Anemarrhena Research Articles

Plasma pharmacochemistry combined with microdialysis to screen potential bioactive components and their metabolites inAnemarrhena asphodeloidessaponin extract using ultrahigh-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry

Although various techniques have been employed to analyze drug metabolites, the metabolism of multicomponent herbal medicine has seldom been fully addressed. In contrast to chemical drugs, a number of compounds in herbal medicine could get into circulation and then be metabolized. Metabolism study on active constituents in herbal medicine is a good way for us to explain and predict a variety of events related to the efficacy and toxicity of herbal medicine. The present work aims to elucidate the multicomponent metabolic characteristics of a herbal medicine by the combination of plasma pharmacochemistry and microdialysis sampling. Anemarrhena asphodeloides, a well-known traditional Chinese medicine, was chosen as a model. After oral administration of A. asphodeloides saponin extract to rats, microdialysis samples were collected continuously in the jugular vein and analyzed by ultrahigh-performance LC/quadrupole-TOF MS. The identification of compounds in biosamples was achieved by accurate mass measurement and detailed fragmentation pathway analysis. The results showed that unbound constituents in blood circulation of the rat included seven parent saponins and six metabolites, which might be the potential active components in vivo. Among which, three metabolites have not been previously reported and were identified in this study. It is the first report on systemic metabolism of total saponins of A. asphodeloides in mammalian plasma.

Synthesis of Naturally Occurring Norlignan (±)-Nyasol

The norlignans including nyasol 1, hinokiresinol 2, agatharesinol 3, sequirin C 4 and sugiresinol 5 have received widespread interest as pharmaceutically valuable compounds, mainly because of their various clinically important biological activities: e.g. antiplasmodial activity, antifungal activity, anticancer activity, antimalarial activity, anti-oomycete activity, and estrogen receptor binding activity. Nyasol is one of the naturally occurring norlignans, which constitute abundant classes of phenylpropanoids. Nyasol has been isolated from various species of plants (Asparagus africanus, Anemarrhena asphodeloides, Asparagus officinalis, and Asparagus cochinchinensis). Nyasol and hinokiresinol are geometrical isomers and the confusion concerning the nomenclature of the two compounds nyasol and hinokiresinol was settled by Oketch-Rabah et al., who agreed that the Z-isomer should be named nyasol and the Eisomer hinokiresinol. In particular, nyasol shows various biological activities. For example, nyasol stimulated the proliferation of estrogendependent T47D breast cancer cells, and their stimulatory effects were blocked by an estrogen antagonist. In addition, nyasol showed binding affinity for the bovine uterine estrogen receptor, but (+)-nyasol was found to bind approximately seven times more strongly than (−)-nyasol. It was reported that (−)-nyasol increases hexobarbital sleeping time in mice. Nyasol was also reported to possess antifungal activity and inhibit testosterone 5α-reductase. Furthermore, nyasol was found to exhibit antioxidant and antiatherogenic activities. These results have encouraged us to complete adaptable and scalable total synthesis of these classes of natural products for further pharmacological study. So far, hinokiresinol derivatives have been easily prepared from chalcones by using reduction and dehydration. However, few successful synthetic procedures for nyasol have been reported other than the recent report by the Hoveyda group, which limits a scalable synthesis of nyasol. Quan et al. also reported an approach to synthesis of di-O-methyl ethers of (+)-nyasol and related norlignans, however, they might fail deprotection of the methyl ethers to obtain the final nyasol since there

Determination of mangiferin in Baihe Zhimu decoction by HPCE

Objective To establish HPCE method for determining the mangiferin in Baihe Zhimu decoction.Methods Rutin was used as internal standard.An uncoated fused silica capillary (77 cm×75 μm,effective length of 70 cm) was served as separation gate.The electrolyte buffer was composed of 12 mmol/L borax-MeOH (90 ∶ 10).20 kV voltages were applied and detection wavelength was set at 318nm.Results Mangiferin was successfully separated within 20 rin,the linear response range was 0.06～1.14 mg/ml.The average recovery was 99.0％.Conclusion The method was simple,rapid and well reproducible.It could be used as a reliable tool for the quantity control of Baihe Zhimu decoction and descriptions containing mangiferin. Key words: Baihe Zhimu decoction; Mangiferin; HPCE

