calycosin-7-O-β-d-glucoside Research Articles

Simple and efficient approach for enrichment of major isoflavonoids from Astragalus membranaceus with macroporous resins and their nephroprotective activities

Isoflavonoids are abundant in Astragalus membranaceus and exhibit multiple pharmacological activities, including nephroprotective activity. In the present study, a simple, green and effective method for the simultaneous enrichment of four major bioactive isoflavonoids from A. membranaceus with macroporous resin was established. Among ten widely used macroporous resins, AB-8 resin exhibited the highest adsorption capacity and desorption ratio in static tests. The static equilibrium adsorption data fit to the Langmuir isotherm, and the adsorption kinetics followed the pseudo-second order model. Dynamic adsorption and desorption experiments were performed to optimize the separation parameters. After only one cycle of treatment with a stepwise elution on AB-8 resin, the contents of calycosin-7-O-β-d-glucoside (CAG), ononin (ONO), calycosin (CAL) and formononetin (FOR) in the final product were 7.71-, 10.64-, 10.50- and 9.49-fold increased with recovery yields of 70.89, 83.14, 85.01 and 74.62%, respectively. Furthermore, the target isoflavonoid-enriched product exhibited better nephroprotective activity than the crude extract, and both the purity of the isoflavonoids and their bioactivities were significantly enhanced after enrichment with macroporous resin. The developed method is a promising foundation for the scaled-up preparation of bioactive isoflavonoids for their wide use in the pharmaceutical industry.

Quality evaluation for Radix Astragali based on fingerprint, indicative components selection and QAMS

Radix Astragali (RA) is one of the most widely used Chinese herbs prescribed in many Chinese formulas to reinforce 'Qi' and treat vital energy deficiency. This study combined fingerprinting with quantitative analysis multi-components by a single marker (QAMS) to improve the quality control standard for RA on the basis of existing quality control methods of traditional Chinese medicinal materials. UPLC-ESI-TOF-MS technique was used to evaluate the quality of RA by fingerprinting and QAMS. Using the anti-inflammatory, anti-oxidation and anti-anoxic activities to screen characteristic components of RA, the calycosin-7-O-β-d-glucoside (CG), ononin, astragaloside IV, astragaloside II, calycosin and astrageloside I significantly inhibited ear edema in mice, the calycosin and CG had good antioxidant activity and the astragaloside I had a significant anti-hypoxia activity. Astragaloside I, astragaloside II, astragaloside IV, ononin, calycosin and CG had significant pharmacological actions. These components were comprehensively used as the indicative components for the quality control of RA. Astragaloside I was used as the internal standard of the relative correction factors of CG (13.45), ononin (0.51), calycosin (12.08), astragaloside IV (0.73) and astragaloside II (0.81). Astragaloside I and CG were used as internal standards of the relative correction factors of the flavonoids and saponins of ononin (1.11), calycosin (0.04), astragaloside IV (0.73) and astragaloside II (0.81). The study combined fingerprinting with QAMS to improve the quality control standard for RA.

Multiple biological defects caused by calycosin-7-O-β-d-glucoside in the nematode Caenorhabditis elegans are associated with the activation of oxidative damage.

Calycosin-7-O-β-d-glucoside (CG) is an important active isoflavone compound in Radix Astragali that has many bioactivities. However, the toxicological effects and related toxicological mechanism of CG have been rarely documented. The purpose of the present study was to evaluate the toxicity effects of CG on the model organism Caenorhabditis elegans. Some characteristics of the nematode, including lifespan, movement behavior and reproductive capacity, were used to detect the toxic effects of CG on C. elegans. The results showed that CG could shorten the lifespan of C. elegans by up to 25.3% and severely damage the movement of N2 larvae compared with the control group. Moreover, CG could prolong the generation times and reduce the brood sizes. Furthermore, CG promoted the formation of reactive oxygen species (ROS), which caused oxidative stress, increased the mRNA expression of sod-1, sod-2, sod-3, sod-5, ctl-1, ctl-2 and ctl-3, and induced the antioxidant enzymes activities of superoxide dismutase and catalase to scavenge free radicals. However, antioxidant treatment experiments showed that Trolox could reduce the level of ROS caused by CG to the normal state of the control. These results suggested that the generation and elimination of ROS could not restore normal homeostasis in C. elegans treated by CG. These findings indicated that the activation of oxidative damage is one of the most important toxic mechanisms of CG in C. elegans.

