Functional characterization of a cycloartenol synthase and four glycosyltransferases in the biosynthesis of cycloastragenol-type astragalosides from Astragalus membranaceus

Astragalosides are the main active constituents of traditional Chinese medicine Huang-Qi, of which cycloastragenol-type glycosides are the most typical and major bioactive compounds. This kind of compounds exhibit various biological functions including cardiovascular protective, neuroprotective, etc. Owing to the limitations of natural sources and the difficulties encountered in chemical synthesis, re-engineering of biosynthetic machinery will offer an alternative and promising approach to producing astragalosides. However, the biosynthetic pathway for astragalosides remains elusive due to their complex structures and numerous reaction types and steps. Herein, guided by transcriptome and phylogenetic analyses, a cycloartenol synthase and four glycosyltransferases catalyzing the committed steps in the biosynthesis of such bioactive astragalosides were functionally characterized from Astragalus membranaceus. AmCAS1, the first reported cycloartenol synthase from Astragalus genus, is capable of catalyzing the formation of cycloartenol; AmUGT15, AmUGT14, AmUGT13, and AmUGT7 are four glycosyltransferases biochemically characterized to catalyze 3-O-xylosylation, 3-O-glucosylation, 25-O-glucosylation/O-xylosylation and 2'-O-glucosylation of cycloastragenol glycosides, respectively. These findings not only clarified the crucial enzymes for the biosynthesis and the molecular basis for the structural diversity of astragalosides in Astragalus plants, also paved the way for further completely deciphering the biosynthetic pathway and constructing an artificial pathway for their efficient production.

Research advance of pharmacological effects of astragalosides on nervous system diseases / 中国中药杂志

Astragali Radix is a traditional Chinese herbal medicine with a long history, which has the functions of tonifying Qi and promoting urination and granulation. Astragalosides are the main effective components of Astragali Radix, and more than 40 triterpenoid saponins have been obtained from Astragalus membranaceus and its related plants, mainly including astragalosides Ⅰ-Ⅷ, isoastragalosides Ⅰ, Ⅱ, and Ⅳ, acetylastragalosides, and soyasaponins. Astragalosides have a wide range of biological activities, such as immunomodulation, antioxidation, and neuroprotection. Nervous system diseases seriously affect people's quality of life, threaten human physical and mental health, and impose a burden on families and society. As natural drugs, astragalosides have good preventive and therapeutic effects on central nervous system diseases. This paper reviews the main pharmacological effects and mechanisms of astragalosides in the treatment of multiple sclerosis, Parkinson's disease, Alzheimer's disease, and cerebral ischemic stroke and proposes the research prospects and potential problems, aiming to provide reference for the clinical application and basic research of astragalosides.

Bioadhesive pellets of astragalosides: preparation and adhesive evaluation based on the physical properties of wet mass / 药学学报

Bioadhesive preparation can be attached to specific sites to control drug release rate, increase drug concentration and increase efficacy, which is based on natural or synthetic polymer material. In this paper, based on the physical properties of wet mass, a method for screening adhesion formulation was proposed, which was different from conventional way of screening optimal formulation, and astragalosides loaded bioadhesive pellets were prepared by extrusion-spheronization method (extrusion speed 30 r·min-1, spheronization speed 808 r·min-1, spheronization time 7.5 min) based on this formulation screening method, small living animal imaging technology and mucin from porcine stomach model were used to evaluate the in vivo and invitro adhesiveness behaviour of the pellets. According to the relationship between the physical properties of wet mass and the formability and adhesiveness of bioadhesive pellets, five key physical properties hardness (Ha), adhesiveness (Ad), springiness (Sp), cohesiveness (Co), chewiness (Ch) were selected as the index of screening optimal formulation, therefore a comprehensive evaluation model was established, which based on principal component analysis, to did digital ranking for these proposed adhesion formulation, the optimal formulation was determined: microcrystalline cellulose: (chitosan∶Carbomer 940 = 2∶1), the adhesive material dosage accounted for 20% of the excipient dosage, and the ratio of drugs to excipients was 1 : 4. All animal experiments have been approved by Ethics Committee of Shanghai University of Traditional Chinese Medicine. The in vivo and in vitro adhesive evaluation results showed the pellets had a clear advantage in intestinal adhesion over normal pellets, its also proved the scientificity and reliability of the method of screening bioadhesive formulation.