Structure characterization and identification of steroidal saponins from the rhizomes of Anemarrhena asphodeloides by ultra performance liquid chromatography and hybrid quadrupole time-of-flight mass spectrometry

In this work, ultra performance liquid chromatography (UPLC) coupled with a hybrid quadrupole time-of-flight mass spectrometry (Q-TOF MSE) was used to separate and identify steroidal saponins from the crude extracts of Anemarrhena asphodeloides. The fragmentation patterns of seven known steroidal saponins, which could be classified into six types, were investigated by UPLC/Qtof MSE technique in both negative and positive ion modes. By using this strategy, both intact precursor and fragment ion information were obtained from a single injection. Based on the exact mass, fragment ions, and retention times of peaks, and comparison with reference standards and known compounds in the literature, sixty-seven steroidal saponins were identified or tentatively identified from A. asphodeloides. This included sixteen groups of isomers. Fourteen of these saponins were identified as new compounds. This study provides a good example for the rapid identification of chemical constituents in complex samples such as TCM by UPLC/Qtof MSE, in the mean time, avoiding the time-consuming and tedious purification of compounds from the crude extracts.

Isolation, Purification, Characterization and Effect upon HepG2 Cells of Anemaran from Rhizome Anemarrhena.

The rhizome of Anemarrhena asphodeloides is used as food and traditional Chinese medicine for its hypoglycemic effect. The aim of this study was to investigate the isolation, purification and hypoglycemic activity of Anemaran as the active component. The influence factors (isolation duration, ratio of residuals to water and extracting times) during the isolation process were evaluated. The optimal conditions for NA and AA were extraction temperature 90ºC and 100ºC, duration 1h and 1.5 h, extraction time 3 and 3, and the solid-liquor ratio 1:20 and 1:15, respectively. Neutral and acid Anemaran (NA and AA) were isolated from the rhizome of Anemarrhena asphodeloides. Five fractions of NA-1, NA-2, NA-3, AA-1 and AA-2 were obtained after crude neutral and acid Anemaran purified through DEAE- 52 cellulose anion-exchange column. The characterizations of Anemaran and its different fractions were both analyzed by Fourier transform infrared spectroscopy (FT-IR) and scanning electron micrographs (SEM). Structural properties of different fractions were examined by FT-IR. Strong characteristic absorption peaks were observed at around 1744 cm(-1)and 1650 cm(-1) caused by the C=O group of uronic acids, and the band between 1440 cm(-1) and 1395 cm(-1) associated with the stretching vibration of C-O of galacturonic acid. Neither the crude neutral, nor the acid anemaran significantly inhibited the growth of HepG2 cells in-vitro, which indicated the low cytotoxicity of the anemaran. Furthermore, both neutral and acid anemaran showed hypoglycemic effect. The hypoglycemic effect of neutral anemaran was much higher than that of acid anemaran.

Simultaneous determination of steroidal saponins in Anemarrhena asphodeloides Bge. by ultra high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Simultaneous determination of steroidal saponins in Anemarrhena asphodeloides Bge. by ultra high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Timosaponin-BII inhibits the up-regulation of BACE1 induced by Ferric Chloride in rat retina