SGLT-1 Transport and Deglycosylation inside Intestinal Cells Are Key Steps in the Absorption and Disposition of Calycosin-7-O-β-d-Glucoside in Rats.

Hydrolysis by lactase-phloridzin hydrolase (LPH) is the first and critical step in the absorption of isoflavonoid glucosides. However, the absorption characteristics of calycosin-7-O-β-d-glucoside (CG) slightly differ from other isoflavonoid glucosides. In this study, we used the rat intestinal perfusion model and performed pharmacokinetic studies and in vitro experiments to determine the factors influencing CG absorption and disposition. After oral administration of isoflavonoid glucosides, LPH was found to play minimal or no role on the hydrolysis of CG, in contrast to that of daidzin. CG was mainly transported into the small intestinal cells by sodium-dependent glucose transporter 1 (SGLT-1) as intact. This pathway could be the main mechanism underlying the high permeability of CG in the small intestine. CG was likely to be hydrolyzed in enterocytes to its aglycone calycosin by broad-specific β-glucuronides (BSβG) and glucocerebrosidase or rapidly metabolized. Calycosin was also rapidly and extensively metabolized to 3'-glucuronide in the enterocytes and liver, and the glucuronidation rates of calycosin and CG were much higher in the former. The metabolites were also transported into lumen by breast cancer resistance protein and multidrug resistance-associated protein 2. In conclusion, the enterocytes could be an important site for CG absorption, deglycosylation, and metabolism in rats. This study could contribute to the theoretical foundation and mechanism of absorption and disposition of flavonoid compounds.

Calycosin‑7‑O‑β‑D‑glucoside attenuates ischemia‑reperfusion injury in vivo via activation of the PI3K/Akt pathway.

The aim of the present study was to investigate the effects and mechanisms of calycosin-7-O-β-d-glucoside (CG) on ischemia-reperfusion (I/R) injury in vivo. Hemodynamic parameters, including ejection fraction (EF), fractional shortening (FS), left ventricular end-systolic pressure (LVESP) and left ventricular end-diastolic pressure (LVEDP) were monitored using an ultrasound system, and infarct size was measured using Evans blue/tetrazolium chloride double staining. The activities of serum creatine kinase (CK), lactate dehydrogenase (LDH) and superoxide dismutase (SOD), and the levels of malondialdehyde (MDA) were determined to assess the degree of myocardial injury and oxidative stress-induced damage. The protein expression levels of cleaved-caspase-3, cleaved-caspase-9, phosphorylated (p)-phosphatidylinositol 3-kinase (PI3K) p85, PI3K p85, p-Akt and Akt were determined using western blotting. The results demonstrated that pretreatment with high dose (H)-CG markedly improved cardiac function, as evidenced by upregulated EF, FS and LVESP, and downregulated LVEDP. In addition, administration of CG resulted in significant decreases in infarct size in the I/R+low dose-CG and I/R+H-CG groups, compared with the I/R group. The activities of CK and LDH, and the levels of MDA in the I/R+H-CG group were reduced, compared with those in the I/R group, whereas SOD activity was elevated. Treatment with CG inhibited the cleavage and activity of caspase-3 and caspase-9, and enhanced the phosphorylation of PI3K p85 and Akt. Notably, administration of the PI3K inhibitor, LY294002, markedly lowered the levels of p-PI3K p85/p-Akt, and eradicated the inhibitory effects of H-CG on infarct size, myocardial injury and oxidative stress-induced damage. Taken together, the results suggested that CG may alleviate I/R injury by activating the PI3K/Akt signaling pathway.

Triterpene and Flavonoid Biosynthesis and Metabolic Profiling of Hairy Roots, Adventitious Roots, and Seedling Roots of Astragalus membranaceus.

