Simultaneous separation and determination of four main isoflavonoids in Astragali Radix by an isocratic LC/ESI-MS method

A simple, reliable and rapid isocratic liquid chromatography(LC)-mass spectrometric detection(MS) coupled with electrospray ionization(ESI) method for simultaneous separation and determination of calycosin-7-O-β-D-glucoside, ononin, calycosin and formonometin in Astragali Radix was developed. After the samples were extracted with ethanol, the optimum separation conditions for these analytes were achieved using water and acetonitrile(70:30, v/v) containing 0.2%(v/v) acetic acid as a mobile phase and a 2.0 mm×150 mm Hypersil-Keystone C18 column. Selective ion monitoring(SIM) mode and [M+H]+ ions at m/z 447, 431, 285 and 269 were used for quantitative analysis of four main active components above mentioned. The calibration curves were linear in the range of 0.4-175.0 μg/mL for calycosin-7-O-β-D-glucoside, 0.2-146.0 μg/m L for ononin, 0.4-210.0 μg/mL for calycosin and 0.5-217.0 μg/mL for formonetion, respectively. The limits of quantification(LOQ) and detection(LOD) were 0.4 μg/mL and 0.08 μg/m L for calycosin-7-O-β-D-glucoside, 0.2 μg/mL and 0.06 μg/m L for ononin, 0.4 μg/mL and 0.1 μg/mL for calycosin, 0.5 μg/m L and 0.1 μg/m L formonetion, respectively. The standard recoveries were in the range of 96.5%-104.7%. The developed method has successfully been used for the determination of four main flavonoids in Astragali Radix from various sources and can be used for identification, differentiation and quality evaluation of Astragali Radix.

New isoflavonoids from Erythrina arborescens and structure revision of anagyroidisoflavone A

Five hitherto unknown isoflavonoids,namely erythrinins D-H(1-5),were isolated from the ethanol extract of Erythrina arborescens.Their structures were elucidated on the basis of extensive spectroscopic studies.In addition,the structure of anagyroidisoflavone A(6a)has been revised as 1″-O-methylerythrinin F(6)by re-analysis of the original spectroscopic data.

Antimicrobial activity and DNA-fragmentation effect of isoflavonoids isolated from seeds of Millettia ferruginea,an endemic legume tree in Ethiopia

Objective:To investigate the seeds of Millettia ferruginea(M.ferruginea)to unravel its antibacterial,antifungal,antitubercular,and antileishmanial potential for the first time.Methods:M.ferruginea seeds were refluxed separately with chloroform,methanol and water to prepare the three extracts,which were tested against the reference strains of Gram-positive and Gram-negative bacteria,yeast cells,Mycobacterium tuberculosis and Leishmania donovani(L.donovani)promastigotes.Next,the seeds were chemically analysed to isolate three constituent compounds,viz.,barbigerone,calopogonium isoflavone-A and durmillone,which were purified,characterised and evaluated for antibacterial and antileishmanial activity.Further,Comet assay was conducted to observe DNA fragmentation effects on human peripheral blood mononuclear cells pretreated with the isoflavonoid compounds.Results:The chloroform and methanol extracts of M.ferruginea seeds exhibited antibacterial and antileishmanial activity.The pure compounds also showed inhibitory activity against Gram-negative ATCC strains(minimum inhibitory concentration~0.5μmol/L),and L.donovani promastigotes(IC_(50)8.2-87.3μg/mL).However,they had little or no activity against yeast cells and tubercle bacilli.The DNA fragmentation study showed that the isoflavonoid constituents of M.ferruginea seeds were safe at therapeutic doses.Conclusions:The antibacterial efficacy of the non-aqueous extracts of M.ferruginea seed was observed against both Gram-positive and Gram-negative ATCC strains.Moreover,the constituents isoflavonoids,viz.,barbigerone,calopogonium isoflavone-A and durmillone,exhibited inhibitory activity against Gram-negative ATCC strains and L.donovani promastigotes.The comet assay showed that the compounds were safe to be considered for human consumption.