BackgroundOur previous studies indicated that oxidative stress up-regulated the expression of β-amyloid precursor protein cleavage enzyme-1 (BACE1) in rat retina. Pharmacological reports have shown Timosaponin-BII, a purified extract originating from Chinese medical herb Rhizoma Anemarrhenae, is characterized as an antioxidant. Our present study aimed to determine whether Timosaponin-BII affected the expression of BACE1, β-amyloid precursor protein cleavage production of Aβ1-40 and β-C-terminal fragment (β-CTF) in rat retina, which were pre-treated with the oxidizing agent (solution of FeCl3).ResultsFew distinctions of BACE1 distribution were observed among all groups (normal control group, model group, Timosaponin-BII treated and vehicle control groups). Rat retinas in model group and vehicle control group manifested an apparent up-regulation of BACE1 expression. Meanwhile, the level of malonaldehyde (MDA), Aβ1-40 and β-CTF were increased. However, when comparing with the vehicle control group, the retinas in Timosaponin-BII treated group showed significantly less BACE1 (p<0.05) and accumulated less Aβ1-40 or β-CTF (p<0.05). It also showed significantly decreased level of MDA (p<0.05) and prolonged partial thromboplastin time (p<0.05).ConclusionOur data suggested that Timosaponin-BII remarkably inhibited the up-regulation of BACE1 and reduced the over-production of β-CTF and Aβ in rat retina, which was induced by FeCl3. The mechanism of Timosaponin-BII on BACE1 expression may be related to its antioxidant property.

A UFLC‐MS/MS method for simultaneous quantitation of spinosin, mangiferin and ferulic acid in rat plasma: application to a comparative pharmacokinetic study in normal and insomnic rats

Suan-Zao-Ren (SZR) decoction, consisting of Ziziphi Spinosae Semen, Poria, Chuanxiong Rhizoma, Anemarrhenae Rhizoma and Glycyrrhizae Radix Et Rhizoma, is a Traditional Chinese Medicine prescription, clinically used for the treatment of insomnia. The objective of this study was to develop a sensitive and reliable UFLC-MS/MS method for simultaneous quantitation of spinosin, mangiferin and ferulic acid, the main active ingredients in SZR decoction, and to compare the pharmacokinetics of these active ingredients in normal and insomnic rats orally administrated with the prescription. Analytes and IS were separated on a Shim-pack XR-ODS column (75 mm × 3.0 mm, 2.2 µm particles) using gradient elution with the mobile phase consisting of methanol and 0.1% formic acid in water at a flow rate of 0.4 mL/min. The detection of the analytes was performed on 4000Q UFLC-MS/MS system with turbo ion spray source in the negative ion and multiple reaction-monitoring mode. The lower limits of quantification were 1, 6 and 1 ng/mL for spinosin, mangiferin and ferulic acid, respectively. Intra- and inter-day precision and accuracy of analytes were well within acceptance criteria (15%). The mean extraction recoveries of analytes and IS from rats plasma were all more than 85.0%. The validated method has been successfully applied to comparing pharmacokinetic profiles of analytes in rat plasma. The results indicated that no significant difference in pharmacokinetic parameters of ferulic acid was observed between two groups, while absorptions of spinosin and mangiferin in insomnic group were significantly lower than those in normal group.

Elucidation of steroid glycosides in Anemarrhena asphodeloides extract by means of comprehensive two‐dimensional reversed‐phase/polyamine chromatography with mass spectrometric detection

In the work presented here, a novel approach to comprehensive two-dimensional liquid chromatography is evaluated. Reversed-phase liquid chromatography was employed for the first-dimension separation and polyamine chromatography was chosen for the second-dimension separation mode. The two dimensions are highly orthogonal and the separation efficacy of the developed octadecylsilica × polyamine was tested by separating an extract from Anemarrhena asphodeloides. The steroid glycosides identified by comprehensive two-dimensional liquid chromatography in this experiment were compared to those obtained for monodimensional liquid chromatography. The comprehensive two-dimensional liquid chromatography system, thanks to the complementary separation selectivity and enhanced peak capacity provided by the two columns, allowed to distribute five compounds of low amounts otherwise unachievable by monodimensional liquid chromatography. In addition, four steroid isomers with similar fragmentation characteristics in MS/MS spectra, were newly separated based on their different chemical structures.