Effect of borneol combined with astragaloside IV and Panax notoginseng saponins on permeability of blood-brain barrier after cerebral ischemia-reperfusion / 中国药理学通报

Aim To probe the effect of borneol combined with astragalosides IV (AST I V) and Panax notoginseng saponins (PNS) on blood-brain barrier (BBB) after cerebral ischemia-reperfusion. Methods Rats with cerebral ischemia-reperfusion were administered by gavage, the symptom of nerve function defect was observed, the water content of brain tissues, the permeability of BBB, and the expression of zonula cccludens 1 (ZO-1), ZO-2, Occludin and Claudin-5 were detected. Results After cerebral ischemia-reperfusion, the neurological deficit symptom appeared, the neurological function score and brain water content increased, and BBB was destroyed. Each drug could relieve the above pathological changes to various degrees, and the effect of borneol + AST IV + PNS was the best, which was stronger than that of single drug and AST IV + PNS. The expressions of ZO-1, ZO-2, Occludin, Claudin-5 proteins decreased after cerebral ischemia-reperfusion. All drugs inhibited the decrease of ZO-1, except AST I V, and increased Occludin expression; Borneol + AST IV + PNS also up-regulated ZO-2, and the increase in ZO-1, ZO-2, Occludin was greater than that of each drug alone and AST IV + PNS. Conclusions Borneol, AST IV and PNS can relieve the increase of BBB permeability, reduce brain edema and antagonize brain injury to various degrees after cerebral ischemia-reperfusion, which are enhanced by the combination of three drugs. The mechanism may be related to the synergistic inhibition of the down-regulation of ZO-1, ZO-2 and Occludin and the protection of BBB after cerebral ischemia.

Screen astragalosides from Huangqi injections by LC-TOF-MS-based mass defect filtering approach / 中国中药杂志

The samples of Huangqi injection (HI) were analyzed by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-TOF-MS), and both positive and negative ion modes were employed to obtain the LC-TOF-MS analysis information of chemical compounds in HI. Then the mass defect filtering (MDF) approach, which was developed based on the previously published articles, was utilized to rapidly screen the astragalosides from the obtained LC-TOF-MS data. Each screened astragaloside was confirmed by the presence of no less than 2 quasi-molecular ions. All the screened astragalosides were then tentatively assigned according to the parent ion and daughter ion information. Finally, a total of 62 astragalosides were screened and characterized from the HI samples, including 15 new detected ones. The identification results indicated that acetylation, hydrogenation, dehydrogenation, methoxylation and hydration might be the major conversion reactions involved in the formation of the astragalosides. The LC-TOF-MS-based MDF approach was proved to be a feasible and efficient tool to screen the chemical constituents in complex matrices such as herbal medicines.

Protective effects of astragalosides on pancreatic tissue in diabeticrats / 国际中医中药杂志