Astragalus membranaceus is an important traditional Chinese herb with various medical applications. Astragalosides (ASTs), calycosin, and calycosin-7-O-β-d-glucoside (CG) are the primary metabolic components in A. membranaceus roots. The dried roots of A. membranaceus have various medicinal properties. The present study aimed to investigate the expression levels of genes related to the biosynthetic pathways of ASTs, calycosin, and CG to investigate the differences between seedling roots (SRs), adventitious roots (ARs), and hairy roots (HRs) using quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR study revealed that the transcription level of genes involved in the AST biosynthetic pathway was lowest in ARs and showed similar patterns in HRs and SRs. Moreover, most genes involved in the synthesis of calycosin and CG exhibited the highest expression levels in SRs. High-performance liquid chromatography (HPLC) analysis indicated that the expression level of the genes correlated with the content of ASTs, calycosin, and CG in the three different types of roots. ASTs were the most abundant in SRs. CG accumulation was greater than calycosin accumulation in ARs and HRs, whereas the opposite was true in SRs. Additionally, 40 metabolites were identified using gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS). Principal component analysis (PCA) documented the differences among SRs, ARs, and HRs. PCA comparatively differentiated among the three samples. The results of PCA showed that HRs were distinct from ARs and SRs on the basis of the dominant amounts of sugars and clusters derived from closely similar biochemical pathways. Also, ARs had a higher concentration of phenylalanine, a precursor for the phenylpropanoid biosynthetic pathway, as well as CG. TCA cycle intermediates levels including succinic acid and citric acid indicated a higher amount in SRs than in the others.

Transcriptional Profiling and Molecular Characterization of Astragalosides, Calycosin, and Calycosin-7-O-β-D-glucoside Biosynthesis in the Hairy Roots of Astragalus membranaceus in Response to Methyl Jasmonate.

We used the next-generation Illumina/Solexa HiSeq2000 platform on RNA analysis to investigate the transcriptome of Astragalus membranaceus hairy roots in response to 100 μM methyl jasmonate (MeJA). In total, 77,758,230 clean reads were assembled into 48,636 transcripts (average length of 1398 bp), which were clustered into 23,658 loci (genes). Of these, 19,940 genes were annotated by BLASTx searches. In addition, DESeq analysis showed that 2127 genes were up-regulated, while 1247 genes were down-regulated by MeJA. Seventeen novel astragaloside (AST) biosynthetic genes and seven novel calycosin and calycosin-7-O-β-D-glucoside (CG) biosynthetic genes were isolated. The accumulation of ASTs, calycosin, and CG increased significantly in MeJA-treated hairy roots compared with control hairy roots. Our findings will provide a valuable resource for molecular characterization of AST, calycosin, and CG biosynthetic pathways and may lead to new approaches to maximize their production and biomass productivity in the hairy roots of A. membranaceus.

The Presystemic Interplay between Gut Microbiota and Orally Administered Calycosin-7-O-β-D-Glucoside.

Presystemic interactions with gut microbiota might play important roles in the holistic action of herbal medicines in their traditional oral applications. However, research interests usually focus on biologic activities of the in vivo available herb-derived components and their exposure in circulation. In this study, we illustrated the importance of studying the presystemic interplay with gut microbiota for understanding the holistic actions of medicinal herbs by using calycosin-7-O-β-D-glucoside (C7G), the most abundant flavonoid and chemical marker in Astragali Radix, as a model compound. When C7G was orally administrated to rats, calycosin-3'-O-glucuronide (G2) was the major circulating component in the blood together with a minor calycosin but not C7G. Rat gut microbiota hydrolyzed C7G in vitro rapidly and produced its aglycone calycosin. Calycosin exhibited higher permeability than C7G and further underwent extensive glucuronidation to yield 3'-glucuronide as the dominant metabolite. Bioactivity assays revealed that G2 exhibited similar or more potent proangiogenic effects than calycosin in human umbilical vein endothelial cells in vitro and in the vascular endothelial growth factor receptor tyrosine kinase inhibitor II-induced blood vessel loss model in zebrafish. More interestingly, the incubation of C7G with gut microbiota from both normal and colitic rats showed a probiotics-like effect through stimulating the growth of the beneficial bacteria Lactobacillus and Bifidobacterium. In conclusion, C7G interacts reciprocally with gut microbiota after oral dosing, which makes it not only an angiogenic prodrug but also a modulator of gut microbiota.

Evidence of calycosin-7-O-β-d-glucoside's role as a major antioxidant molecule of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao plants under freezing stress

It was previously demonstrated that a significant increase in calycosin-7-O-β-d-glucoside (CG), the major isoflavonoid in roots of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao, is induced under low temperatures. To reveal its physiological functions under stress, seedlings with different isoflavonoid levels were established using a phenylalanine ammonia lyase (PAL) enzyme inhibitor, l-α-aminooxy-β-phenylpropionic acid (AOPP). Changes in the content of malondialdehyde (MDA), activities of antioxidant enzymes and levels of isoflavonoids were monitored in leaves and roots of this plant during 72h freezing and H2O2 treatments. Cold pretreatment significantly enhanced the freezing tolerance of this species. The plants with higher CG content showed both lower MDA content and a lower percentage of injury under freezing stress. A marked decrease in CG level over a 72h freezing treatment in all experimental groups strongly indicated that CG was transformed and might play a key role against freezing stress. At the same time, plants with a higher level of CG also showed more superoxide dismutase (SOD) and catalase (CAT) activity. These results implied that the significant promotion of antioxidant capacity in this species might be associated with the remarkable accumulation of CG after cold pretreatment. Our results provided the first evidence that a type of isoflavonoid, CG, might play a very important role against freezing stress in vivo. This report also provides a basis for future studies to reveal the mechanisms of CG's role in freezing tolerance.

