Ganoderic acids alleviate atherosclerosis by inhibiting macrophage M1 polarization via TLR4/MyD88/NF-κB signaling pathway.

BACKGROUND AND AIMS: Atherosclerosis (AS) is a chronic inflammatory disease characterized by lipid infiltration and plaque formation in blood vessel walls. Ganoderic acids (GA), a class of major bioactive compounds isolated from the Chinese traditional medicine Ganoderma lucidum, have multiple pharmacological activities. This study aimed to determine the anti-atherosclerotic effect of GA and reveal the pharmacological mechanism. METHODS: ApoE-/- mice were fed a high-cholesterol diet and treated with GA for 16 weeks to induce AS and identify the effect of GA. Network pharmacological analysis was performed to predict the anti-atherosclerotic mechanisms. An invitro cell model was used to explore the effect of GA on macrophage polarization and the possible mechanism involved in bone marrow dereived macrophages (BMDMs) and RAW264.7 cells stimulated with lipopolysaccharide or oxidized low-density lipoprotein. RESULTS: It was found that GA at 5 and 25 mg/kg/d significantly inhibited the development of AS and increased plaque stability, as evidenced by decreased plaque in the aorta, reduced necrotic core size and increased collagen/lipid ratio in lesions. GA reduced the proportion of M1 macrophages in plaques, but had no effect on M2 macrophages. In vitro experiments showed that GA (1, 5, 25 µg/mL) significantly decreased the proportion of CD86+ macrophages and the mRNA levels of IL-6, IL-1ß, and MCP-1 in macrophages. Experimental results showed that GA inhibited M1 macrophage polarization by regulating TLR4/MyD88/NF-κB signaling pathway. CONCLUSIONS: This study demonstrated that GA play an important role in plaque stability and macrophage polarization. GA exert the anti-atherosclerotic effect partly by regulating TLR4/MyD88/NF-κB signaling pathways to inhibit M1 polarization of macrophages. Our study provides theoretical basis and experimental data for the pharmacological activity and mechanisms of GA against AS.

The ROS/SIRT1/STAR axis as a target for melatonin ameliorating atrazine-induced mitochondrial dysfunction and steroid disorders in granulosa cells.

The granulosa cells (GCs) of birds are essential for the reproduction and maintenance of populations in nature. Atrazine (ATR) is a potent endocrine disruptor that can interfere with reproductive function in females and Diaminochlorotriazine (DACT) is the primary metabolite of ATR in the organism. Melatonin (MT) is an endogenous hormone with antioxidant properties that plays a crucial role in development of animal germ cells. However, how ATR causes mitochondrial dysfunction, abnormal secretion of steroid hormones, and whether MT prevents ATR-induced female reproductive toxicity remains unclear. Thus, the purpose of this study is to investigate the protective effect of MT against ATR-induced female reproduction. In the present study, the GCs of quail were divided into 6 groups, as follows: C (Serum-free medium), MT (10 µM MT), A250 (250 µM ATR), MA250 (10 µM MT+250 µM ATR), D200 (200 µM DACT) and MD200 (10 µM MT+200 µM DACT), and were cultured for 24 h. The results revealed that ATR prevented GCs proliferation and decreased cell differentiation. ATR caused oxidative damage and mitochondrial dysfunction, leading to disruption of steroid synthesis, which posed a severe risk to GC's function. However, MT supplements reversed these changes. Mechanistically, our study exhibited that the ROS/SIRT1/STAR axis as a target for MT to ameliorate ATR-induced mitochondrial dysfunction and steroid disorders in GCs, which provides new insights into the role of MT in ATR-induced reproductive capacity and species conservation in birds.

Covalent immobilization of tyrosinase on magnetic multi-walled carbon nanotubes for inhibitor screening.