Objective To investigate the protective effects of astragalosides on pancreatic tissue in diabetic rats.Methods The experimental diabetic rat models were made by feeding with high fat and sugar and injecting STZ, and according to blood glucose level, the rat models were randomly devided into five groups: a diabetic model control group, an astragalosides low (30 mg/kg), a medium (60 mg/kg), a high (120 mg/kg) dose treated groups and a metformin hydrochloride 200 mg/kg treated group (n=16), and selected another 16 same-aged rats as a normal control group. 72 h after the models were made, the drugs were given by intragastric administration for 4 weeks, once a day. Before and after the drugs were given 7, 14, 21, 28 days, the level of plasma glucose (FPG) and insulin (FINS) were determined, and the ISI and HOMA-IR were calculated, the level of T-AOC and the content of ROS in serum were detected, the activity of SOD, CAT and the content of MDA in pancreatic tissue were detected, the histopathological changes of pancreatic tissue and pancreatic cells apoptosis were observed, and the apoptosis index were analyzed.Results Four weeks later, compared with the diabetic model control group, the FINS (9.34 ± 1.67 mIU/ml, 10.46 ± 1.51 mIU/mlvs.7.37 ± 1.39 mIU/ml) and the level of ISI (6.82 ± 0.96, 6.52 ± 0.79vs.7.35 ± 0.84) in astragalosides medium and high dose treated groups were significantly increased (P<0.05 orP<0.01), the level of HOMA-IR (43.62 ± 5.26, 29.37 ± 4.51vs.61.70 ± 5.85) were significantly decreased (P<0.05 orP<0.01). The level of FPG (9.32 ± 2.57 mmol/Lvs.17.15 ± 2.54 mmol/L) in astragalosides high dose treated group was significantly decreased (P<0.01), the level of T-AOC (12.20 ± 3.36 U/L vs.9.34 ± 2.59 U/L) in serum was significantly increased (P<0.01). The activity of SOD (11.52 ± 2.31 U/mg, 15.29 ± 2.50 U/mgvs.7.53 ± 1.86 U/mg) in serum of astragalosides medium and high dose treated groups were significantly increased (P<0.05 orP<0.01). The content of MDA (2.83 ± 0.37 nmol/mg, 1.92 ± 0.31 nmol/mgvs. 4.08 ± 0.42 nmol/mg) in pancreatic tissue of astragalosides medium, high dose treated groups were significantly decreased (P<0.05 orP<0.01); the activity of SOD (11.52 ± 2.31 U/mg, 15.29 ± 2.50 U/mgvs.7.53 ± 1.86 U/mg) and CAT (392.28 ± 38.05 U/g, 461.73 ± 42.52 U/gvs. 280.46 ± 29.61 U/g) were significantly increased (P<0.05 orP<0.01).Conclusion Astragalosides had protective effects on pancreatic tissue in diabetic rats.

Inhibitory Effects of Astragalosides on Proliferation of Lung Adenocarcinoma SPCA-1 Cells / 中国药房

OBJECTIVE:To investigate the inhibitory effect of astragalosides on proliferation of lung adenocarcinoma SPCA-1 cells. METHODS:After the cells were cultured in 0 (blank control),7.8,15.6,31.2,62.5,125.0 and 250.0 μg/ml for 48 h, MTT method was used to determine cell viability and median inhibitory concentration(IC50)was calculated,and terminal deoxynu-cleotidyl transferase-mediated dUTP-biotin nick end labeling assay(TUNEL)and flow cytometry were employed to detect apopto-sis. RESULTS:Compared to the blank control,after the cells were cultured in 7.8,15.6,31.2,62.5,125.0 and 250.0 μg/ml as-tragalosides for 48 h,the cell viability was poorer(P<0.01),and IC50 was 61.75 μg/ml;after the cells were cultured in 15.6,31.2, 62.5,125.0 and 250.0 μg/ml astragalosides for 48 h,the apoptosis rate was higher. CONCLUSIONS:Astragalosides have an anti-tumor effect to some degree by a mechanism which may be related to inhibiting cancer cell proliferation and accelerating apoptosis.

Transformation of astragalosides from radix astragali under acidic, neutral, and alkaline extraction conditions monitored by LC-ESI-TOF/MS / 中国天然药物

AIM@#To reveal the profile of astragalosides for better quality evaluation of Radix Astragali, this study was aimed to investigate the transformation of astragalosides under different conditions.@*METHOD@#Seven major astragalosides were selected for evaluation under acidic, neutral and alkaline conditions. The transformation in real plant samples was also examined and the products were characterized by LC-ESI-TOF/MS.@*RESULTS@#In weak acidic solution, all of the astragalosides are stable. In addition, the transformation ratios of the astragalosides under neutral and alkaline conditions were also obtained.@*CONCLUSION@#In neutral solution, malonylastragaloside I was transformed to astragaloside I; and in alkaline solution, substituent group(s) in the xylose moiety of all the astragalosides were eliminated. Since astragalosdie IV is the basic skeleton structure of the astrgalosides, it is a common transformation product of other astragalosides.