(−)-Nyasol, isolated from Anemarrhena asphodeloides suppresses neuroinflammatory response through the inhibition of I-κBα degradation in LPS-stimulated BV-2 microglial cells

Microglial activation has been associated with neurodegenerative diseases by inducing the neuroinflammatory mediators such as nitric oxide (NO), TNF-α and IL-1β. (−)-Nyasol, a norlignan isolated from a medicinal plant Anemarrhena asphodeloides, showed anti-inflammatory potential in lipopolysaccharide (LPS)-activated BV-2 microglial cells. (−)-Nyasol inhibited the production of NO and prostaglandin E2 (PGE2) and also the expression of inducible nitric oxide synthase and cyclooxygenase-2, which are responsible for the respective production of NO and PGE2. It also suppressed the mRNA levels of TNF-α and IL-1β in activated microglial cells. These effects of (−)-nyasol were correlated with the inactivation of p38 MAPK and the suppression of LPS-induced I-κBα degradation. Taken together, these results suggest that (−)-nyasol can be a modulator in neuroinflammatory conditions induced by microglial activation.

Metabolism profile of timosaponin B‐II in urine after oral administration to rats by ultrahigh‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry

Timosaponin B-II (TB-II) is one of the major bioactive steroid glycosides isolated from Anemarrhena asphodeloides Bge. (Fam. Liliaceae). It has been regarded as a potential lead compound, which may be further developed into a promising new drug for preventing dementia. To fully understand the action mechanism of TB-II, it is important to study the metabolism profile of this compound in vivo. Herein, a rapid and sensitive method based on ultrahigh-performance liquid chromatography (UHPLC)/quadrupole-time-of-flight mass spectrometry (QTOFMS) was established to comprehensively investigate the metabolism of TB-II in Sprague-Dawley rat urine following oral administration of a single dose of TB-II at 500.4 mg·kg(-1). A total of twelve metabolites were detected and identified by means of comparing molecular mass, retention time and spectral pattern of the analytes with those of the parent drug. A possible metabolic pathway on the biotransformation of TB-II was also investigated and proposed. Oxidation, deglycosylation and E-ring cleavage were found to be the major metabolic processes of the compound in rat. It is the first report on a mammalian metabolism study of timosaponin, a common member of steroid glycosides, in rat urine.

Simultaneous determination of nine components in Anemarrhena asphodeloides by liquid chromatography‐tandem mass spectrometry combined with chemometric techniques

A novel quantitative method using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry was developed for simultaneous determination of the important active constituents including four steroidal saponins, two xanthone glycosides, two isoflavonoids, and one anthraquinone in different parts of Anemarrhena asphodeloides from different habitats. Hierarchical clustering analysis and principal components analysis were performed to differentiate and classify the samples. The separation was performed on a C(18) column with acidified aqueous acetonitrile gradients. Quantification of the analytes was achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. Multiple-reaction monitoring scanning was employed with switching electrospray ion source polarity between positive and negative modes in a single run. The validation results of the method indicated that the method was simple, rapid, specific, and reliable. The results demonstrated that the quantitative difference in content of nine active compounds was useful not only for chemotaxonomy of many samples from different sources but also for the standardization and differentiation of many similar samples. Simultaneous quantification of bioactive components by HPLC-ESI-MS coupled with chemometric techniques would be a well-acceptable strategy to comprehensively control the quality of A. asphodeloides.

Inhibition of UVB-induced wrinkle formation and MMP-9 expression by mangiferin isolated from Anemarrhena asphodeloides

Chronic exposure of human skin to solar ultraviolet (UV) radiation causes photoaging. Naturally occurring phytochemicals are known to have anti-photoaging effects. The present study examined the effect of mangiferin isolated from Anemarrhena asphodeloides on wrinkle formation, skin thickness, and changes in collagen fibers in hairless mice. The in vitro effects and possible mechanism of mangiferin on UVB irradiation were determined in human keratinocyte (HEKa) cells. In vitro results showed that mangiferin reduced UVB-induced matrix metalloproteinase (MMP)-9 protein expression and enzyme activity and subsequent attenuation of UVB-induced phosphorylation of mitogen-activated protein kinase kinase1 (MEK) and extracellular signal-regulated kinase (ERK). In the in vivo studies, mangiferin inhibited UVB-induced mean length and mean depth of skin wrinkle based on skin replica, epidermal thickening, and damage to collagen fiber. Taken together, these results indicate that mangiferin exerts anti-photoaging activity in UVB-irradiated hairless mice by regulating MMP-9 expression through inhibition of MEK and ERK.