The inhibition of tyrosinase is considered to be a common therapeutic strategy for some hyperpigmentation disorders. Screening of tyrosinase inhibitors is of great significance to the treatment of pigmentation diseases. In this study, tyrosinase was covalently immobilized on magnetic multi-walled carbon nanotubes for the first time, and the immobilized tyrosinase was applied for ligand fishing of tyrosinase inhibitors from complex medicinal plants. The immobilized tyrosinase was characterized by transmission electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, vibrating sample magnetometry, and thermo-gravimetric analyzer, which indicated that tyrosinase was immobilized onto magnetic multi-walled carbon nanotubes. The immobilized tyrosinase showed better thermal stability and reusability than the free one. The ligand was fished out from Radix Paeoniae Alba and identified as 1,2,3,4,6-pentagalloylglucose by ultra-performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry. 1,2,3,4,6-pentagalloylglucose was found to be a tyrosinase inhibitor with similar half maximal inhibitory concentration values of 57.13 ± 0.91 µM compared to kojic acid (41.96 ± 0.78 µM). This work not only established a new method for screening tyrosinase inhibitors but also holds considerable potential for exploring the new medicinal value of medicinal plants.

C:N:P stoichiometric characteristics of mosses in Picea crassifolia forest in Helan Mountains, Ningxia, China.

To explore the stoichiometric characteristics of C, N and P and adaptive mechanism of mosses in mountain forest ecosystems, we set up 15 plots along the altitude gradient in Picea crassifolia forest in Helan Mountains, Ningxia. We analyzed the C:N:P stoichiometry of moss aboveground tissues and its relationship with environmental factors. The results showed the mean values of C, N and P concentration in moss aboveground tissues were 336.67, 20.31 and 0.66 mg·g-1, respectively. The mean value of aboveground tissue N:P was 33.4, indicating that the growth of mosses was limited by P. The C concentration in the aboveground tissues of mosses was positively correlated with soil total nitrogen concentration and negatively correlated with soil total phosphorus concentration. The N concentration in aboveground tissues of mosses was significantly negatively correlated with soil organic carbon and soil total nitrogen concentrations. Results of redundancy analysis showed that the interpretation rate of environmental factors on the stoichiometry was 48.5%, with canopy closure, soil total nitrogen and soil total phosphorus as the main factors. Canopy closure was the main environmental factor affecting the growth of mosses in P. crassifolia forest in Helan Mountains. High canopy closure facilitated the growth of mosses.

Anti-osteoporosis effect of Semen Cuscutae in ovariectomized mice through inhibition of bone resorption by osteoclasts.

ETHNOPHARMACOLOGICAL RELEVANCE: Semen Cuscutae, called Tu-si-zi in Chinese, is a kind of dried mature seed in the Convolvulaceae family. It mainly distributes in China, Korea, Pakistan, Vietnam, India and Thailand. It is used as a kidney-tonifying drug for treatment of aging related diseases such as osteoporosis in traditional Chinese medicine. However, the exact mechanisms on bone resorption are poorly studied. AIM OF THE STUDY: The aim of this study was to investigate the potential effect of Semen Cuscutae on ovariectomy (OVX)-induced osteoporosis in mice and clarify the exact mechanisms by which Semen Cuscutae exert the anti-osteoporosis effect. MATERIALS AND METHODS: Qualitative and quantitative analyses of Semen Cuscutae were performed by UPLC-Q-TOF-MS and HPLC-MS/MS, respectively. Changes in bone mineral density (BMD) induced by OVX in mice were measured by dual-energy X-ray absorptiometry and micro-computed tomography (µCT). Tartrate-resistant acid phosphatase (TRAP) staining as well as hematoxylin and eosin (HE) staining were used to observe bone microarchitectural changes. ELISA kits were used to assess the therapeutic effects of Semen Cuscutae on the serum levels of osteoprotegerin (OPG), tartrate-resistant acid phosphatase 5b (TRACP-5b), and receptor activator of nuclear factor-κB (RANKL). The effect of Semen Cuscutae on primary cell viability was assessed using CCK-8 and anti-tartrate phosphatase assays. TRAP staining and actin ring staining were used to observe the effect of Semen Cuscutae on osteoclast differentiation. Western blotting was used to measure the effects of Semen Cuscutae on expressions of NFATC1, c-Src kinase, and c-fos. RESULTS: Results from UPLC-Q-TOF-MS showed that the main components of Semen Cuscutae were flavonoid compounds that included quercitrin, quercetin, hyperoside, caffeic acid, rutin, chlorogenic acid, luteolin, apigenin, kaempferol, isoquercetin, cryptochlorogenic acid, isorhamnetin-3-O-glucoside, and astragalin. After the Semen Cuscutae extract was orally administered to OVX mice, bone density increased (P < 0.01) and bone microstructure was significantly improved (P < 0.01 or 0.05). Additionally, Semen Cuscutae exhibited a significant descending effect in the levels of serum TRACP-5b and RANKL, while there was a significant increase in OPG in the Semen Cuscutae group compared with the OVX group, especially at high doses. Moreover, we found that increasing of c-fos, c-Src kinase, and NFATC1 protein expressions were reversed by Semen Cuscutae in vitro and in vivo. CONCLUSIONS: Our results showed that Semen Cuscutae exhibited anti-osteoporosis effects through the c-fos/c-Src kinase/NFATC1 signaling pathway.