Effects of Astragalosides on Angiogenesis in Myocardium Infarction Rats / 中国基层医药

Objective To investigate the effects of astragalosides(AST)on angiogenesis of myocardium in rats after myocardial infarction.Methods Myocardial infarction(MI)was induced by ligation of the proximal left anterior descending coronary artery,30 postoperative rats were randomly divided into three same-size groups,i.e,medical group A(AST 2.5 mg · kg-1 · d-1),medical group B(AST 10mg · kg-1 · d-1)and control group(physiological saline).All of three groups were treated with intraperitoneal injection of 2ml dose for 4 weeks.The pathological changes of the heart tissue were observed by H-E staining and the micro-vascular count(MVC)/micro-vascular density (MVD)were calculated by CD34-staining.Results HE staining showed cardiac fabric disarrangement,granulation tissue generation,and fibroblast proliferation;The change of medical groups was less obvious than the control group; the change of group B with higher dose was less obvious than group A.CD34 staining showed that regeneration of neovascularization at the margin of myocaardium infarction was seen in all of three groups;for the MVC/MVD,medical groups were significantly higher than the control group,while group B is significantly higher than group A (all P ＜0.01).Conclusion AST can improve myocardial ischemia of rats after myocardial infarction.AST can promote angiogenesis in ischemic myocardium of rats,and the effect is positively correlated with AST dose.

Neuroprotective effects of astragalosides against amyloid β-protein and DEX-induced neurotoxicity in rat hippocampal neurons / 中草药

Objective: To study the effect of astragaloside (AST) on the injury induced by amyloid β-protein (Aβ) plus Dexamethasone (DEX) in rat hippocampal neurons. Methods: In vitro, the effects of AST on hippocampal neurons cell death with Aβ plus DEX were detected by MTT assay and intracellular calcium ([Ca2+]i); The effects of AST on phospho-tau (P-tau) protein were analyzed to explore the mechanisms responsible for DEX enhanced Aβ-induced cell death in hippocampal neurons. Results: AST (10, 20, and 40 μg/mL) could protect hippocampal neurons against DEX (10 μmol/L) plus Aβ25-35 (5 μmol/L) - induced hippocampal neuronal injury of felal rat in vitro (P<0.01). AST could inhibit the increased levels of [Ca2+]i and P-tau protein level induced by DEX (10 μmol/L) plus Aβ25-35 (5 μmol/L) (P < 0.05). Conclusion: AST could protect hippocampal neuron against synergistic neurotoxicity of Aβ and DEX.

Study on Preparation Technology for Pingfenggujin Granule / 中国中医药信息杂志

Objective To optimize a preparation technique of Pingfenggujin Granule. Methods The technique was determined by the orthogonal design with content of Astragalosides, polysaccharides and the rate of dried extracts. The contents of polysaccharides and Astragalosides were determined by UV and ELSD-HPLC respectively. Results The optimum water-extraction technique was to steep 2.5 h, ten times of water as much as weigh of total drugs and extract three times, 2 h each. Conclusion The optimum technique is feasible, and the method of content determination is accurate and reliable.

Effect of Astragalosides on proliferation and collagen production of hepatic stellate cells in vitro / 中国药理学通报

AIM To study the effect of astragalosides (AST) on pr ol iferation and collagen production of hepatic stellate cells (HSC). METHO DS The proliferation and collagen production of rat hepatic stellate cell s, HSC-T6, stimulated with Kupffer cell-conditioned medium (KCCM), mixed sera containing new bovine serum (NBS) and rat serum (RS) were measured with 3H -TdR and 3H-proline incorporation. RESULTS Both of the p roliferation and collagen production of HSC-T6 stimulated with KCCM at the dilu tion of 1∶4 were suppressed after being treated with AST (16, 32, 64, 128 and 2 56 mg?L -1 ). When HSC-T6 cells were stimulated with mixed sera containing 10% NBS and 3% RS, their proliferation was suppressed after being treated with AST(32, 64, 128 and 256 mg?L -1 )for 48 h and so was the collagen produ ction after being treated with AST(16, 32, 64, 128 and 256 mg?L -1 )for 72 h. CONCLUSION The suppression of hepatic stellate cell prolif eration and collagen production by astragalosides may be one of the mechanisms f or the depression of hepatic fibrosis by astragalosides.