P01.20. The effect of WIN-34B on cartilage protection and regeneration by chondrogenesis from subchondral bone in vitro and in vivo

Purpose WIN-34B is a butanol fraction extract from the mixture of two oriental herbs, the dried Lonicera japonica flowers and the root of Anemarrhena asphodeloides. In previous studies, we indentified that WIN-34B has analgesic, anti-inflammatory, gastroprotective and safety effects. In this study, we measured the major components and investigated the efficacy of WIN-34B on cartilage protection and regeneration through the chondrognesis of mesenchymal progenitor cells of subchondral bone for the treatment of osteoarthritis and development of new medicines.

P04.13. Population-based case-control study of Chinese herbal products containing ephedra and cardiovascular disease risk

Results There were 1,120 case subjects and 41,409 control subjects in the final analysis. There was a significant reduction of CVD development for consuming ephedrarelated CHP (OR=0.183, p<0.001). Only three items (Xiao Xu Ming Shang OR=2.212, p<0.001; Gui Qi Shao Yao Zhi Mu Shang OR=1.701, p<0.001; She Gan Ma Huang Shang OR=1.441, p=0.006) out of a total of 24 ephedra-related CHP were associated with the risk of CVD development. No statistically linear dose-response relationship was observed with the prescribed dose of ephedra.

Antifungi Efficacy of the Compound Preparation of &lt;i&gt;Anemarrhena asphodeloides&lt;/i&gt; Bunge on the Drug Resistant Strain of Candida Albicans

To investigate the efficacy of the compound preparation named KZY-2 which include the effective group formula of anemarrhena asphodeloides bunge and rhizoma coptidis on the fluconazole and ifraconazo resistant strains of Candida albicans respectively in vitro. The fluconazole and ifraconazo resistant strains of Candida albicans were sieved by drug sensitive scrip test and the diameter of inhibition zone was measured adopting the method of agar plate diffusion, reference to the M27-A recommended by the United States National Laboratory Standards Committee (NCCLS), the minimal inhibitive concentration(MIC,mg/m1) of KZY-2 against the the standard strain(ATCC32354) and the fluconazole and ifraconazo resistant strains of candida albicans was measured respectively. Results showed that the 12 fluconazole and ifraconazo resistant strains which named RC1 to RC12 were sieved from the 20 strains Candida albicans isolated from clinic, the diameters of inhibited zone of KZY-2 against ATCC32354 was 32mm and against RC1～RC12 was between 12mm and 28mm, the MIC of KZY-2 against ATCC32354 was 312.5μg/m1 and against RC1～RC12 was between 625μg /m1 and 5000μg /m1. These results indicate that KZY-2 has good antifungi efficacy on the standard strain and the fluconazole and ifraconazo resistant strains of Candida albicans in vitro.

(−)‐Nyasol, isolated from Anemarrhena asphodeloides suppresses neuroinflammatory response through the inhibition of I‐[kappa]B[alpha]degradation in LPS‐stimulated BV‐2 microglial cells

Activation of microglia has been associated with pathological hallmarks of several neurodegenerative diseases. The regulation of microglial activation by inhibiting the production of pro‐inflammatory molecules can be useful therapeutic approach against neurodegenerative disorders.Anemarrhena asphodeloides Bunge (Liliaceae) has been used as an anti‐pyretic, anti‐inflammatory and anti‐diabetic agent in traditional medicine in Korea, China and Japan. (−)‐Nyasol, a norlignan isolated from Anemarrhena asphodeloides, showed anti‐inflammatory potential in LPS‐activated BV‐2 microglial cells. (−)‐Nyasol suppressed the mRNA levels of tumor necrosis factor α (TNF‐α) and Interleukin 1β (IL‐1β) in activated microglial cells. It also inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) by down‐regulation of inducible nitric oxide synthase (i‐NOS) and cyclooxygenase‐2 (COX‐2), respectively. These inhibitory effects were correlated with the suppression of LPS‐induced nuclear factor κB (NF‐κB) nuclear translocation through the inactivation of p38 mitogen‐activated protein kinase (MAPK). These results suggest that (−)‐nyasol can be a modulator in neuro‐inflammatory conditions induced by microglial activation.