Supportive psychological intervention on psychological disorders in clinical medicine students with English Learning Difficulties: A protocol of systematic review.

BACKGROUND: This study aims to examine the effect of supportive psychological intervention (SPI) on psychological disorders (PD) in clinical medicine students (CMS) with English Learning Difficulties (ELD). METHODS: We will perform a comprehensive literature search from the following databases: Cochrane Library, MEDLINE, EMBASE, Allied and Complementary Medicine Database, Chinese Biomedical Literature Database, and China National Knowledge Infrastructure. All databases will be performed from their inception to the present without language limitation by 2 independent reviewers. We will also look for grey literature, such as conference proceedings, dissertations or theses. Newcastle-Ottawa Scale will be used to assess study quality, and RevMan 5.3 software will be applied to carry out statistical analysis. RESULTS: This study will summarize the most recent evidence to assess the effect of SPI on PD in CMS with ELD. CONCLUSION: This study may provide helpful evidence of SPI on PD in CMS with ELD. OSF REGISTRATION NUMBER:: osf.io/tah2s.

Approach selection of radiofrequency catheter ablation for ventricular arrhythmias originating from the left ventricular summit: potential relevance of Pseudo Delta wave, Intrinsicoid deflection time, maximal deflection index.

BACKGROUND: Ventricular arrhythmias (VAs) originating from the left ventricular summit is a challenge for radiofrequency catheter ablation (RFCA). The present study aimed to investigate the appropriate RFCA strategy for VAs originating from the left ventricular summit. METHODS: Forty-five consecutive patients with VAs arising from the left ventricular summit were successfully ablated at our cardiac electrophysiology center and reviewed in the study. RESULTS: Thirty-two cases of VAs were eliminated in the left ventricular endocardium by retrograde transaortic (n = 22, 22/45, 48.9%) or antegrade transseptal (n = 10, 10/45, 22.2%) approaches, the other 13 cases were eliminated in the left ventricular epicardium by distal great cardiac vein (DGCV) approach (n = 13, 13/45, 28.9%). Though these VAs were similar in electrocardiographic (ECG) morphology, the pseudo delta waves (PDW), intrinsicoid deflection time (IDT), maximal deflection index (MDI) differed among them, PDW >53 ms, IDT > 74 ms, MDI > 0.45 strongly indicated that ablating left ventricular summit VAs by DGCV approach. During mean follow-up of 19.5 ± 13.2 (range, 3-60) months, 2 (4.4%) patients experienced VAs recurrence. CONCLUSION: This retrospective study showed that VAs of left ventricular summit origin can be effectively cured with RFCA. For these VAs, prolonged PdW, IDT, MDI indicating RFCA by DGCV approach can be attempted firstly.

Origins location of the outflow tract ventricular arrhythmias exhibiting qrS pattern or QS pattern with a notch on the descending limb in lead V1.