Calycosin-7-O-β-d-glucoside regulates nitric oxide /caveolin-1/matrix metalloproteinases pathway and protects blood–brain barrier integrity in experimental cerebral ischemia–reperfusion injury

Ethnopharmacology relevanceAstragali Radix (AR) has been used for thousands years to treat ischemic stroke. Calycosin and its glycoside form calycosin-7-O-β-d-glucoside (CG) are two representative isoflavones in Astragali Radix. However, its neurological effects and related molecular mechanisms are largely unknown. The present study aims to evaluate the neuroprotective effects of CG on blood–brain barrier (BBB) integrity of ischemic brain tissue and explore the relevant signaling mechanisms. Material and methodMale adult Sprague-Daweley rats were subjected to 2h of middle cerebral artery occlusion (MCAO) plus 24h or 14 days of reperfusion. CG (26.8 mg/kg) was intraperitoneally administered into the rats at 15min before onset of ischemia. The neuroprotective effects of CG were evaluated by measuring infarct volume, histological damage and BBB permeability. Furthermore, the effects of CG on scavenging nitric oxide (NO), and modulating matrix metalloproteinases (MMPs) and caveolin-1 (cav-1) were investigated with in vitro cultured brain microvascular endothelial cells treated with NO donor or oxygen–glucose deprivation (OGD) and/or in vivo rat model of MCAO cerebral ischemia–reperfusion injury. ResultsCG treatment significantly reduced infarct volume, histological damage and BBB permeability in the in vivo MCAO ischemia–reperfusion rat model. CG treatment remarkably inhibited the expression and activities of MMPs, and secured the expression of cav-1 and tight junction proteins in the microvessels isolated from ischemic rat cortex. Furthermore, CG was revealed to scavenge NO, inhibit the activities of MMP-2 and MMP-9, and attenuate cell death in the in vitro cultured brain microvascular endothelial cells under OGD condition. ConclusionCG could protect BBB integrity in experimental cerebral ischemia–reperfusion injury via regulating NO/cav-1/MMPs pathway.

Ultraviolet irradiation induces accumulation of isoflavonoids and transcription of genes of enzymes involved in the calycosin-7-O-β-d-glucoside pathway in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao

Isoflavonoids are a group of phenolic secondary metabolites found almost exclusively in leguminous plants. Formononetin, calycosin and calycosin-7-O-β-d-glucoside (CG) are isoflavonoid products in the CG pathway. Accumulation of the three isoflavonoids plus daidzein and expression of six genes of enzymes involved in the CG pathway were studied in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao with ultraviolet (UV) irradiation. Our results showed that (1) main isoflavonoids in roots, stems and leaves were CG, daidzein and calycosin, respectively; they accumulated significantly under the induction of UV irradiation during 8 days but their content declined later; (2) expression of six genes of enzymes involved in the CG pathway was inhibited slightly at early stage but the expression was increased greatly afterward; (3) chalcone synthase, chalcone reductase and chalcone isomerase were expressed to their individual maximum level within shorter hours than were cinnamate 4-hydroxylase, isoflavone synthase (IFS) and isoflavone 3'-hydroxylase and (4) more calycosin but less daidzein accumulated in leaves. IFS was highly expressed in leaves, which might lead to high accumulation of the common precursor of daidzein and 2,7-dihydroxy-4'-O-methoxy-isoflavanone, the latter of which would be converted to formononetin, calycosin and CG via a series of reactions. Little daidzein accumulated in leaves, which suggested that rather than be converted to daidzein, the 2,7,4'-trihydroxyisoflavanone was probably more easily caught by 2-hydroxyisoflavanone 4'-O-methyltransferase and hence provided more precursors for formononetin. The findings were discussed in terms of the influence of UV irradiation in the accumulation of isoflavonoids.