified in the screening test, chemical investigation of the active fraction of L. cuneata led to the isolation of nine compounds including four lignanosides (1-4), three flavonoid glycosides (5-7), and two phenolics (8-9). Cytotoxicity and the molecular mechanism were examined by methyl thiazolyl tetrazolium (MTT) assay and Western blot analysis. Of the isolated compounds, (-)-9'-O-(α-l-rhamnopyranosyl)lyoniresinol (3) demonstrated the strongest effect in suppressing A2780 human ovarian carcinoma cell proliferation in a dose-dependent manner, with an IC50 value of 35.40 ±â€¯2.78 µM. Control A2780 cells had normal morphology, whereas cell blebbing, shrinkage, and condensation were observed after treatment with compound 3. Western blotting analysis showed that compound 3 inhibited A2780 human ovarian cancer cell viability by activating caspase-8, caspase-3, and PARP, which contributed to apoptotic cell death. These results suggest that (-)-9'-O-(α-l-rhamnopyranosyl)lyoniresinol (3) has potent anticancer activities against A2780 human ovarian carcinoma cells through the extrinsic apoptotic pathway. Therefore, (-)-9'-O-(α-l-rhamnopyranosyl)lyoniresinol is an excellent candidate for the development of novel chemotherapeutics.

Lespedeza bicolor ameliorates endothelial dysfunction induced by methylglyoxal glucotoxicity.

BACKGROUND: Lespedeza species have been used as a traditional medicine to treat nephritis, azotemia, inflammation, energy depletion, diabetes, and diuresis. PURPOSE: The purpose of this study is to screen the most potent Lespedeza species against methylglyoxal (MGO)-induced glucotoxicity, and to elucidate the mechanisms of action. Also, we will attempt to identify small chemical metabolites that might be responsible for such anti-glucotoxicity effects. METHODS: Firstly, the protective effect of 26 different Lespedeza species against MGO-induced toxicity in human umbilical vein endothelial cells was investigated. The chemical metabolites of the most potent species (Lespedeza bicolor 1 (LB1) were identified by high pressure liquid chromatography quadrupole time-of-flight tandem mass spectrometry (HPLC-Q-TOF-MS/MS), then quantified by HPLC. The effects of LB1 on MGO-induced apoptosis were measured by annexin V-FITC staining and western blot. Inhibitory effects of LB1 on MGO-induced ROS generation, and effect of LB1 on advanced glycation end products (AGEs) inhibitor or a glycated cross-link breaker are also measured. RESULTS: Among different Lespedeza species, LB1 extract was shown to reduce intracellular reactive oxidative species, exhibit anti-apoptotic effects, strongly inhibit all the mitogen-activated protein kinase signals, inhibit MGO-induced AGEs formation, and break down preformed AGEs. We tentatively identified 17 chemical constituents of LB1 by HPLC-Q-TOF-MS/MS. Among those, some components, such as genistein and quercetin, significantly reduced the AGEs formation and increased the AGEs-breaking activity, resulting in the reduction of glucotoxicity. CONCLUSION: LB1 extract has shown to be effective in preventing or treating MGO-induced endothelial dysfunction.