BACKGROUND: Ventricular outflow tract(VOT) ventricular arrhythmias(VAs) presenting qrS pattern or QS pattern with a notch on the descending limb in lead V1 were consistently thought of arising from the commissure between left and right coronary cusp (L-RCC) by previous studies. However, we found they could originate from other anatomic structures in VOT. This study aimed to investigate the exact origin of this kind VAs. METHODS: Forty-nine patients of VOT premature ventricular contrations/ventricular tachycardia(PVCs/VT) with lead V1 presenting qrS pattern or QS pattern with a notch on the descending limb undergoing successful radiofrequency catheter ablation(RFCA) in our center were analyzed. RESULTS: 12-lead electrocardiogram(ECG) of these PVCs/VT were summarized. Among these PVCs/VT, 37 cases exhibited qrS morphology in lead V1, 12 cases presented QS pattern with a notch on the descending limb in the same lead. Based on the successful ablation sites, these PVCs/VT were divided into 2 groups: (1)Right ventricular outflow tract(RVOT) group (26 cases), and (2) Left ventricular outflow tract (LVOT) group(23 cases, 4 cases originating from the left coronary cusp(LCC), 2 from the right coronary cusp(RCC), 16 from the L-RCC, 1 from the area inferior to LCC(ILCC)). The ECG characteristics of each PVCs/VT were analyzed. Among these PVCs/VT, applying the precordial transitional zone index(TZ index) < 0 to predict LVOT origin was demonstrated with sensitivity of 95.65%, specificity of 96.15%, positive predicting value(PPV) of 95.65% and negative predicting value(NPV) of 96.15%. In LVOT group, further applying the r, R, m,or Rs morphology in lead I to predict L-RCC and RCC origin was demonstrated with sensitivity of 94.44%, specificity of 60.00%, PPV of 89.47% and NPV of 75.00%. CONCLUSIONS: Ventricular outflow tract PVCs/VT with lead V1 presenting qrS pattern or QS pattern with a notch on descending limb not only arising from L-RCC, but also RVOT, LCC, RCC and ILCC. Combining TZ index and QRS morphology in lead I to predict origin site of these kind VAs is a convenient, simple and reliable method and facilitates the RFCA procedure.

Minerals and Trace Elements Intakes and Food Consumption Patterns of Young Children Living in Rural Areas of Tibet Autonomous Region, P.R. China: A Cross-Sectional Survey.

BACKGROUND AND OBJECTIVES: Several studies revealed clinical signs of stunting and rickets among rural populations of Tibet Autonomous Region (T.A.R.), and especially amid children. Further, these populations are affected by a bone disease named Kashin-Beck disease (KBD). However, little is known about the dietary status of this population. This survey aimed to assess the usual intakes of young Tibetan children living in rural areas around Lhasa for energy, water, and ten minerals and trace elements (Na, K, Ca, P, Mg, Fe, Zn, Cu, Mn, and Se) involved in bone metabolism. DESIGN: A cross-sectional survey was designed. Totally, 250 pre-school children aged 3-5 years living in rural areas were enrolled. The 24-h food recall method was used to collect the intakes for two days, during two different seasons (September 2012 and April 2013). Because Tibetan foods are mainly derived from local agriculture and artisanal production, a combination of food composition tables was compiled, including specific and local food composition data. RESULTS: The Chinese dietary recommended intakes are not met for most of the elements investigated. Intake of sodium is much too high, while usual intakes are too low for K, Ca, Zn, Cu, and Se. Bioavailability of Ca, Fe, and Zn may be of concern due to the high phytic acid content in the diet. CONCLUSION: These nutrient imbalances may impact growth and bone metabolism of young Tibetan children. The advantages of the implementation of food diversification programs are discussed as well as the relevance of supplements distribution.

Pharmacokinetic comparison between quercetin and quercetin 3-O-ß-glucuronide in rats by UHPLC-MS/MS.