Investigation of the Interaction between Isoflavonoids and Bovine Serum Albumin by Fluorescence Spectroscopy

The interactions of bovine serum albumin （BSA） with three structurally related isoflavonoids, genistein, puerarin and daidzein, were studied under physiological conditions by fluorescence spectroscopic technique. The quenching mechanism of these compounds with BSA was suggested as static quenching and the binding constants were determined at different temperatures based on the fluorescence quenching results. The transfer efficiency of energy and distance between the acceptor and BSA were investigated on the basis of the mechanism of the Forster energy transference. According to the thermodynamic parameters it has been suggested that the acting force be mainly hydrophobic force. The comparison of binding potency of the three isoflavonoids to BSA showed that the substitution by 5-OH and 8-Glc could enhance the binding affinity. All these obtained in the work can make us better understand the mode of the action and pharmacological activities of the isoflavonoids.

Chemical Constituents from Pueraria lobata

Pueraria lobata ( Wild. ) Ohwi ( Ye-Ge in Chinese ) is a perennial herb ofthe genus Pueraria, which belongs to the Leguminosae family. It is commonly employed to relievefever and dysentery, promote the production of body fluid, reduce stiffness and pain of the nape,and for the treatment of cardiovascular diseases, e. g. hypertension, myocardial infarction, andarrhythmia. Previous phytochemical studies on P. lobata reported a number of bioactive isoflavones,e. g. daidzein, daidzin, and puerarin. Further investigation of its root has led to isolation offourteen compounds and their structures were identified as daidzein, ononin, daidzin, 3'' -methoxypuerarin, puerarin, pueroside B, daidzein-8-C-apiosyl- (1-6)-glucoside, 3''-hydroxy-puerarin,puerarinxyloside, daidzein-7, 4'' -O-glucoside, puerarin-4''-O-glucoside, mirificin-4''-O-glucoside,sissotorin, and pueroside C. Compounds 11 and 13 were isolated from the root of P. lobata for thefirst time.

Semisynthetic Derivatives of Sissotrin Isolated from Trifolium baccarinii Chiov. (Fabaceae) and Evaluation of Their Antibacterial Activities

Starting from sissotrin (1), a natural isoflavonoid isolated from Trifolium baccarinii (Fabaceae), one new semisynthetic derivative, 6-nitrobiochanin A (1b) and two known derivatives, 8-nitrobiochanin A (1a) and 2",3",4",6"-tetraacetylsissotrin (1c) have been obtained after performing nitration and acetylation reactions. Their structures were assigned after interpretation of their spectrometric (HR-ESI-MS) and spectroscopic (NMR 1D and 2D) data and by comparison with those reported in the literature. The substrate as well as the semisynthetic derivatives were evaluated for their antibacterial activities against six strains. The results reveal that they are inactive or weakly active on the strains tested with the exception of 8-nitrobiochanin A (1a) which showed moderate activity (MIC = 62.5 μg∙mL−1) on Staphylococcus aureus ATCC 43300.

Chemical and microbiological characterization of tinctures and microcapsules loaded with Brazilian red propolis extract

The aim of this study was to characterize tinctures and microcapsules loaded with an ethanol extract of red propolis through chemical, physicochemical and microbiological assays in order to establish quality control tools for nutraceutical preparations of red propolis. The markers(isoflavonoids, chalcones, pterocarpans,flavones, phenolic acids, terpenes and guttiferones) present in the tinctures A and B were identified and confirmed using LC/ESI/FTMS/Orbitrap. Four compositions(A, B, C and D) were prepared to contain B tincture of the red propolis with some pharmaceutical excipients and submitted to two drying processes, i. e.spray-drying and freeze-drying to obtain microcapsules loaded with the red propolis extract. The tinctures and microcapsules of the red propolis were submitted to the total flavonoid content and antioxidant activity tests.The antibacterial activity and minimum inhibitory concentration(MIC) were tested using Staphylococcus aureus ATCC 25293 and Pseudomonas aeruginosa ATCC 27853 strains. The tinctures and microcapsules presented high flavonoid quantities from 20.50 to 40.79 mg/100 mg of the microcapsules. The antioxidant activity and IC50 were determined for the tinctures A and B(IC50: 6.95 μg/m L and 7.48 μg/m L), the spray-dried microcapsules(IC50: 8.89–15.63 μg/m L) and the freeze-dried microcapsules(IC50: 11.83–23.36 μg/m L). The tinctures and microcapsules were proved to be bioactive against gram-positive and gram-negative bacteria with inhibition halos superior to 10 mm at concentration of 200 μg/m L and MIC values of 135.87–271.74 μg/m L using gram-positive strain and 271.74–543.48 μg/m L using gram-negative strain. The tinctures and microcapsules of the red propolis have a potential application for nutraceutical products.

