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.

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.

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.

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.

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.

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.

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.

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.

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.

异戊烯基转移酶催化异黄酮类的异戊烯基化研究进展

异戊烯基异黄酮具有优良的生物活性,广泛应用于医药、食品等领域,异戊烯基是其药效基团之一。化学法进行异戊烯基化反应存在化学、区域选择性差,且保护羟基与去保护步骤多,限制了该反应的研究和开发。在植物体内,异戊烯基转移酶催化使异戊烯基供体转移到异黄酮底物上。植物来源的异戊烯基转移酶是膜蛋白,催化效率低,分离纯化比可溶性蛋白困难。真菌来源的异戊烯基转移酶是可溶的,具有广泛的底物和供体杂泛性,具有成为生物催化剂的潜力,并且能接受异黄酮作为底物。本论文对近20年关于异戊烯基异黄酮的结构特点、酶催化异黄酮的异戊烯基化的研究进展进行综述。

New Isoflavonoid Glycosides from the Rhizomes of Iris leptophylla Lingelsh.

Two new isoflavonoid glucosides, 5-hydroxy-6,7-methylenedioxy-isoflavone-4＇-O-D-glucopyranosyl （2→〉l）-L-rhamnoside （irilone-bioside） （compound 1） and 5,4＇-methoxy-6,7-methylenedioxyisoflavone-3＇-O-β-D-glucoside （irisleptophyllidin） （compound 2）, together with five known compounds, nigricanin- 4＇-O-β-D-glucoside （compound 3）, irifloside （compound 4）, irigenin （compound 5）, 5, 3＇, 4＇-trimethoxy-6,7-methylenedioxyisoflavone （compound 6）, and nigricanin （compound 7） were isolated from the alcoholic extract of rhizomes of Iris leptophylla Lingelsh. Their structures were elucidated by spectroscopic methods.

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.

药用植物异黄酮类化学成分及其药理作用的研究进展

异黄酮类化合物的基本母核为3-苯基色原酮,具有良好的药理作用。对2013—2022年国内外报道的新异黄酮类化合物的化学结构进行综述;归纳其氢谱核磁共振及碳谱核磁共振数据的特点,对其δH-2位进行了重新归属;并对其抗肿瘤、抗氧化、抑菌等方面的药理作用进行总结,以期为异黄酮类化合物的开发应用提供理论参考。

超高效液相色谱法分析淡豆豉配方颗粒中3种异黄酮

建立超高效液相色谱法同时测定淡豆豉配方颗粒中3种异黄酮(大豆苷元、黄豆黄素、染料木素)的含量。选择Eclipse Plus C18RRHD色谱柱(100 mm×2.1 mm,1.8μm),流动相为乙腈-0.1%乙酸溶液,梯度洗脱,流量为0.3mL/min,柱温为30℃,检测波长为260 nm。大豆苷元、黄豆黄素、染料木素的质量浓度分别在18.41~184.14、0.48~4.82、30.65~306.46μg/mL范围内线性关系良好(r≥0.999 9)。样品测定结果的相对标准偏差分别为1.80%、2.20%、1.71%(n=6),平均加标回收率分别为96.49%、100.93%、98.53%。该方法快速,准确、可靠,可为淡豆豉配方颗粒的质量控制提供参考。

茉莉酸甲酯调控牛大力有效成分积累及相关基因表达

目的:研究外源茉莉酸甲酯(methyl Jasmonate,MeJA)对牛大力有效成分含量的影响,探究其对异黄酮生物合成以及茉莉酸(jasmonate,JA)信号通路关键酶基因的调控模式。方法:以牛大力幼苗为材料,采用HPLC法分析MeJA对有效成分含量的影响,利用Illumina HiSeq平台进行转录组测序,并利用qRT-PCR技术验证相关基因的表达量。结果:外源MeJA诱导提高了牛大力有效成分芒柄花素与高丽槐素的含量。基于牛大力参考基因组数据库,鉴定出20727个差异基因(DEGs),其中9091个为上调基因,主要富集在植物激素信号转导、淀粉和蔗糖代谢、异黄酮生物合成等代谢通路,qRT-PCR结果验证了转录组测序结果准确可靠。外源MeJA激活了部分异黄酮生物合成和JA信号通路关键酶基因的表达,其中异黄酮生物合成途径基因CHS、HIDs、I3′Hs、VR、PTR、CYP81E7、7IOMT、PTS等呈上调表达;JA信号通路基因LOXs、AOSs、OPR、ACXs、JAZs和MYCs等呈上调表达。结论:牛大力在响应外源MeJA诱导的过程中可激活CHS、HID-1、VR等异黄酮生物合成途径和LOX、MYC等JA信号通路关键酶基因,从而正向调控其有效成分芒柄花素与高丽槐素含量。

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.

膜荚黄芪毛状根中异黄酮成分的反相高效液相色谱分析

对膜荚黄芪毛状根中６种异黄酮成分进行了反相高效液相色谱法测定。色谱柱为ＮｕｃｌｅｏｓｉｌＣ１８柱，流动相为甲醇—水（３∶２和３∶１，ｖ／ｖ），检测波长为２５４ｎｍ和２８０ｎｍ。６种异黄酮成分为：１０羟基３，９二甲氧基紫檀烷，（３Ｒ）８，２’二羟基７，４’二甲氧基异黄烷，芒柄花素（７羟基４’甲氧基异黄酮），８，３’二羟基７，４’二甲氧基异黄酮，２’羟基３’，４’二甲氧基异黄烷７Ｏ葡萄吡喃糖甙，毛蕊异黄酮（７，３’二羟基４’甲氧基异黄酮）。异黄酮浓度在２５～１２５μｇ范围内与峰面积有良好的线性关系；加样回收率为９６４７％～１０３３３％；精密度试验相对标准偏差为２５７％～６５２％；测得黄芪毛状根中６种异黄酮成分的含量在００００５％～０００６５％之间。

槐果皮中的异黄酮甙类成分

目的 :对槐果皮进行化学成分研究。方法 :采用波谱和化学方法对 6 6个异黄酮甙类成分进行了鉴定。结果 :从槐 (Sophorajaponica)果皮中分离得到 5个异黄酮甙类成分。它们的结构确定为染料木素 7,4′ 二葡萄糖甙 (genistein 7,4′ di O β D glucoside ,Ⅰ ) ,槐属双甙 (sophorabioside ,Ⅱ ) ,樱黄素 4′ O β D 葡萄糖甙 (prunetin4′ O β D glucoside ,Ⅲ ) ,槐属甙 (sophororicoside ,Ⅳ ) ,染料木甙 (genistin ,Ⅴ )。 结论 :化合物Ⅰ。

射干的化学成分研究(Ⅰ)

目的 :研究中药射干的化学成分。方法 :采用硅胶柱层析及SephadexLH 2 0等色谱技术分离纯化化合物 ,根据理化性质及光谱数据鉴定结构。结果 :从射干乙醇提取物的乙醚萃取部分分得 8个化合物 ,其中 6个异黄酮 ,分别为德鸢尾素 (irilone ,Ⅰ )、染料木素 (genistein ,Ⅱ )、鸢尾甙元 (tectorigenin ,Ⅲ )、野鸢尾黄素 (irigenin ,Ⅳ )、二甲基鸢尾甙元 (dimethyltectorigenin ,Ⅴ )、次野鸢尾黄素 (irisflorentin ,Ⅵ ) ;另外 2个化合物为 β 谷甾醇 (Ⅶ )和豆甾醇 (Ⅷ )。结论