Quercetin is a natural flavonoid widely distributed in human diet and functional foods. Quercetin 3-O-ß-glucuronide (Q3G) is present in wine and some medicinal plants. Quercetin and Q3G may be metabolized from each other in vivo. While quercetin has been the subject of many studies, the pharmacokinetic profiles of quercetin and Q3G (in animals) have not yet been compared. Herein, we prepared a column-based method for rapid isolation of Q3G from Nelumbo nucifera. Then, we developed an UHPLC-MS/MS method to compare the pharmacokinetics of quercetin and Q3G. Our results showed that the plasma concentration-time curves of quercetin and Q3G show two maxima (Tmax1 ≈ 0.75 h, Tmax2 ≈ 5 h). After oral administration of 100 mg/kg quercetin or 100 mg/kg Q3G in rats, predominantly Q3G was detected in plasma with AUC at 39529.2 ± 6108.2 mg·h·L-1 or 24625.1 ± 1563.8 mg·h·L-1, 18-fold higher than quercetin with AUC at 1583.9 ± 583.3 mg·h·L-1 or 1394.6 ± 868.1 mg·h·L-1, respectively. After intravenous injection of 10 mg/kg in rats, Q3G showed extensive tissue uptake in kidney (409.2 ± 118.4 ng/g), liver (166.1 ± 52.9 ng/g), heart (97.7 ± 22.6 ng/g), and brain (5.8 ± 1.2 ng/g). In conclusion, we have shown that Q3G is a major active component in plasma and tissue for oral administration of quercetin or Q3G.

Inhibitory effect of Zanthoxylum bungeanum seed oil on ovalbumin­induced lung inflammation in a murine model of asthma.

The present study aimed to investigate the therapeutic efficacy of Zanthoxylum bungeanum seed oil (Z. seed oil) to alleviate airway inflammation in asthmatic mice. The asthmatic mice were treated with vehicle, ovalbumin (OVA), or OVA + Z. seed oil (2 g/kg) for between 24 h and 14 days. Following treatment, inflammatory cell infiltration and pulmonary tissue damage were assessed by hematoxylin and eosin staining, and immunohistochemistry. The expression levels of pro­inflammatory cytokines, chemokines, adhesion molecules and mitogen activated protein kinase signaling proteins were measured by enzyme­linked immunosorbent assays, reverse transcription quantitative­polymerase chain reaction and western blot analysis. In asthmatic mice, administration of Z. seed oil attenuated lung tissue injury and airway remodeling, and inhibited the infiltration of leukocytes and eosinophils into the airway by reducing the expression levels of inflammatory cytokines and chemokines compared with OVA­treated mice (P<0.05). Z. seed oil also reduced the levels of inflammatory chemokine and adhesion molecules via downregulation of extracellular signal­regulated kinase and activation of c­JUN N­terminal kinase in the Z. seed­treated mice compared with OVA­treated mice (P<0.05). Thus, data from the present study indicates that Z. seed oil can suppress pulmonary inflammation and tissue injury during asthma, and suggests that it may be used to effectively treat allergen­induced asthma.

Alpha-Actinin-4 is a Possible Target Protein for Aristolochic Acid I in Human Kidney Cells In Vitro.

Aristolochic acid I (AA-I) is a strong nephrotoxin, carcinogen, and mutagen found in plants such as the Aristolochia species. The mechanisms underlying AA-I toxicity in the kidneys are poorly understood. In this study, we aimed to gain insight into the mechanism of AA-I nephrotoxicity by analyzing the uptake, subcellular distribution, and intracellular targets of AA-I in the human kidney cell line HK-2 using immunocytochemistry, immunoprecipitation, and LC-MS/MS. In HK-2 cells incubated with 20[Formula: see text][Formula: see text]g/mL AA-I for different periods of time (up to 12[Formula: see text]h), AA-I was detected by a specific monoclonal antibody (MAb) against AA-I, both in the cytoplasm and nuclei. Nuclear localization depended on the exposure time. A protein with the molecular weight of 100 kDa was immunoprecipitated with the anti-AA-I MAb from the AA-I-treated cell lysates and was identified by LC-MS/MS as [Formula: see text]-actinin-4 after digestion of the protein, and was confirmed by immunoblotting with a specific anti-[Formula: see text]-actinin-4 MAb. This evidence shows, for the first time, that [Formula: see text]-actinin-4 is a protein targeted by AA-I in kidney cells. Our findings strongly suggest an association between [Formula: see text]-actinin-4 and AA-I nephrotoxic activity.