Effects of Rare Earth Elements on Promoting Isoflavonoid Production and Release of Pueraria Lobata Hairy Roots in Vitro

The effects of various rare earth elements on growth and isoflavonoid production in hairy root cultures of Pueraria lobata (Willd.) Ohwi (strain TR2) cultured in 500 ml flasks were studied. After 32 days of culture, the biomass of hairy roots increase 15 times and reach 3.2 g dry weight. Hairy root growth was inhibited by Y_2O_3, NaSeO_3 and Sm^(3+) because of brown formation. But significant promoting effect on root growth due to callus formation was observed in La^(3+) treatments. It is the most noteworthy that the production of total isoflavonoids and puerarin was enhanced greatly by La^(3+) treatment. A major portion of increased total isoflavonoids and puerarin was released into medium in La^(3+) treatment while the hairy root viabilities were preserved. Some specific secondary metabolite release processes could be induced by La^(3+) and their possible mechanism is discussed.

Severe &Moderate BPH Symptoms in Mid-Aged Men Improve with Isoflavonoid-Equol Treatment: Pilot Intervention Study

Benign prostatic hyperplasia (BPH) is the pathological cellular progression of glandular proliferation associated with aging. Current available treatment options for BPH have limitations and various adverse effects. Equol is a polyphenolic/isoflavonoid molecule derived from intestinal metabolism, dairy and dietary plant sources. It has the unique characteristic to bind specifically 5α-dihydrotestosterone (5α-DHT) by sequestering 5α-DHT from the androgen receptor, thus decreasing androgen hormone actions to improve prostate health by acting as a selective androgen modulator (SAM). It also has affinity for estrogen related receptor gamma (ERR-γ) and estrogen receptor beta (ER-β) within the prostate that is known to improve male health via selective estrogen receptor modulatory (SERM) activities to decrease inflammation, cellular proliferation and carcinogenesis. We investigated the possible clinical efficacy of equol on the symptoms associated with benign prostatic hyperplasia (BPH) in this study. Materials and Methods: We performed a pilot intervention study evaluating the effects of low dose oral equol supplement (6 mg, twice a day with meals) for 4 weeks in a total of 18 men (49 - 60 years old) with moderate or severe BPH. Subjects included in the study: gave informed consent, underwent a physical examination and verified their BPH symptoms as measured by the International Prostate Symptom Scores (IPSS) and then were assigned to the moderate or severe BPH groups based upon their total IPSS index. All adverse events were reported. The primary efficacy measure was the IPSS parameters comparing baseline to 2 and 4 week IPSS indices. Blood samples were collected at the baseline and 4th week visits that served as secondary efficacy parameters that included testosterone, 5α-DHT and general blood chemistries along with cardiac and hepatic function panels. Results: Low dose equol positively improved moderate to severe BPH symptoms according to the IPSS indices. In moderately symptomatic men (n = 10) 5 out of 7 of the IPSS parameters significantly improved by 4 weeks of equol treatment. In severely symptomatic men (n = 8) all 7 of the IPSS parameters significantly improved with 4 weeks of equol treatment. There were no significant changes in androgen levels, general blood chemistries or cardiac and hepatic function parameters. Although, 5α-DHT levels declined by 21% in severely symptomatic men (from baseline vs. 4 week values). Conclusion: These findings suggest that equol may provide a well tolerated and rapid beneficial therapy for BPH that can be used alone or in combination with current pharmaceutical therapies. The beneficial clinical efficacy of equol observed in this study may be due to the multiple positive biological actions that are not present in current pharmaceutical treatments.

Derrisisoflavones H-K and One Isoflavan Derivative from Derris robusta

Four hitherto unknown prenylated isoflavonoids,named derrisisoflavones H-K(1-4)and one new isoflavan,namely 6-hydroxyisosativan(5),were isolated from the ethanol extract of Derris robusta.Their structures were elucidated on the basis of extensive spectroscopic studies.To our knowledge,derrisisoflavones J(3)and K(4)are the first examples of hydroxyethylated isoflavonoid.