Selective inhibition of MMP-9 gene expression by mangiferin in PMA-stimulated human astroglioma cells: Involvement of PI3K/Akt and MAPK signaling pathways

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases which play a key role in invasion, migration, and angiogenesis of astrogliomas and other malignant tumors. Thus, controlling MMPs has been considered an important therapeutic strategy for prevention and/or treatment of gliomas. However, most MMP inhibitors developed so far are broad spectrum inhibitors; thus, it is necessary to develop a selective MMP inhibitor to minimize potential side effects. In the present study, we found that mangiferin, a glucosylxanthone isolated from Anemarrhena asphodeloides, specifically inhibited MMP-9 gene expression in phorbol myristate acetate (PMA)-stimulated human astroglioma U87MG, U373MG, and CRT-MG cells. However, it did not affect other MMPs, such as MMP-1, -2, -3, and -14. Mangiferin suppressed MMP-9 expression at the promoter, mRNA, and protein levels and additionally inhibited MMP-9 enzymatic activity. The Matrigel-invasion assay showed that mangiferin suppresses the in vitro invasiveness of glioma cells, which appears to be correlated with mangiferin-mediated MMP-9 inhibition. Further mechanistic studies demonstrated that mangiferin inhibits the binding of NF-κB and AP-1 to the MMP-9 promoter and suppresses the PMA-induced phosphorylation of Akt and MAP kinases, which are upstream signaling molecules in MMP-9 expression. Thus, the specific inhibition of MMP-9 by mangiferin may provide a valuable pharmacological tool for treatment of gliomas.

Determination of two major xanthone glycosides in rhizome of Anemarrhena asphodeloides using high performance capillary electrophoresis

Anemarrhena rhizome known as “Zhi-Mu” is an important traditional Chinese medicine (TCM). In this study, an accurate, rapid and reproducible high performance capillary electrophoresis (HPCE) method with ultraviolet (UV) detection has been established for the simultaneous determination of two major bioactive xanthone glycosides, mangiferin and neomangiferin in rhizome of Anemarrhena asphodeloides (Zhi-Mu). The running buffer was 30 mM borate (pH 9.3) containing 15% methanol. The separation voltage was 30 kV and temperature was 25°C. Gentiopicroside was adopted as an internal standard (IS) and detection wavelength was 254 nm. Within 15 min, the contents of mangiferin and neomangiferin can be simultaneously assayed. The results also indicate that it is important to assay the xanthone content in quality control of “Zhi-Mu” for its great variation among different samples. Key words: High performance capillary electrophoresis (HPCE), Anemarrhena asphodeloides, mangiferin, neomangiferin.

Influence of Aconiti Lateralis Radix Praeparata on asthenia cold syndrome rats with whole genome gene expression of liver by gene chip technique

To study the influence of Aconiti Lateralis Radix Praeparata on asthenia cold syndrome rats with whole genome gene expression of liver by gene chip technique. The asthenia cold syndrome rat models were established by administering traditional Chinese medicine raw Gypsum Fibrosum, Gentianae Radix, Phellodendri Chinensis Cortex and Anemarrhenae Rhizoma. After treated with Aconiti Lateralis Radix Praeparata, the rats' liver gene expressions were detected using gene chip. Differential expression genes were screened for gene function annotation, and some genes were selected to check the accuracy of the results by RT-PCR. Compared with the asthenia cold model group, the asthenia cold treatment group showed 212 differential expression genes, mainly involving function of immune response and oxidoreductase activity. Aconiti Lateralis Radix Praeparata is proved to have an effect on up-regulating immune response-related genes and oxidizing oxidoreductase activity-related genes of asthenia cold syndrome rats and may be a molecular mechanism for classical warm-nature medicine Aconiti Lateralis Radix Praeparata in warming meridians and dissipating cold.