[UPLC/Q-TOF MS and NMR plant metabolomics approach in studying the effect of growth year on the quality of Polygala tenuifolia].

Growth year is one of the important factors for the quality of Polygala tenufolia. In this study, primary metabolites and secondary metabolites were compared in 1, 2 and 3 years old P. tenufolia cultivated in Shaanxi Heyang. The samples were subjected to ultra-high performance liquid chromatography (UPLC)-quadrupole time-of-flight mass spectrometry (Q-TOF MS) and nuclear magnetic resonance (NMR) analysis, and the obtained data were analyzed using principal component analysis (PCA) and other statistical analysis methods. In addition, content and correlation of different metabolites were also calculated. The results showed no significance between main component contents in 2 year-old and 3 year-old P. Tenufolia, but 1 year-old was statistically different. The contents of primary metabolites, such as fructose, sucrose, and choline increased as time goes on, while glycine and raffinose decreased. The contents of secondary metabolites, such as onjisaponin Fg, polygalasaponin XXVIII, polygalasaponin XXXII increased, while polygalaxanthone III and parts of oligosaccharide multi-ester including tenuifoliose A, tenuifoliose C, tenuifoliose C2 and tenuifoliose H decreased with the extension of the growth years. Growth years has important impact on the quality of P. tenuifolia and the existing growing years of commodity P. tenuifolia have its scientific evidence. This study supplied a new method for the quality evaluation of Chinese medicinal materials.

Localization of aristolochic acid in mouse kidney tissues by immunohistochemistry using an anti-AA-I and AA-II monoclonal antibody.

Aristolochic acids (AAs) are found in herbal medicines of Aristolochiaceae plants, including Aristolochia and Asarum species. AAs are associated with a rapidly progressive interstitial nephritis, which is called aristolochic acid nephropathy (AAN). However, the in-situ localization of AAs in the target organ, the kidney, has not been investigated yet. In the present study, the accumulation of aristolochic acid I (AA-I) in mouse kidney was revealed by immunoperoxidase light microscopy as well as colloidal gold immunoelectron microscopy (IEM) based on an anti-AA-I and AA-II monoclonal antibody (mAb). Male BALB/c mice were treated with 1.25 or 2.50 mg kg(-1) of AA-I per day for 5 days. Paraffin sections and ultra-thin sections of kidney tissue were respectively prepared. Under light microscopy, the apical surface of proximal tubules was strongly stained for AA-I, whereas no obvious immunostaining was found in the distal tubules and glomerulus, which remained relatively intact. Under electron microscopy, epithelial cells of the proximal tubules, distal tubules and collecting tubules were broken to various degrees. Gold labeling in the proximal and distal tubules was stronger than that in the collecting tubules. In renal tubules, immunogold signals of AA-I tended to accumulate in the mitochondria and peroxisomes, though the signals could be observed all over the cell. Gold signals were also found in the erythrocytes of glomeruli. The MAb against AA-I and AA-II provides a clue for the identification of proteins or factors which might interact with AA-I and thus induce targeted damage of kidney.

Performance of arrhenotokous and thelytokous Thrips tabaci (Thysanoptera: Thripidae) on onion and cabbage and its implications on evolution and pest management.

Onion thrips, Thrips tabaci Lindeman (Thysanoptera: Thripidae), is an important pest on onion and cabbage. Two reproductive modes--arrhenotoky and thelytoky--are found in this species and co-occur in the field. We compared life table traits between arrhenotokous and thelytokous T. tabaci on cabbage and onion. Experiments were conducted in cages to determine which reproductive mode is more competitive. Additionally, host adaption of the arrhenotokous and thelytokous T. tabaci between onion and cabbage was investigated. On onion, arrhenotokous T. tabaci performed better than thelytokous T. tabaci, while on cabbage the opposite occurred. When comparing life table and demographic growth parameters (net reproductive rates R(o), mean generation time T, the intrinsic rate of natural increase r(m), finite rate of increase A, and population doubling time T(d)) on different host plants, we found that arrhenotokous T. tabaci performed better on onion than on cabbage, whereas thelytokous T. tabaci performed better on cabbage than on onion. Host-related performance differences in this species suggest that the divergence between two reproductive modes might be associated with host adaption. Pest management strategies for this global pest should recognize that the two reproductive modes can impact population dynamics on different crops.