Review: Anti-Oxidant and Anti-Aging Properties of Equol in Prostate Health (BPH)

Benign prostatic hyperplasia (BPH) is the pathological cellular progression of glandular proliferation associated with aging. The primary changes in prostate disorders are mediated by the conversion of the principle androgen, testosterone, to its more potent metabolite, 5α-dihydrotestosterone (5α-DHT). However, recent evidence suggests that estrogen hormonal actions via estrogen receptor subtypes also play an important role in BPH. Current pharmaceutical options for BPH have advantages, limitations and adverse effects. Complementary and Alternative Medicine (CAM) treatments for BPH include botanicals such as polyphenols and isoflavones. Equol is a polyphenolic/isoflavonoid molecule derived from intestinal metabolism, dairy and dietary plant sources. Equol has potent anti-oxidant and anti-aging properties to decrease prostatic irritation and potentially neoplastic growth. It has the unique characteristic to bind specifically 5α-DHT by sequestering 5α-DHT from the androgen receptor (AR), thus decreasing androgen hormone actions to improve prostate health by acting as a selective androgen modulator (SAM). It also has affinity for estrogen related receptor gamma (ERR-γ) and estrogen receptor beta (ER-β) within the prostate that is known to improve male health via selective estrogen receptor modulatory (SERM) activities to decrease inflammation, cellular proliferation and carcinogenesis. The possible clinical efficacy of equol on the symptoms associated with BPH is presented and the reviewed findings suggest that equol may provide a well-tolerated and rapid beneficial therapy for BPH that can be used alone or in combination with current pharmaceutical therapies. The beneficial clinical efficacy of equol observed may be due to the multiple positive biological actions that are not present in current pharmaceutical treatments.

Decoding the biochemical dialogue:metabolomic insights into soybean defense strategies against diverse pathogens

Soybean,a crucial global leguminous crop,confronts persistent threats from diverse pathogens,exerting a profound impact on global yields.While genetic dimensions of soybean-pathogen interactions have garnered attention,the intricate biochemical responses remain poorly elucidated.In this study,we applied targeted and untargeted liquid chromatography coupled to mass spectrometry(LC-MS)metabolite profiling to dissect the complex interplay between soybeans and five distinct pathogens.Our analysis uncovered 627 idMS/MS spectra,leading to the identification of four main modules,encompassing flavonoids,isoflavonoids,triterpenoids,and amino acids and peptides,alongside other compounds such as phenolics.Profound shifts were observed in both primary and secondary metabolism in response to pathogenic infections.Particularly notable were the bidirectional changes in total flavonoids across diverse pathogenic inoculations,while triterpenoids exhibited a general declining trend.Noteworthy among the highly inducible total flavonoids were known representative antipathogen compounds(glyceollin I),backbone forms of isoflavonoids(daidzein,genistein,glycitein,formononetin),and newly purified compounds in this study(prunin).Subsequently,we delved into the biological roles of these five compounds,validating their diverse functions against pathogens:prunin significantly inhibited the vegetative growth and virulence of Phytophthora sojae;genistein exhibited a pronounced inhibitory effect on the vegetative growth and virulence of Phomopsis longicolla;daidzein and formononetin displayed significant repressive effects on the virulence of P.longicolla.This study underscores the potent utility of metabolomic tools,providing in-depth insights into plant-pathogen interactions from a biochemical perspective.The findings not only contribute to plant pathology but also offer strategic pathways for bolstering plant resistance against diseases on a broader scale.

The GmSTF1/2–GmBBX4 negative feedback loop acts downstream of blue-light photoreceptors to regulate isoflavonoid biosynthesis in soybean