Antitumor activity of AST and its mechanism of action / 中国药理学通报

AIM To study the antitumor activity of astragalosides(AST) and its mechanism of action. METHODS By using two experimental models of hepatoma(HepA) and Sarcoma 180 in mice, the rate of inhibition of tumor weight AST on the growth of HepA and S180 tumor cells were tested. The growth inhibition of AST on Hela cells was detected by MTT assay. The effect of AST on cell cycle and apoptosis was analyzed by flow cytometry and TUNEL. RESULTS AST inhibited the growth of tumor cells of HepA and S180 in mice. AST inhibited the growth of Hela cell in concentration dependent manner with IC 50 of 80 4 mg?L -1 . Flow cytomety analgsis showed that G 0/G 1 phase rate was increased but S phase rate was decreased. The apoptosis rate of Hela cells treated with AST( 80 and 160 mg?L -1 ) was significantly higher than that of control. CONCLUSION AST can inhibit the growth of tumor cells of HepA and S180 in mice and the growth of HeLa cells in vitro . Causing cell cycle arrest and apoptosis is probably one of the mechanisms of antitumor effect by AST.

Effects of the total extract of astragalosides on the growth, AFP secretion and γ-GT activity of human hepatoma cells / 中国临床药理学与治疗学

AimTo study the effect of the total extract of astragalosides(TEA) on the growth, AFP secretion and γ-GT activity of human hepatoma cells invitro.Methods The human hepatoma HepG2 and Bel-7404 cells were cultured with TEA 5～160 mg · L-1 for 6 days. Anti-hepatoma activity was measured by MTT method. AFP was assaysed by ELISA and γ-GT was determined by enzyme-substrate method. Results The growth of the human hepatoma HepG2 and Bel-7404 cells and the secretion of AFP of HepG2 cells were reduced significantly by TEA 5～160 mg· L-1. The γ-GT activity of HepG2 cells was inhibited and its ALB content was increased by TEA 20, 40 and 80 mg · L-1. The γ-GT activity of Bel-7404 cell was inhibited and its ALB content was increased byTEA 40 and 80 mg · L-1 Conclusion These results suggest that TEA has inhibitory effects on the growth, AFP secretion and γ-GT activity of human hepatoma cells.

Anti-inflammatory effect of astragalosides and its mechanisms of action / 中国临床药理学与治疗学

AimTo study the anti-inflammatory effect of astragalosides(AST) and its mechanisms of action. Methods The exudate volume, neutrophil count and protein content in exudate were measuredinthe carrageenan-induced air pounch model in rats. The content of PGE2 was assayed by radioimmunoassay,the activity of PLA2 by microacid titration assay, IL-8 by ELISA, and NO by nitrate reductase assay. The production of O2 in neutrophil was determined by cytochrome C assay. Results AST(40, 80 mg· kg -1) could markedly reduce the exudate volume, neutrophil count, protein content, the content of IL-8, and the production of O2.AST lowered PLA2 activity of neutrophil and accellular component in exudate, and it also decreased the contents of PGE2 and NO in exudate. Conclusion AST has an obvious anti-inflammatory effect on carrageenan-induced acute inflammation.Its mechanisms may be related to the inhibition of vascular permeability and leukocyte migration, as well as to the suppression of PLA2 activity and the reduction of IL-8, PGE2, NO and O2 production.

Effect of Astragalosides on Gastrin Receptor in Gastric Parietal Cells of Rats with Spleen Deficiency / 广州中医药大学学报

[Objective] To observe the regulatory effect of astragalosides (AST) on the binding capacity of gastrin receptor in gastric parietal cells (GPC) of rats with spleen deficiency. [Methods] SD rats were given gastric gavage of Radix et Rhizoma Rhei (Dahuang) decoction 10 g/kg to induce spleen deficiency. Gastric parietal cells were isolated and purified by Lewin gastric sac method and Percoll density gradient separation method. 2 mL suspension of GPC from rat models was incubated in vitro with AST 20 ?L at the concentrations of 10, 20 and 30 g/L respectively. Suspension of GPC from rat models incubated with phosphate buffered saline (PBS) served as the model group and suspension of GPC from the normal rats incubated with PBS as the normal group. The binding capacity of gastrin receptor in GPC was detected by radioligand receptor assay. [Results] The binding sites were 262.29?60.80 in the model group, much lower than 443.93?133.58 in the normal group (P