[Comparison of HPLC fingerprints of Xanthium sibiricum and Xanthium mongolicum fruits].

OBJECTIVE: To compare and establish the HPLC fingerprint of Xanthium sibiricum and Xanthium mongolicum fruits for the quality control. METHODS: Sample was separated on C18 column (250 mm x 4.6 mm, 5 µm) with the mobile phase consisting of methanol-0.2% phosphoric acid solution by gradient elution. The flow rate was 1.0 mL/min and wavelength was set at 327 nm. RESULTS: HPLC fingerprints of Xanthium sibiricum and Xanthium mongolicum fruits were established, and 17 common peaks were found. The HPLC fingerprints of Xanthium sibiricum fruits from different habitats had good similarities with similaritis over 0.9, but some compositions' relative contents of Xanthium mongolicum fruits were different compared with Xanthium sibiricum. CONCLUSION: This method is accurate, reproducible and stable, which can be used for the quality control of Xanthium sibiricum fruits and distinguishing it with Xanthium mongolicum fruits effectively.

A R2R3-MYB transcription factor, GmMYB12B2, affects the expression levels of flavonoid biosynthesis genes encoding key enzymes in transgenic Arabidopsis plants.

Isoflavones play diverse roles in plant-microbe interactions and are potentially important for human nutrition and health. To study the regulation of isoflavonoid synthesis in soybean, the R2R3-MYB transcription factor GmMYB12B2 was isolated and characterized. Yeast expression experiments demonstrated that GmMYB12B2 showed transcriptional activity. GmMYB12B2 was localized in the nucleus when it was transiently expressed in onion epidermal cells. Real-time quantitative PCR analysis revealed that GmMYB12B2 transcription was increased in roots and mature seeds compared with other organs. The gene expression level in immature embryos was consistent with the accumulation of isoflavones. CHS8 is a key enzyme in plant flavonoid biosynthesis. Transient expression experiments in soybean calli demonstrated that CHS8 was regulated by GmMYB12B2 and produced more fluorescence. The expression levels of some key enzymes in flavonoid biosynthesis were examined in transgenic Arabidopsis lines. The results showed that the expression levels of PAL1, CHS and FLS in transgenic plants were significantly higher than those in wild type plants. However, the expression level of DFR was lower, and the expression levels of CHI, F3H and F3'H were the same in all lines. GmMYB12B2 expression caused a constitutive increase in the accumulation of flavonoids in transgenic Arabidopsis lines compared with wild type plants.

Development of an Eastern blotting technique for the visual detection of aristolochic acids in Aristolochia and Asarum species by using a monoclonal antibody against aristolochic acids I and II.

INTRODUCTION: Aristolochic acids (AAs) are naturally occurring nephrotoxicants and human carcinogens. Aristolochic acid I (AA-I) and aristolochic acid II (AA-II) are two important AAs with clear toxicity. OBJECTIVE: To obtain a monoclonal antibody (MAb) recognising AA-I and AA-II and develop an Eastern blotting technique for the specific visualisation and easy determination of AA-I and AA-II in plant extracts or tissues of Aristolochia and Asarum species. METHODS: A hybridoma secreting MAb against AAs was prepared by cell fusion with splenocytes derived from a mouse immunised with AA-I-keyhole limpet haemocyanin (KLH) conjugate and the myeloma cell line SP2/0-Ag14. AA-I and AA-II were separated by thin-layer chromatography (TLC) and then blotted onto a positively charged polyethersulphone (PES) membrane using a modified carbodiimide method. The resulting membrane-bound AA-protein conjugates were linked to the newly prepared MAb and then to the secondary antibody labelled with peroxidase. 4-Chloro-1-naphthol was then added as the peroxidase substrate for staining. RESULTS: MAb 2A10-10B showed a high specificity for AA-I (100%) and AA-II (69.3%) and low cross reactivity (≤ 2.2%) toward analogues that may disrupt detection of AA-I and AA-II in plants. An established Eastern blotting method was applied to the immunohistolocalisation of AA-I and AA-II in dry plant tissues, and this analysis showed that the phelloderm, cortex and phloem of Aristolochia manshuriensis stem may contain higher amounts of total AA-I and AA-II as compared with the pith and xylem. CONCLUSION: This method was extremely useful for the visual screening of AA-I and AA-II among easily mistaken herbal medicines.