Isoflavonoids,secondary metabolites derived from the phenylalanine pathway,are predominantly bio-synthesized in legumes,especially soybean(Glycine max).They are not only essential for plant responses to biotic and abiotic stresses but also beneficial to human health.In this study,we report that light signaling controls isoflavonoid biosynthesis in soybean.Blue-light photoreceptors(GmCRY1s,GmCRY2s,GmPHOT1s,and GmPHOT2s)and the transcription factors GmSTF1 and GmSTF2 promote isoflavonoid accumulation,whereas the E3 ubiquitin ligase GmCOP1b negatively regulates isoflavonoid biosynthesis.GmPHOT1s and GmPHOT2s stabilize GmSTF1/2,whereas GmCOP1b promotes the degradation of these two proteins in soybean.GmSTF1/2 regulate the expression of approximately 27.9%of the genes involved in soybean isoflavonoid biosynthesis,including GmPAL2.1,GmPAL2.3,and GmUGT2.They also repress the expression of GmBBX4,a negative regulator of isoflavonoid biosynthesis in soybean.In addition,GmBBX4 physically interacts with GmSTF1 and GmSTF2 to inhibit their transcriptional activation activity toward target genes related to isoflavonoid biosynthesis.Thus,GmSTF1/2 and GmBBX4 form a negative feedback loop that acts downstream of photoreceptors in the regulation of isoflavonoid biosynthesis.Our study provides novel insights into the control of isoflavonoid biosynthesis by light signaling in soybean and will contribute to the breeding of soybean cultivars with high isoflavonoid content through genetic and metabolic engineering.

Quality evaluation of Radix Astragali from different sources in China

Radix Astragali （Huangqi） is one of the most valuable traditional Chinese medicinal herbs. It is used in the traditional Chinese medicine to reinforce ＇qi＇, and it has immunostimulant, tonic, and antioxidant activities. There are many different sources of Huangqi in the market. In this study, the quality of Huangqi was evaluated by the measurement of four flavonoids, calycosin-7-O-β-D-glucopyranoside, ononin, calycosin and formononetin with HPLC-UV, as well as astragaloside IV with HPLC-ELSD. Samples included different plant species, different places of cultivation, different ages of plants, different seasons of collection and different commercial specifications. The results showed that the contents of isoflavonoids and astragaloside IV varied significantly in different sources. Our study provided useful information for the quality evaluation of Radix Astragali.

Isolation and structural elucidation of an unstable isoflavonoid glycoside malonate from Radix hedysari

Based on the stability results of Radix hedysari extract, an unstable compound was extracted using acidic methanol and purified by rapid chromatographic methods successfully from Radix hedysari for the first time guided by HPLC analysis. Its structure was identified as formononetin 7-O-β-D-（6＂-O-malonyl）-glucopyranoside by spectroscopic analyses.

New dual functional CYP450 gene involves in isoflavone biosynthesis in Glycine max L.

Glycine max L.accumulates a large amount of isoflavonoid compounds,which is beneficial for plant defense,plant-microbe symbiotic interactions,and human health.Several CYP450 subfamily genes are involved in the flavonoid biosynthetic pathway in plants.In the present study,we found 24 CYP82 subfamily genes were differentially expressed in various tissues of soybean,in Phytophthora sojae-infected soybean varieties and in soybean hairy roots treated with cell wall glucan elicitor.Six of them(GmCYP82A2,GmCYP82A3,GmCYP82A4,GmCYP82A23,GmCYP82C20 and GmCYP82D26)were co-expressed with other known isoflavonoid pathway genes in soybean.Their enzymatic activity in yeast feeding assays showed that only GmCYP82D26 was able to convert naringenin to daidzein with both aryl migration and dehydration function.When GmCYP82D26 was over-expressed in soybean hairy roots,the contents of the two major isoflavonoid aglycones in soybean(daidzein and genistein)were reduced,but total flavonoids were not affected.When GmCYP82D26 was suppressed by RNAi in the hairy roots,daidzein content was decreased but genistein content was increased,with unchanged total flavonoid content.GmCYP82D26 was found to be localized in the endoplasmic reticulum at subcellular level when transiently expressed in tobacco leaf epidermis.GmCYP82D26 gene was preferentially expressed in roots,with low expression level in other tissues in soybean.Homology modeling and molecular docking showed that GmCYP82D26 could form hydrogen bond with both HEM and naringenin at C5-OH and C4 carbonyl.All these results indicated that GmCYP82D26 possesses new and dual enzymatic activity,which bridges the two branches(daidzein and genistein branch)of isoflavonoid pathway in soybean.