Effects of the total extract of astragalosides on the growth,AFP secretion and ?-GT activity of human hepatoma cells / 中国临床药理学与治疗学

Aim To study the effect of the total extract of astragalosides (TEA) on the growth, AFP secretion and ?_GT activity of human hepatoma cells in vitro. Methods The human hepatoma HepG2 and Bel_7404 cells were cultured with TEA 5～160 mg?L-1 for 6 days. Anti_hepatoma activity was measured by MTT method. AFP was assaysed by ELISA and ?_GT was determined by enzyme_substrate method. Results The growth of the human hepatoma HepG2 and Bel_7404 cells and the secretion of AFP of HepG2 cells were reduced significantly by TEA 5～160 mg?L-1.The ?_GT activity of HepG2 cells was inhibited and its ALB content was increased by TEA 20,40 and 80 mg?L-1. The ?_GT activity of Bel_7404 cell was inhibited and its ALB content was increased by TEA 40 and 80 mg?L-1.Conclusion These results suggest that TEA has inhibitory effects on the growth, AFP secretion and ?_GT activity of human hepatoma cells.

Effects of total astragalosides on hepatoma cells apoptosis and wtp53 expression / 中国药理学通报

AIM\ To study whether total astragalosides(TA) induces apoptosis in human hepatoma cells and its mechanism of action. METHODS\ The human hepatoma HepG2 and Bel 7404 were cultured with TA (20,40 and 80 mg?L -1 ) for 6 days and DNA fragmentation and the expression of wtp53 protein were determined by flow cytometry. RESULTS\ HepG2 and Bel 7404 cell apoptosis induced by TA(20,40 and 80 mg?L -1 ) were found by flow cytometry. The apoptotic peak of HepG2 cells increased in dose dependent manner in the range from 4 1% to 74 5%; The apoptotic peak of Bel 7404 cells also increased from 4 5% to 50 5%. The expression of wtp53 in HepG2 cells and Bel 7404 cells treated with TA (80 mg?L -1 ) were upregulated. CONCLUSION\ TA may induce apoptosis in HepG2 cells and Bel 7404 cells, its mechanism of action might be associated with upregulating the expression of wtp53.

Effects of astragalosides on the expression of BDNF,TrkB and p75NTR mRNA against focal cerebral ischemia-reperfusion injury / 中国药理学通报

Aim To observe the neurological protective effects of astragalosides(AST) on focal cerebral ischemia-reperfusion(I/R) injury in rats and to explore its possible mechanism.Methods Male SD rats received right middle cerebral artery occlusion for 120 min,and were decapitated 1,3,7,and 14 days after reperfusion.AST(40 mg?kg-1) was orally administered after I/R.Neurological deficit score was daily determined,the expressions of BDNF and p75NTR mRNA were detected by RT-PCR,and the expression of TrkB mRNA was detected by real-time PCR.Results AST reduced the neurological deficit score on days 3,increased the expression of BDNF mRNA on days 3,7 and 14,decreased p75NTR mRNA and increased TrkB mRNA on days 3 and 7.Conclusions AST improves the neurological deficits after I/R in rats.The mechanism may be related with increasing BDNF,and TrkB mRNA,and decreasing p75NTR mRNA.

Effect of astragalosides on schistosomal hepatic fibrosis in vitro / 中国药理学通报

Aim To observe the effect of astragalosides on proliferation and collagen synthesis of HSC-T6 cells driven using schistosoma japonicum soluble egg antigen-activated macrophage conditioned medium(SEA-MCM).Methods SEA-MCM was prepared by injection of Schistosoma japonicum SEA via mice peritoneal.The proliferation and collagen synthesis of HSC-T6 cells stimulated with SEA-MCM were measured using MTT colorimetric assay and()~3H-proline incorporation respectively.Results The proliferation and collagen production of HSC-T6 cells were significantly promoted by SEA-MCM.They were significantly suppressed after being treated with AST(32.5、65、130 mg?L~(-1)) showing concentration-dependent effect.Conclusion:Astragalosides may inhibit HSC-T6 cells proliferation and collagen production that was driven by SEA activated MCM in vitro.