Comparative study of the contents of analogues of aristolochic acid in two kinds of Aristolochiae Fructus by high-performance liquid chromatography.

Aristolochiae Fructus ("Madouling") is derived from the fruits of Aristolochia contorta and A. debilis (Aristolochiaceae). These two species contain potentially nephrotoxic constituents, but are officially used in China. Distinction of constituents and toxicity between these two species remains unclear. A high-performance liquid chromatography method was developed and validated for the simultaneous determination of seven analogues of aristolochic acid (aristolochic acids I, II, IIIa, IVa and VIIa), as well as aristololactams I and II in Aristolochiae Fructus. Chromatographic separation was achieved on a Zorbax SB-C(18) column with a gradient mobile phase comprising acetonitrile and 1 % acetic acid-30 mM triethylamine (20:1, v/v) buffer. Analytes were detected with a diode array detector at 250 and 260 nm. The contents of seven constituents in samples (11 batches of A. contorta fruits, 15 batches of A. debilis fruits and 33 commercial samples of Madouling) were determined. The content of aristolochic acid IVa was higher than that of aristolochic acid VIIa in A. contorta fruits, whereas the opposite was true in A. debilis fruits. This feature can be used to distinguish the two species from each other and identify the resource plant of Madouling. Through a morphological method and a newly found principle based on the ratio AA-IVa/AA-VIIa, we found that the 33 commercial samples collected from 12 provinces in China were all derived from the fruits of A. contorta.

Comparative studies of saponins in 1-3-year-old main roots, fibrous roots, and rhizomes of Panax notoginseng, and identification of different parts and growth-year samples.

Notoginsenosides R1, R4, Fa, and K (N-R1, N-R4, N-Fa, and N-K), as well as ginsenosides Rg1, Rb1, Rd, Re, Rf, Rg2 and Rh1 (G-Rg1, G-Rb1, G-Rd, G-Re, G-Rf, G-Rg2 and G-Rh1) in 47 Notoginseng samples including 1-, 2- and 3-year-old main roots, rhizomes and fibrous roots of Panax notoginseng were determined by high-performance liquid chromatography-diode array detection method. Total contents (%) of the 11 saponins were 9.82-14.57 for 2-year old and 14.20-16.00 for 3-year-old rhizomes; 2.72-4.50 for 2-year-old and 1.98-4.92 for 3-year-old fibrous roots; 1.75-3.05 for 1-year-old whole roots; and 3.71-8.98 for 2-year-old and 7.03-11.23 for 3-year-old main roots. Contents of most saponins and total content of 11 saponins were in the order 3- >2- >1-year-old main root samples. G-Rf content, sum of G-Rf and G-Rh1 were, respectively, 0.08-0.18 and 0.14-0.32 for 2- or 3-year-old rhizomes, and 0.01-0.07 and 0.03-0.10 for 2- or 3-year-old main roots. Combined contents of N-R1, G-Rg1 and G-Rb1 were 5.78-9.37 in 3-year-old main roots, and 2.99-7.13 in 2-year-old main roots, of which nearly one-third of samples were lower than the limit (5 %) in the Chinese Pharmacopoeia. Those of 2- or 3-year-old fibrous roots (1.47-3.83) and 1-year-old whole roots (1.41-2.44) were much lower than the limit, and were considered not suitable for use as Notoginseng. Two-year-old main roots are not appropriate for collection as Notoginseng. Different parts and growth years of P. notoginseng can be identified from each another according to differences in saponin content.