Chemical Constituents from Dried Aerial Parts of Eclipta prostrata

Abstract: Objective To study the constituents in the dried aerial parts of Eclipta prostrata. Methods The constituents wereisolated and purified by column chromatography and their structures were elucidated by spectroscopic methods (1D,2D NMR, UV, IR, and HRESI-TOF-MS) and chemical analyses. Results Eight compounds were isolated andidentified as 7-O-methylorobol-4′-O-β-D-glucopyranoside (1), 3′-hydroxybiochanin A (2), echinocystic acid28-O-β-D-glucopyranoside (3), ecliptasaponin A (4), eclalbasaponin I (5), eclalbasaponin IV (6), echinocystic acid(7), and 3-oxo-16α-hydroxy-olean-12-en-28-oic acid (8). Conclusion Compound 1 is a new compound andcompound 3 is obtained from this genus for the first time.

A New Isoflavonoid from Belamcanda chinensis （L.） DC.

A new isoflavonoid, 5, 6, 7, 3＇-terahydroxy-8, 4＇, 5＇-trimethoxyisoflavone （1）, along with 10 known isoflavonoids, namely 5, 6, 7, 4＇-tetrahydroxy-8-methoxyisoflavone （2）, irilone （3）, genistein （4）, tectorigenin （5）, irigenin （6）, irisflorentin （7）, dichotomitin （8）, dimethyltectorigenin （9）, iridin （10）, and tectoridin （11）, was isolated from the alcohol extract of the rhizomes of Belamcanda chinensis （L.） DC. The structures of these compounds were elucidated on the basis of results of spectroscopic analysis.

Effects of a novel isoflavonoid from the stem bark of Alstonia scholaris against fructose-induced experimental cataract

Objective: The present study investigated the anticataract activity of a novel isoflavonoid, isolated from stem bark of Alstonia scholaris, against fructose-induced experimental cataract.Methods: The bioactivity of fractions extracted from A. scholaris, an isolated isoflavonoid(ASII) was screened using in vitro(goat lens) and in vivo(albino rats) experimental cataract models. For the in vivo evaluation, albino rats(12–15 weeks old) were divided into five groups(n = 6). Group I(normal)received 0.3% carboxymethyl cellulose solution(10 m L/[kgád], p.o.). Group II(control) received 10%(w/v)fructose solution in their drinking water. Groups III–V received ASII at three different doses, 0.1, 1.0 and10 mg/(kgád), concurrently with 10%(w/v) fructose solution. Treatment was given daily for 8 consecutive weeks. During the protocol, systolic blood pressure, diastolic blood pressure, blood glucose level and lenticular opacity were monitored at 2-week intervals. Pathophysiological markers(catalase, superoxide dismutase, glutathione peroxidase, reduced glutathione and malondialdehyde) in eye lenses were examined at the end of the 8-week treatment period.Results: The results of in vitro study showed that A. scholaris extract and the active fraction(A3) reduced the lenticular opacity as compared to toxic control group. The in vivo study showed that 8-week administration of ASII(0.1, 1.0 and 10 mg/[kgád], p.o.) led to significant reduction in blood pressure and blood glucose level and retarded the initiation and evolution of cataractogenesis, compared to the fructoseinduced cataract model control. Additionally, ASII treatment led to significant improvement in lens antioxidants(catalase, superoxide dismutase, glutathione peroxidase and reduced glutathione) and decreased lens malondialdehyde, compared to the control group(group II).Conclusion: Results revealed that administration of ASII played a crucial role in the reduction of cataract formation in diabetic and hypertensive models.

Pharmacological and therapeutic properties of the Red Clover(Trifolium pratense L.):an overview of the new findings

OBJECTIVE:To review beneficial effects of red clover(Trifolium pratense L.)on biological processes.METHODS:A systematic review was carried out according tothe PRISMA checklist.Using Me SH keywords,385 articles were extracted from national and international databases(Pub Med,Science Direct,SID,and Google Scholar search engine)without time limit.After removing duplicates,80 articles were reviewed.RESULTS:Our findings revealed that the compositions of red clover promote anti-oxidant and anti-cancer effects.Furthermore,they exude beneficial effects on cardiovascular functionand improvesymptoms of menopause.Also,these compounds can regulate blood glucose and lipid markers.The effects of the herb on have also been investigated on various tissues including endometrium,breast,skin and reproductive system.CONCLUSION:In recent studies,the anti-cancer effects of clover(Trifolium)extract co-administrated with standard drugs have been reportedin different cancers.